| 1. |
van Selm S,
van Cann LM,
Kolkman MA,
van der Zeijst BA,
van Putten JP,
( 2003 ) Genetic basis for the structural difference between Streptococcus pneumoniae serotype 15B and 15C capsular polysaccharides. PMID : 14573636 : DOI : 10.1128/iai.71.11.6192-6198.2003 PMC : PMC219561 Abstract >>
In a search for the genetic basis for the structural difference between the related Streptococcus pneumoniae capsular serotypes 15B and 15C and for the reported reversible switching between these serotypes, the corresponding capsular polysaccharide synthesis (cps) loci were investigated by keeping in mind that at the structural level, the capsules differ only in O acetylation. The cps locus of a serotype 15B strain was identified, partially PCR amplified with primers based on the related serotype 14 sequence, and sequenced. Sequence analysis revealed, among other open reading frames, an intact open reading frame (designated cps15bM) whose product, at the protein level, exhibited characteristics of previously identified acetyltransferases. Genetic analysis of the corresponding region in a serotype15C strain indicated that the same gene was present but had a premature stop in translation. Closer analysis indicated that the serotype 15B gene contained a short tandem TA repeat consisting of eight TA units. In serotype 15C, this gene contained nine TA units that resulted in a frameshift and a truncated product. Genetic analysis of 17 serotype 15B and 15C clinical isolates revealed a perfect correlation between the serotype and the length of the short tandem repeat in the putative O-acetyltransferase gene. The number of TA repeating units varied between seven and nine in the various isolates. Together, the data strongly suggest that the structural difference between serotypes 15B and 15C is based on variation in the short tandem TA repeat in the O-acetyltransferase gene and that the transition between serotypes is due to slipped-strand mispairing with deletion or insertion of TA units in the cps15bM gene.
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2. |
Martin C,
Sibold C,
Hakenbeck R,
( 1992 ) Relatedness of penicillin-binding protein 1a genes from different clones of penicillin-resistant Streptococcus pneumoniae isolated in South Africa and Spain. PMID : 1396576 : PMC : PMC556892 Abstract >>
Penicillin-resistant strains of Streptococcus pneumoniae have been common in South Africa and Spain for several years. Multilocus enzyme electrophoresis identified one clone of capsular type 6B which was prevalent in Spain and another clone of type 23F that was present in both countries. Genes for penicillin-binding proteins (PBPs) in penicillin-resistant strains are often mosaics where parts of the pneumococcal genes are replaced by homologous genes from other species. We have compared the mosaic structures of the PBP 1a genes from the two clones as well as from genetically distinct South African isolates. Four classes of mosaic PBP 1a genes were found that contained blocks of sequences divergent by 6-22% from those of sensitive genes; two classes contained sequences coming from more than one external source. Data are presented showing that the PBP 1a genes from the 23F and the 6B clone are related, and that the two PBP 1a genes from the South African isolates are also related. We suggest that the type 23F clone originated in Spain prior to distribution into other continents.
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3. |
Martín-Galiano AJ,
Balsalobre L,
Fenoll A,
de la Campa AG,
( 2003 ) Genetic characterization of optochin-susceptible viridans group streptococci. PMID : 14506029 : DOI : 10.1128/aac.47.10.3187-3194.2003 PMC : PMC201122 Abstract >>
Two clinical isolates of viridans group streptococci (VS) with different degrees of susceptibility to optochin (OPT), i.e., fully OPT-susceptible (Opt(s)) VS strain 1162/99 (for which the MIC was equal to that for Streptococcus pneumoniae, 0.75 micro g/ml) and intermediate Opt(s) VS strain 1174/97 (MIC, 6 micro g/ml) were studied. Besides being OPT susceptible, they showed characteristics typical of VS, such as bile insolubility; lack of reaction with pneumococcal capsular antibodies; and lack of hybridization with rRNA (AccuProbe)-, lytA-, and pnl-specific pneumococcal probes. However, these VS Opt(s) strains and VS type strains hybridized with ant, a gene not present in S. pneumoniae. A detailed characterization of the genes encoding the 16S rRNA and SodA classified isolates 1162/99 and 1174/97 as Streptococcus mitis. Analysis of the atpCAB region, which encodes the c, a, and b subunits of the F(0)F(1) H(+)-ATPase, the target of optochin, revealed high degrees of similarity between S. mitis 1162/99 and S. pneumoniae in atpC, atpA, and the N terminus of atpB. Moreover, amino acid identity between S. mitis 1174/97 and S. pneumoniae was found in alpha helix 5 of the a subunit. The organization of the chromosomal region containing the atp operon of the two Opt(s) VS and VS type strains was spr1284-atpC, with spr1284 being located 296 to 556 bp from atpC, whereas in S. pneumoniae this distance was longer than 68 kb. In addition, the gene order in S. pneumoniae was IS1239-74 bp-atpC. The results suggest that the full OPT susceptibility of S. mitis 1162/99 is due to the acquisition of atpC, atpA, and part of atpB from S. pneumoniae and that the intermediate OPT susceptibility of S. mitis 1174/97 correlates with the amino acid composition of its a subunit.
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4. |
Díaz E,
López R,
García JL,
( 1992 ) Role of the major pneumococcal autolysin in the atypical response of a clinical isolate of Streptococcus pneumoniae. PMID : 1355082 : DOI : 10.1128/jb.174.17.5508-5515.1992 PMC : PMC206493 Abstract >>
The autolytic enzyme (an N-acetylmuramyl-L-alanine amidase) of a clinical isolate, strain 101/87, which is classified as an atypical pneumococcus, has been studied for the first time. The lytA101 gene coding for this amidase (LYTA101) has been cloned, sequenced, and expressed in Escherichia coli. The LYTA101 amidase has been purified and shown to be similar to the main autolytic enzyme (LYTA) present in the wild-type strain of Streptococcus pneumoniae, although it exhibits a lower specific activity, a higher sensitivity to inhibition by free choline, and a modified thermosensitivity with respect to LYTA. Most important, in contrast with the LYTA amidase, the activity of the LYTA101 amidase was inhibited by sodium deoxycholate. This property is most probably responsible of the deoxycholate-insensitive phenotype shown by strain 101/87. Phenotypic curing of strain 101/87 by externally adding purified LYTA or LYTA101 amidase restored in this strain some typical characteristics of the wild-type strain of pneumococcus (e.g., formation of diplo cells and sensitization to lysis by sodium deoxycholate), although the amount of the LYTA101 amidase required to restore these properties was much higher than in the case of the LYTA amidase. Our results indicate that modifications in the primary structure or in the mechanisms that control the activity of cell wall lytic enzymes seem to be responsible for the characteristics exhibited by some strains of S. pneumoniae that have been classically misclassified and should be now considered atypical pneumococcal strains.
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5. |
del Solar G,
Espinosa M,
( 1992 ) The copy number of plasmid pLS1 is regulated by two trans-acting plasmid products: the antisense RNA II and the repressor protein, RepA. PMID : 1371181 : DOI : 10.1111/j.1365-2958.1992.tb00840.x Abstract >>
The promiscuous plasmid pLS1 encodes two transacting elements that regulate its copy number: protein RepA and antisense RNA II. In vitro transcription showed that RNAs for both repressors are synthesized from two promoters, PAB and PII. From PAB, genes encoding RepA (transcriptional repressor) and RepB (initiator of replication) are cotranscribed, the target of RepA being located within PAB. Mutants in repA or in PAB are still sensitive to RepA. However, cloning of the repA gene in a compatible replicon did not result in incompatibility towards pLS1. From PII, the 50-nucleotide RNA II is synthesized. The main incompatibility determinant towards pLS1 corresponds to the coding sequence for RNA II. The RNA II target could be reduced to 21 nucleotides, including the RepB initiation of translation signals. We propose that plasmids of the pLS1 family (pE194, pADB201, and pLB4) share functional and structural characteristics for the regulation of their copy numbers.
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6. |
Gentry DR,
Ingraham KA,
Stanhope MJ,
Rittenhouse S,
Jarvest RL,
O'Hanlon PJ,
Brown JR,
Holmes DJ,
( 2003 ) Variable sensitivity to bacterial methionyl-tRNA synthetase inhibitors reveals subpopulations of Streptococcus pneumoniae with two distinct methionyl-tRNA synthetase genes. PMID : 12760849 : DOI : 10.1128/aac.47.6.1784-1789.2003 PMC : PMC155832 Abstract >>
As reported previously (J. R. Jarvest et al., J. Med. Chem. 45:1952-1962, 2002), potent inhibitors (at nanomolar concentrations) of Staphylococcus aureus methionyl-tRNA synthetase (MetS; encoded by metS1) have been derived from a high-throughput screening assay hit. Optimized compounds showed excellent activities against staphylococcal and enterococcal pathogens. We report on the bimodal susceptibilities of S. pneumoniae strains, a significant fraction of which was found to be resistant (MIC, > or =8 mg/liter) to these inhibitors. Using molecular genetic techniques, we have found that the mechanism of resistance is the presence of a second, distantly related MetS enzyme, MetS2, encoded by metS2. We present evidence that the metS2 gene is necessary and sufficient for resistance to MetS inhibitors. PCR analysis for the presence of metS2 among a large sample (n = 315) of S. pneumoniae isolates revealed that it is widespread geographically and chronologically, occurring at a frequency of about 46%. All isolates tested also contained the metS1 gene. Searches of public sequence databases revealed that S. pneumoniae MetS2 was most similar to MetS in Bacillus anthracis, followed by MetS in various non-gram-positive bacterial, archaeal, and eukaryotic species, with streptococcal MetS being considerably less similar. We propose that the presence of metS2 in specific strains of S. pneumoniae is the result of horizontal gene transfer which has been driven by selection for resistance to some unknown class of naturally occurring antibiotics with similarities to recently reported synthetic MetS inhibitors.
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7. |
Chesnel L,
Pernot L,
Lemaire D,
Champelovier D,
Croizé J,
Dideberg O,
Vernet T,
Zapun A,
( 2003 ) The structural modifications induced by the M339F substitution in PBP2x from Streptococcus pneumoniae further decreases the susceptibility to beta-lactams of resistant strains. PMID : 12923202 : DOI : 10.1074/jbc.M305948200 Abstract >>
PBP2x is a primary determinant of beta-lactams resistance in Streptococcus pneumoniae. Altered PBP2x with multiple mutations have a reduced "affinity" for the antibiotics. An important polymorphism is found in PBP2x sequences from clinical resistant strains. To understand the mechanism of resistance, it is necessary to identify and characterize the relevant substitutions. Many similar PBP2x sequences from resistant isolates have the previously studied T338A mutation, adjacent to the active site Ser337. We report here the structural and functional analysis of the M339F substitution that is found in a subset of these sequences, originating from highly resistant strains. The M339F mutation causes a 4-10-fold reduction of the reaction rate with beta-lactams, depending on the molecular context. In addition, release of the inactivated antibiotic from the active site is up to 3-fold faster as a result from the M339F mutation. These effects measured in vitro are correlated with the level of beta-lactam resistance in vivo conferred by several PBP2x variants. Thus, a single amino acid difference between similar PBP2x from clinical isolates can strongly modulate the degree of beta-lactam resistance. The crystal structure of the double mutant T338A/M339F solved to a resolution of 2.4 A shows a distortion of the active site and a reorientation of the hydroxyl group of the active site Ser337, which can explain the kinetic effects of the mutations.
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8. |
Obregón V,
García P,
López R,
García JL,
( 2003 ) VO1, a temperate bacteriophage of the type 19A multiresistant epidemic 8249 strain of Streptococcus pneumoniae: analysis of variability of lytic and putative C5 methyltransferase genes. PMID : 12705678 : DOI : 10.1089/107662903764736292 Abstract >>
A temperate bacteriophage (VO1) has been isolated from the Streptococcus pneumoniae type 19F multiresistant epidemic 8249 strain (South African strain). Structural analysis of the specific integration site, protein composition, restriction patterns, and molecular dissection of the lytic system of this phage revealed high sequence similarity with MM1, a temperate phage from the Spain23F-1 strain of pneumococcus, another multiresistant epidemic clone. The different pneumococcal strains sequenced so far exhibit an identical and single attB located in the same site of the genome. Remarkably, the LytA amidase coded by VO1 showed clear differences with that of the host bacterium in contrast with the situation previously documented for bacterial- and phage-coded amidases of pneumococcus. In addition, a new gene (orfmet) putatively coding for a C5 methyltransferase has been identified. A noticeable variability affecting the presence (or absence) of this supernumerary gene(s) in the same region of the genomes of three otherwise highly similar phages (i.e., VO1, MM1, and HB-3) suggests frequent recombinational events leading to introduce variability in this genome region. The peculiarities of genes like lytA and orfmet in VO1 provide interesting insights on mechanisms of horizontal transfer and lysogenic state co-evolution.
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9. |
Price AC,
Rock CO,
White SW,
( 2003 ) The 1.3-Angstrom-resolution crystal structure of beta-ketoacyl-acyl carrier protein synthase II from Streptococcus pneumoniae. PMID : 12837788 : DOI : 10.1128/jb.185.14.4136-4143.2003 PMC : PMC164876 Abstract >>
The beta-ketoacyl-acyl carrier protein synthases are members of the thiolase superfamily and are key regulators of bacterial fatty acid synthesis. As essential components of the bacterial lipid metabolic pathway, they are an attractive target for antibacterial drug discovery. We have determined the 1.3 A resolution crystal structure of the beta-ketoacyl-acyl carrier protein synthase II (FabF) from the pathogenic organism Streptococcus pneumoniae. The protein adopts a duplicated betaalphabetaalphabetaalphabetabeta fold, which is characteristic of the thiolase superfamily. The two-fold pseudosymmetry is broken by the presence of distinct insertions in the two halves of the protein. These insertions have evolved to bind the specific substrates of this particular member of the thiolase superfamily. Docking of the pantetheine moiety of the substrate identifies the loop regions involved in substrate binding and indicates roles for specific, conserved residues in the substrate binding tunnel. The active site triad of this superfamily is present in spFabF as His 303, His 337, and Cys 164. Near the active site is an ion pair, Glu 346 and Lys 332, that is conserved in the condensing enzymes but is unusual in our structure in being stabilized by an Mg(2+) ion which interacts with Glu 346. The active site histidines interact asymmetrically with Lys 332, whose positive charge is closer to His 303, and we propose a specific role for the lysine in polarizing the imidazole ring of this histidine. This asymmetry suggests that the two histidines have unequal roles in catalysis and provides new insights into the catalytic mechanisms of these enzymes.
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10. |
Balsalobre L,
Ferrándiz MJ,
Liñares J,
Tubau F,
de la Campa AG,
( 2003 ) Viridans group streptococci are donors in horizontal transfer of topoisomerase IV genes to Streptococcus pneumoniae. PMID : 12821449 : DOI : 10.1128/aac.47.7.2072-2081.2003 PMC : PMC161831 Abstract >>
A total of 46 ciprofloxacin-resistant (Cip(r)) Streptococcus pneumoniae strains were isolated from 1991 to 2001 at the Hospital of Bellvitge. Five of these strains showed unexpectedly high rates of nucleotide variations in the quinolone resistance-determining regions (QRDRs) of their parC, parE, and gyrA genes. The nucleotide sequence of the full-length parC, parE, and gyrA genes of one of these isolates revealed a mosaic structure compatible with an interspecific recombination origin. Southern blot analysis and nucleotide sequence determinations showed the presence of an ant-like gene in the intergenic parE-parC regions of the S. pneumoniae Cip(r) isolates with high rates of variations in their parE and parC QRDRs. The ant-like gene was absent from typical S. pneumoniae strains, whereas it was present in the intergenic parE-parC regions of the viridans group streptococci (Streptococcus mitis and Streptococcus oralis). These results suggest that the viridans group streptococci are acting as donors in the horizontal transfer of fluoroquinolone resistance genes to S. pneumoniae.
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11. |
Smith AM,
Klugman KP,
( 2003 ) Site-specific mutagenesis analysis of PBP 1A from a penicillin-cephalosporin-resistant pneumococcal isolate. PMID : 12499220 : DOI : 10.1128/aac.47.1.387-389.2003 PMC : PMC149026 Abstract >>
Our mutagenesis study has investigated all amino acid mutations in the penicillin-binding domain of PBP 1A from Hungarian pneumococcal isolate 3191 to determine the importance of every mutation in the development of penicillin and cefotaxime resistance. Our data reveal that mutations at amino acid positions 574 to 577 and position 539 cause penicillin and cefotaxime resistance.
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12. |
Abeyta M,
Hardy GG,
Yother J,
( 2003 ) Genetic alteration of capsule type but not PspA type affects accessibility of surface-bound complement and surface antigens of Streptococcus pneumoniae. PMID : 12496169 : DOI : 10.1128/iai.71.1.218-225.2003 PMC : PMC143148 Abstract >>
The Streptococcus pneumoniae capsular polysaccharides and pneumococcal surface protein A (PspA) are major determinants of virulence that are antigenically variable and capable of eliciting protective immune responses. By genetically switching the pspA genes of the capsule type 2 strain D39 and the capsule type 3 strain WU2, we showed that the different abilities of antibody to PspA to protect against these strains was not related to the PspA type expressed. Similarly, the level of specific antibody binding to PspA, other surface antigens, and surface-localized C3b did not depend on the PspA type but instead was correlated with the capsule type. The type 3 strain WU2 and an isogenic derivative of D39 that expresses the type 3 capsule bound nearly identical amounts of antibody to PspA and other surface antigens, and these amounts were less than one-half the amount observed with the type 2 parent strain D39. Expression of the type 3 capsule in D39 also reduced the amount of C3b deposited and its accessibility to antibody, resulting in a level intermediate between the levels observed with WU2 and D39. Despite these effects, the capsule type was not the determining factor in anti-PspA-mediated protection, as both D39 and its derivative expressing the type 3 capsule were more resistant to protection than WU2. The specific combination of PspA and capsule type also did not determine the level of protection. The capsule structure is thus a major determinant in accessibility of surface antigens to antibody, but certain strains appear to express other factors that can influence antibody-mediated protection.
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13. |
Yokota S,
Sato K,
Kuwahara O,
Habadera S,
Tsukamoto N,
Ohuchi H,
Akizawa H,
Himi T,
Fujii N,
( 2002 ) Fluoroquinolone-resistant Streptococcus pneumoniae strains occur frequently in elderly patients in Japan. PMID : 12234869 : DOI : 10.1128/aac.46.10.3311-3315.2002 PMC : PMC128788 Abstract >>
We identified and genetically characterized seven fluoroquinolone-resistant Streptococcus pneumoniae strains among 293 clinical strains isolated from 1999 to 2001 in Japan. The resistant strains were isolated only from adults, and 7 of 31 isolates (22.6%) were from patients more than 20 years old. Resistant strains were not found in 262 isolates from children under age 10.
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14. |
Dicuonzo G,
Gherardi G,
Gertz RE,
D'Ambrosio F,
Goglio A,
Lorino G,
Recchia S,
Pantosti A,
Beall B,
( 2002 ) Genotypes of invasive pneumococcal isolates recently recovered from Italian patients. PMID : 12354862 : DOI : 10.1128/jcm.40.10.3660-3665.2002 PMC : PMC130901 Abstract >>
We examined 73 recent invasive pneumococcal isolates within selected areas of Italy for genotypic variability. Thirty-three genomic macrorestriction types were found, three of which represented multiple serotypes. Restriction fragment patterns of pbp2b, pbp2x, and pspA were conserved within the majority of isolates that shared macrorestriction types. Of the nine macrorestriction types found among the 22 penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) isolates, seven comprised isolates with allelic profiles showing five to seven allelic matches to profiles in the multilocus sequence typing database (www.mlst.net); however, three of the seven profiles represented serotypes not previously associated with these clonal clusters. Two PNSP macrorestriction types represented new clones with unique allelic profiles. Allelic profiles obtained from isolates of 3 of the 25 macrorestriction types found among the 51 penicillin-susceptible S. pneumoniae (PSSP) isolates were closely related to previously described profiles. One PSSP isolate was a novel type 24F isolate related to the multiresistant clone France(9V)-3. This work reports new PNSP strains and new serotype-clone associations.
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15. |
Sá-Leão R,
Vilhelmsson SE,
de Lencastre H,
Kristinsson KG,
Tomasz A,
( 2002 ) Diversity of penicillin-nonsusceptible Streptococcus pneumoniae circulating in Iceland after the introduction of penicillin-resistant clone Spain(6B)-2. PMID : 12232837 : DOI : 10.1086/343769 Abstract >>
After the introduction and extensive dissemination of the multidrug-resistant Streptococcus pneumoniae clone Spain(6B)-2 between 1989 and the early to mid-1990s, the prevalence of pneumococcal isolates expressing intermediate resistance to penicillin, mainly of capsular types 6, 19, and 23, also began to increase in Iceland. The purpose of this study was to investigate whether these isolates originated in Iceland or represented strains imported to the country. Isolates were characterized by pulsed-field gel electrophoresis; multilocus sequence typing; determination of pbp1a, pbp2b, and pbp2x gene restriction patterns; and partial sequencing of these pbp genes. The results indicate that, although singular events suggesting horizontal transfer of pbp genes (and capsular genes) were detected, the majority of clones circulating in the country had genetic backgrounds also detected abroad. The major mechanism of dissemination of penicillin resistance in Iceland appears to be the repeated introduction of multiple lineages, followed by clonal spread.
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16. |
Robinson DA,
Briles DE,
Crain MJ,
Hollingshead SK,
( 2002 ) Evolution and virulence of serogroup 6 pneumococci on a global scale. PMID : 12399507 : DOI : 10.1128/jb.184.22.6367-6375.2002 PMC : PMC151942 Abstract >>
To study the evolution and virulence of pneumococcal populations, we used multilocus sequence typing to identify the major clones among 212 carriage and invasive isolates expressing capsular serogroup 6 from 39 countries. The global population consisted of 8 major complexes and 6 minor complexes of related clones and 32 clones of diverse origin. Surprisingly, serotype 6A clones evolved by mutation nearly as often as by recombination, whereas serotype 6B clones evolved almost exclusively by recombination (P = 0.0029). This is the first report of population genetic differences among serotypes of this species. The largest clonal complex was associated with invasive disease (P = 0.019) and included a common ancestor for five previously identified drug-resistant clones. The putative ancestors of the major clonal complexes were represented by a greater proportion of carriage isolates than were their descendents (P = 0.001), and the ancestors tended to be less virulent than their descendents in a mouse model of infection. These data suggested that virulent serogroup 6 clones have evolved multiple times from less-virulent ancestral clones.
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17. |
Pericone CD,
Bae D,
Shchepetov M,
McCool T,
Weiser JN,
( 2002 ) Short-sequence tandem and nontandem DNA repeats and endogenous hydrogen peroxide production contribute to genetic instability of Streptococcus pneumoniae. PMID : 12142409 : DOI : 10.1128/jb.184.16.4392-4399.2002 PMC : PMC135236 Abstract >>
Loss-of-function mutations in the following seven pneumococcal genes were detected and analyzed: pspA, spxB, xba, licD2, lytA, nanA, and atpC. Factors associated with these mutations included (i) frameshifts caused by reversible gain and loss of single bases within homopolymeric repeats as short as 6 bases, (ii) deletions caused by recombinational events between nontandem direct repeats as short as 8 bases, and (iii) substitutions of guanine residues caused at an increased frequency by the high levels of hydrogen peroxide (>2 mM) typically generated by this species under aerobic growth conditions. The latter accounted for a frequency as high as 2.8 x 10(-6) for spontaneous mutation to resistance to optochin and was 10- to 200-fold lower in the absence of detectable levels of H2O2. Some of these mutations appear to have been selected for in vivo during pneumococcal infection, perhaps as a consequence of immune pressure or oxidative stress.
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18. |
Miyaji EN,
Ferreira DM,
Lopes AP,
Brandileone MC,
Dias WO,
Leite LC,
( 2002 ) Analysis of serum cross-reactivity and cross-protection elicited by immunization with DNA vaccines against Streptococcus pneumoniae expressing PspA fragments from different clades. PMID : 12183557 : DOI : 10.1128/iai.70.9.5086-5090.2002 PMC : PMC128265 Abstract >>
Streptococcus pneumoniae is a major cause of disease, especially in developing countries, and cost-effective alternatives to the currently licensed vaccines are needed. We constructed DNA vaccines based on pneumococcal surface protein A (PspA), an antigen shown to induce protection against pneumococcal bacteremia. PspA fragments can be divided into three families, which can be subdivided into six clades, on the basis of PspA amino acid sequence divergence (S. K. Hollingshead, R. Becker, and D. E. Briles, Infect. Immun. 68:5889-5900, 2000). Since most clinical isolates belong to family 1 or family 2, PspA fragments from members of both of these families were analyzed. Vectors encoding the complete N-terminal regions of PspAs elicited significant humoral responses, and cross-reactivity was mainly restricted to the same family. DNA vaccines encoding fusions between PspA fragments from family 1 and family 2 were also constructed and were able to broaden the cross-reactivity, with induction of antibodies that showed reactions with members of both families. At least for the pneumococcal strains tested, the cross-reactivity of antibodies was not reflected in cross-protection. Animals immunized with DNA vaccines expressing the complete N-terminal regions of PspA fragments were protected only against intraperitoneal challenge with a strain expressing PspA from the same clade.
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19. |
du Plessis M,
Bingen E,
Klugman KP,
( 2002 ) Analysis of penicillin-binding protein genes of clinical isolates of Streptococcus pneumoniae with reduced susceptibility to amoxicillin. PMID : 12121904 : DOI : 10.1128/aac.46.8.2349-2357.2002 PMC : PMC127354 Abstract >>
The recent emergence of pneumococcal isolates exhibiting an unusual resistance phenotype of higher amoxicillin MICs in relation to the penicillin MICs prompted an analysis of the pbp genes from three such strains isolated in France. For comparison, three amoxicillin-susceptible strains were included in the study. DNA sequence analysis of the pbp2x, pbp2b, and pbp1a genes revealed extensive sequence divergence in all six isolates compared to the sequences of the genes of penicillin-susceptible strain R6. With the exception of pbp2b, no amino acid mutations were unique to the resistant isolates. Transformation experiments with cloned pbp genes isolated from one of the resistant isolates demonstrated a stepwise development of amoxicillin resistance involving penicillin-binding proteins (PBPs) 2X, 2B, and 1A. Full resistance, equivalent to that of the donor strain, was achieved only when genomic DNA was transformed into R6(2x/2b/1a) mutants, suggesting that full resistance development in this isolate is mediated by a non-PBP determinant. Moreover, the recently identified murMN resistance determinant does not appear to have any impact on resistance in this isolate. This determinant (from the French isolate) was, however, able to transform an R6 mutant harboring pbp2x, pbp2b, and pbp1a genes from a Hungarian clone with an extremely high level of penicillin resistance so that it had increased levels of penicillin resistance. These results indicate that the development of high-level beta-lactam resistance is a complex process and that the involvement of MurMN in penicillin resistance appears to be dependent on specific mutations in PBPs 2X, 2B, and/or 1A. Furthermore, an additional (as yet unidentified) non-PBP-mediated resistance determinant is required for full resistance development in some pneumococci.
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20. |
Overweg K,
Bogaert D,
Sluijter M,
de Groot R,
Hermans PW,
( 2001 ) Molecular characteristics of penicillin-binding protein genes of penicillin-nonsusceptible Streptococcus pneumoniae isolated in the Netherlands. PMID : 11822772 : DOI : 10.1089/10766290152773338 Abstract >>
Recently, a nation-wide molecular epidemiologic survey of penicillin-nonsusceptible Streptococcus pneumoniae has been performed in the Netherlands. In the current study, we analyzed the genes pbp1a, pbp2b, and pbp2x from these clinical isolates at the molecular level, and identified the genetic composition of the penicillin-binding domains. The pneumococcal strains were selected on the basis of differences in restriction fragment length polymorphism (RFLP) patterns of the genes pbp1a, pbp2b, and pbp2x, and represented 8, 7, and 10 distinct patterns, respectively. The genetic heterogeneity observed by sequence analysis of the pbp gene parts was comparable with the heterogeneity of the entire pbp genes as deduced from RFLP analysis. Furthermore, the mutations in the pbp sequences of the Dutch isolates invariably matched with the mutations described in pbp sequences of penicillin-nonsusceptible pneumococci isolated in other countries. Finally, novel mosaic structures were identified indicating horizontal exchange of pbp gene parts among penicillin-nonsusceptible pneumococci.
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21. |
van Selm S,
Kolkman MA,
van der Zeijst BA,
Zwaagstra KA,
Gaastra W,
van Putten JP,
( 2002 ) Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9V. PMID : 12055294 : DOI : 10.1099/00221287-148-6-1747 Abstract >>
The capsular polysaccharide (CPS) synthesis locus of Streptococcus pneumoniae serotype 9V was amplified by long-range PCR and sequenced. The locus was 17368 bp in size and contained 15 ORFs. The genetic organization of the cluster shared many features with other S. pneumoniae capsule loci, including the presence of four putative regulatory genes at the 5' end. Comparative sequence analyses allowed putative functions to be assigned to each of the gene products. The ORFs appeared to encode, besides the four regulatory genes, five glycosyltransferases, two O-acetyltransferases, an N-acetylglucosamine 2-epimerase, a glucose 6-dehydrogenase, an oligosaccharide transporter protein and a polysaccharide repeating unit polymerase. These functions covered the steps proposed in the CPS biosynthesis of serotype 9V. TLC of carbohydrate intermediates formed after incubation of bacterial membrane preparations with 14C-labelled precursors demonstrated that the fifth ORF (cps9vE) encoded a UDP-glucosyl-1-phosphate transferase. This function was confirmed with the help of a cps9vE mutant that carried a deletion of a guanine residue located adjacent to a stretch of adenines. The identification and characterization of the serotype 9V locus is a major step in unravelling the 9V capsule biosynthesis pathway and broadens the insight into the genetic diversity of the S. pneumoniae capsule loci.
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22. |
Obregón V,
García P,
García E,
Fenoll A,
López R,
García JL,
( 2002 ) Molecular peculiarities of the lytA gene isolated from clinical pneumococcal strains that are bile insoluble. PMID : 12089276 : DOI : 10.1128/jcm.40.7.2545-2554.2002 PMC : PMC120542 Abstract >>
The autolytic LytA amidase from 12 bile (deoxycholate)-insoluble streptococcal isolates (formerly classified as atypical Streptococcus pneumoniae) showing different antibiotic resistance patterns was studied. These atypical strains, which autolyze at the end of the stationary phase of growth, contain highly divergent lytA alleles (pairwise evolutionary distances of about 20%) compared to the lytA alleles of typical pneumococci. The atypical LytA amidases exhibit a peculiar deletion of two amino acids responsible for cell wall anchoring in the carboxy-terminal domain and have a reduced specific activity. These enzymes were inhibited by 1% deoxycholate but were activated by 1% Triton X-100, a detergent that could be used as an alternative diagnostic test for this kind of strain. Preparation of functional chimeric enzymes, PCR mutagenesis, and gene replacements demonstrated that the characteristic bile insolubility of these atypical strains was due to their peculiar carboxy-terminal domain and that the 2-amino-acid deletion was responsible for the inhibitory effect of deoxycholate. However, the deletion alone did not affect the specific activity of LytA. A detailed characterization of the genes encoding the 16S rRNA and SodA together with multilocus sequence typing indicated that the strains studied here are not a single clone and, although they cannot be strictly classified as typical pneumococci, they represent a quite diverse pool of organisms closely related to S. pneumoniae. The clinical importance of these findings is underlined by the role of the lytA gene in shaping the course of pneumococcal diseases. This study can also contribute to solving diagnostic problems and to understanding the evolution and pathogenic potential of species of the Streptococcus mitis group.
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23. |
Morona JK,
Morona R,
Miller DC,
Paton JC,
( 2002 ) Streptococcus pneumoniae capsule biosynthesis protein CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase. PMID : 11751838 : DOI : 10.1128/jb.184.2.577-583.2002 PMC : PMC139577 Abstract >>
The first four genes of the capsule locus (cps) of Streptococcus pneumoniae (cpsA to cpsD) are common to most serotypes. We have previously determined that CpsD is an autophosphorylating protein-tyrosine kinase, demonstrated that CpsC is required for CpsD tyrosine-phosphorylation, and shown that CpsB is required for dephosphorylation of CpsD. In the present study we show that CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase that belongs to the PHP (polymerase and histidinol phosphatase) family of phosphoesterases. We also show that an S. pneumoniae strain with point mutations in cpsB, affecting one of the conserved motifs of CpsB, is unencapsulated and appears to be morphologically identical to a strain in which the cpsB gene had been deleted.
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24. |
Campbell HA,
Kent C,
( 2001 ) The CTP:phosphocholine cytidylyltransferase encoded by the licC gene of Streptococcus pneumoniae: cloning, expression, purification, and characterization. PMID : 11786295 : DOI : 10.1016/s1388-1981(01)00174-3 Abstract >>
Streptococcus pneumoniae is a member of a small group of bacteria that display phosphocholine on the cell surface, covalently attached to the sugar groups of teichoic acid and lipoteichoic acid. The putative pathway for this phosphocholine decoration is, in its first two enzymes, functionally similar to the CDP-choline pathway used for phosphatidylcholine biosynthesis in eukaryotes. We show that the licC gene encodes a functional CTP:phosphocholine cytidylyltransferase (CCT). The enzyme has been expressed and purified to homogeneity. Assay conditions were optimized, particularly with respect to linearity with time, pH, Mg(2+), and ammonium sulfate concentration. The pure enzyme has K(M) values of 890+/-240 microM for CTP, and 390+/-170 microM for phosphocholine. The k(cat) is 17.5+/-4.0 s(-1). S. pneumoniae CTP:phosphocholine cytidylyltransferase (SpCCT) is specific for CTP or dCTP as the nucleotide substrate. SpCCT is strongly inhibited by Ca(2+). The IC(50) values for recombinant and native SpCCT are 0.32+/-0.04 and 0.27+/-0.03 mM respectively. The enzyme is also inhibited by all other tested divalent cations, including Mg(2+) at high concentrations. The cloning and expression of this enzyme sets the stage for design of inhibitors as possible antipneumococcal drugs.
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25. |
Bethe G,
Nau R,
Wellmer A,
Hakenbeck R,
Reinert RR,
Heinz HP,
Zysk G,
( 2001 ) The cell wall-associated serine protease PrtA: a highly conserved virulence factor of Streptococcus pneumoniae. PMID : 11728722 : DOI : 10.1111/j.1574-6968.2001.tb10931.x Abstract >>
The surface-associated subtilisin-like serine protease PrtA was identified by screening a genomic expression library from Streptococcus pneumoniae using a convalescent-phase serum. In Western blot analysis two forms of PrtA were detected in whole cell lysate and a truncated form only in culture supernatant suggesting that PrtA is produced as a precursor protein, translocated to the cell surface, truncated, and released into the surroundings. A 5' fragment of the gene was found highly conserved among 78 pneumococcal isolates of clinical relevance. Immunogenicity of PrtA, limited genetic variation, and the involvement in pneumococcal virulence demonstrated in in vivo experiments might identify PrtA as a promising candidate for a protein based vaccine.
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26. |
Martín-Galiano AJ,
Gorgojo B,
Kunin CM,
de la Campa AG,
( 2002 ) Mefloquine and new related compounds target the F(0) complex of the F(0)F(1) H(+)-ATPase of Streptococcus pneumoniae. PMID : 12019076 : DOI : 10.1128/aac.46.6.1680-1687.2002 PMC : PMC127268 Abstract >>
The activities of mefloquine (MFL) and related compounds against previously characterized Streptococcus pneumoniae strains carrying defined amino acid substitutions in the c subunit of the F(0)F(1) H(+)-ATPase were studied. In addition, a series of MFL-resistant (Mfl(r)) strains were isolated and characterized. A good correlation was observed between inhibition of growth and inhibition of the membrane-associated F(0)F(1) H(+)-ATPase activity. MFL was about 10-fold more active than optochin and about 200-fold more active than quinine in inhibiting both the growth and the ATPase activities of laboratory pneumococcal strain R6. Mutant strains were inhibited by the different compounds to different degrees, depending on their specific mutations in the c subunit. The resistant strains studied had point mutations that changed amino acid residues in either the c subunit or the a subunit of the F(0) complex. Changes in the c subunit were located in one of the two transmembrane alpha helices: residues M13, G14, G20, M23, and N24 of helix 1 and residues M44, G47, V48, A49, and V57 of helix 2. Changes in the a subunit were also found in either of the transmembrane alpha helices, helix 5 or 6: residue L186 of helix 5 and residues W206, F209, and S214 of helix 6. These results suggest that the transmembrane helices of the c and a subunits interact and that the mutated residues are important for the structure of the F(0) complex and proton translocation.
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27. |
de las Rivas B,
García JL,
López R,
García P,
( 2001 ) Molecular characterization of the pneumococcal teichoic acid phosphorylcholine esterase. PMID : 11759082 : DOI : 10.1089/10766290152652756 Abstract >>
A search to identify proteins with high affinity for choline-containing pneumococcal cell walls (choline-binding proteins) has permitted the localization, cloning, sequencing, and overexpression of a gene (pce), coding for a protein (Pce) that liberates phosphorylcholine from purified cell walls of Streptococcus pneumoniae. The pce gene of the pneumococcal strain R6 encodes a protein of 627 amino acids with a predicted Mr of 72,104. Pce can remove a maximum of 20% phosphorylcholine residues from the cell wall teichoic acid. In silico analysis of Pce shows a modular organization of the enzyme where the choline-binding domain, involved in cell wall substrate recognition, and the catalytic domain are located at the carboxy- and amino-terminal moieties of the protein, respectively. Remarkably, a long tail of 85 amino acids follows the carboxy-terminal domain, a structural feature that had not been described for the published choline-binding proteins. The carboxy-terminal moiety of Pce is assembled by 10 repeated motifs, and the protein has also a cleavable signal peptide of 25 amino acids that renders after its cleavage a mature protein of 69,426 Da (602 amino acids). The pce gene has been expressed in Escherichia coli, and Pce was active when assayed on pneumococcal walls. We have also found that the signal peptide of Pce was functional in E. coli. Biochemical analysis suggested that Pce is the teichoic acid phosphorylcholine esterase of S. pneumoniae that had been biochemically characterized previously. Construction of two pce pneumococcal mutants (R6D and M31D) by insertion-duplication mutagenesis revealed that these mutants grew at a doubling-time similar to those of the parental strains of the wild-type R6 and the lytA-mutant M31, respectively. R6D and M31D were morphologically indistinguishable from the parental strains when whole-mounted cells were observed under the electron microscope and exhibited levels of competence for genetic transformation slightly lower than those reported for R6 and M31.
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28. |
Smith AW,
Roche H,
Trombe MC,
Briles DE,
Håkansson A,
( 2002 ) Characterization of the dihydrolipoamide dehydrogenase from Streptococcus pneumoniae and its role in pneumococcal infection. PMID : 11972781 : DOI : 10.1046/j.1365-2958.2002.02883.x Abstract >>
In the present study, we have characterized the dihydrolipoamide dehydrogenase (DLDH) of Strepto-coccus pneumoniae and its role during pneumococcal infection. We have also demonstrated that a lack of DLDH results in a deficiency in alpha-galactoside metabolism and galactose transport. DLDH is an enzyme that is classically involved in the three-step conversion of 2-oxo acids to their respective acyl-CoA derivatives, but DLDH has also been shown to have other functions. The dldh gene was virtually identical in three pneumococcal strains examined. Besides the functional domains and motifs associated with this enzyme, analysis of the pneumococcal dldh gene sequence revealed the presence of an N-terminal lipoyl domain. DLDH-negative bacteria totally lacked DLDH activity, indicating that this gene encodes the only DLDH in S. pneumoniae. These DLDH-negative bacteria grew normally in vitro but were avirulent in sepsis and lung infection models in mice, indicating that DLDH activity is necessary for the survival of pneumococci within the host. The lack of virulence was not associated with a loss of 2-oxo acid dehydrogenase activity, as the wild-type pneumococcal strains did not contain activity of any of the known 2-oxo acid enzyme complexes. Instead, studies of carbohydrate utilization demonstrated that the DLDH-negative bacteria were impaired for alpha-galactoside and galactose metabolism. The DLDH mutants lost their ability to oxidize or grow with galactose or melibiose as sole carbon source and showed reduced oxidation and growth on raffinose or stachyose. The bacteria had an 85% reduction in alpha-galactosidase activity and showed virtually no transport of galactose into the cells, which can explain these phenotypic changes. The DLDH-negative bacteria produced only 50% of normal capsular polysaccharide, a phenotype that may be associated with impaired carbohydrate metabolism.
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29. |
Del Grosso M,
Iannelli F,
Messina C,
Santagati M,
Petrosillo N,
Stefani S,
Pozzi G,
Pantosti A,
( 2002 ) Macrolide efflux genes mef(A) and mef(E) are carried by different genetic elements in Streptococcus pneumoniae. PMID : 11880392 : DOI : 10.1128/jcm.40.3.774-778.2002 PMC : PMC120261 Abstract >>
Susceptibilities to macrolides were evaluated in 267 Streptococcus pneumoniae isolates, of which 182 were from patients with invasive diseases and 85 were from healthy carriers. Of the 98 resistant isolates, 20 strains showed an M phenotype and carried mef. Strains that carried both mef(A) and mef(E) were found: 17 strains carried mef(A) and 3 carried mef(E). The characteristics of the strains carrying the mef genes and the properties of the mef-containing elements were studied. Strains carrying mef(A) belonged to serotype 14, were susceptible to all the antibiotics tested except erythromycin, and appeared to be clonally related by pulsed-field gel electrophoresis (PFGE). The three mef(E) strains belonged to different serotypes, showed different susceptibility profiles, and did not appear to be related by PFGE. The sequences of a fragment of the mef-containing element, which encompassed mef and the msr(A) homolog, were identical among the three mef(E)-positive strains and among the three mef(A)-positive strains, although there were differences between the sequences for the two variants at 168 positions. In all mef(A)-positive strains, the mef element was inserted in celB, which led to impairment of the competence of the strains. In line with insertion of the mef(E) element at a different site, the competence of the mef(E)-positive strains was maintained. Transfer of erythromycin resistance by conjugation was obtained from two of three mef(A) strains but from none of three mef(E) strains. Due to the important different characteristics of the strains carrying mef(A) or mef(E), we suggest that the distinction between the two genes be maintained.
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30. |
Muñoz R,
Mollerach M,
López R,
García E,
( 1997 ) Molecular organization of the genes required for the synthesis of type 1 capsular polysaccharide of Streptococcus pneumoniae: formation of binary encapsulated pneumococci and identification of cryptic dTDP-rhamnose biosynthesis genes. PMID : 11902728 : DOI : 10.1046/j.1365-2958.1997.4341801.x Abstract >>
We report here the molecular organization of the capsular locus (cap1) of the type 1 pneumococcus. This locus is located between dexB and aliA and flanked by IS1167 insertion elements. Sequence analysis showed that the cluster contains 11 genes (cap1A to cap1K), which are apparently arranged as a single transcriptional unit. The presence of a functional promoter (cap1p) located upstream of cap1A has been demonstrated and the transcription start point was mapped by primer-extension analysis. A 14.3 kb fragment containing the genes cap1ABCDEFGHIJK and including cap1p was sufficient to allow the synthesis of a type 1 capsule in Streptococcus pneumoniae. An internal deletion of cap1E leads to an unencapsulated phenotype demonstrating that this gene is essential for capsular production. The cap1K gene has been expressed in Escherichia coli resulting in UDP-glucose dehydrogenase (UDP-GlcDH) activity. Moreover, this gene was able to restore the synthesis of type 3 capsule when cloned into a plasmid and introduced by transformation into S. pneumoniae cap3A mutants deficient in UDP-GlcDH. In marked contrast with what was previously thought, recombination between cap1K and cap3A does occur. We provide data on the molecular mechanism that leads to the formation of binary encapsulated pneumococcal cells, i.e. strains that simultaneously produce type 1 and type 3 capsules. Downstream of cap1K, one truncated and three complete open reading frames homologous to those involved in the biosynthesis of dTDP-rhamnose, a monosaccharide that does not participate in the formation of type 1 polysaccharide, have been identified in all the clinical strains of type 1 pneumococcus tested. Our results provide new insights into the generation of capsule diversity in pneumococci.
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31. |
Iannelli F,
Oggioni MR,
Pozzi G,
( 2002 ) Allelic variation in the highly polymorphic locus pspC of Streptococcus pneumoniae. PMID : 11891047 : DOI : 10.1016/s0378-1119(01)00896-4 Abstract >>
PspC, also called SpsA, CbpA, PbcA, and Hic, is a surface protein of Streptococcus pneumoniae studied for its antigenic properties, its capability to bind secretory IgA, C3 and complement factor H, and its activity as an adhesin. In this work we characterized the pspC locus of 43 pneumococcal strains by DNA sequencing of PCR fragments. Using PCR primers designed on two unrelated open reading frames, flanking the pspC locus, it was possible to amplify the pspC locus of each of the 43 strains of S. pneumoniae. In 37 out of 43 strains there was a single copy of the pspC gene, while two tandem copies of pspC were found in the other six strains. The sequence of the pspC locus was different in each of the 43 strains. Insertion sequences were found in the pspC locus of 11 out of 43 strains. Analysis of the deduced amino acid sequence of the PspC variants showed a common organization of the molecules: (i) a 37 amino acid leader peptide which is conserved in all proteins, (ii) an N-terminal portion which is essentially alpha-helical, and is the result of assembly of eight major sequence blocks, (iii) a proline-rich region, and (iv) a C-terminal anchor responsible for the cell surface attachment. By sequence comparison we identified 11 major groups of PspC proteins. Proteins within one group displayed only minor variations of the amino acid sequence. An unexpected finding was that PspC variants could differ in the anchor sequence. While 32 of the PspC proteins displayed the typical choline binding domain of pneumococcal surface proteins, 17 other PspCs showed the LPXTG motif, which is typical of surface proteins of other gram-positive bacteria. This major difference in the anchor region was also observed in the adjacent proline-rich regions which differed considerably in size and composition.
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32. |
Yother J,
Ambrose KD,
Caimano MJ,
( 1997 ) Association of a partial H-rpt element with the type 3 capsule locus of Streptococcus pneumoniae. PMID : 11902721 : DOI : 10.1046/j.1365-2958.1997.4361798.x Abstract >>
N/A
|
33. |
McCool TL,
Cate TR,
Moy G,
Weiser JN,
( 2002 ) The immune response to pneumococcal proteins during experimental human carriage. PMID : 11828011 : DOI : 10.1084/jem.20011576 PMC : PMC2193593 Abstract >>
Colonization of the nasopharynx is the initial step in all infections caused by Streptococcus pneumoniae. The antibody response to carriage was examined in an experimental model of human colonization in healthy adults. Asymptomatic colonization was detected in 6/14 subjects and continued for up to 122 d. Susceptibility to carriage did not correlate with total serum immunoglobulin (Ig)G to the homotypic capsular polysaccharide. All of the colonized subjects, in contrast, developed a serum IgG and secretory IgA response to a 22 kD protein, whereas 7 of 8 subjects who did not become colonized had preexisting antibody to this protein. Analysis of the 22 kD protein identified it as the NH(2)-terminal region of pneumococcal surface protein A (PspA). Our findings provide evidence for the role of antibody to this protein fragment in preventing pneumococcal carriage by humans.
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34. |
Dessen A,
Mouz N,
Gordon E,
Hopkins J,
Dideberg O,
( 2001 ) Crystal structure of PBP2x from a highly penicillin-resistant Streptococcus pneumoniae clinical isolate: a mosaic framework containing 83 mutations. PMID : 11553637 : DOI : 10.1074/jbc.M107608200 Abstract >>
Penicillin-binding proteins (PBPs) are the main targets for beta-lactam antibiotics, such as penicillins and cephalosporins, in a wide range of bacterial species. In some Gram-positive strains, the surge of resistance to treatment with beta-lactams is primarily the result of the proliferation of mosaic PBP-encoding genes, which encode novel proteins by recombination. PBP2x is a primary resistance determinant in Streptococcus pneumoniae, and its modification is an essential step in the development of high level beta-lactam resistance. To understand such a resistance mechanism at an atomic level, we have solved the x-ray crystal structure of PBP2x from a highly penicillin-resistant clinical isolate of S. pneumoniae, Sp328, which harbors 83 mutations in the soluble region. In the proximity of the Sp328 PBP2x* active site, the Thr(338) --> Ala mutation weakens the local hydrogen bonding network, thus abrogating the stabilization of a crucial buried water molecule. In addition, the Ser(389) --> Leu and Asn(514) --> His mutations produce a destabilizing effect that generates an "open" active site. It has been suggested that peptidoglycan substrates for beta-lactam-resistant PBPs contain a large amount of abnormal, branched peptides, whereas sensitive strains tend to catalyze cross-linking of linear forms. Thus, in vivo, an "open" active site could facilitate the recognition of distinct, branched physiological substrates.
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35. |
Pikis A,
Campos JM,
Rodriguez WJ,
Keith JM,
( 2001 ) Optochin resistance in Streptococcus pneumoniae: mechanism, significance, and clinical implications. PMID : 11474432 : DOI : 10.1086/322803 Abstract >>
Traditionally, Streptococcus pneumoniae is identified in the laboratory by demonstrating susceptibility to optochin. Between 1992 and 1998, 4 pneumococcal isolates exhibiting optochin resistance were recovered from patients at Children's National Medical Center. Three of the 4 isolates consisted of mixed populations of optochin-resistant and -susceptible organisms. Both subpopulations had identical antibiograms, serotypes, and restriction fragment profiles. The other isolate was uniformly resistant to optochin. Resistant strains had MICs of optochin 4-30-fold higher than susceptible strains, belonged to different serotypes, and had dissimilar restriction fragment profiles, indicating clonal unrelatedness. Resistance arose from single point mutations in either the a-subunit (W206S) or the c-subunit (G20S, M23I, and A49T) of H(+)-ATPase. There is speculation of a possible association between exposure to antimalarial drugs and evolution of optochin resistance. alpha-Hemolytic streptococci resistant to optochin, particularly invasive isolates, should be tested for bile solubility or with an S. pneumoniae DNA probe before identification as viridans streptococci.
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36. |
Smith AM,
Botha RF,
Koornhof HJ,
Klugman KP,
( 2001 ) Emergence of a pneumococcal clone with cephalosporin resistance and penicillin susceptibility. PMID : 11502545 : DOI : 10.1128/aac.45.9.2648-2650.2001 PMC : PMC90708 Abstract >>
We report two South African serotype 6B pneumococcal isolates with cephalosporin resistance, yet with susceptibility to penicillin. DNA fingerprinting revealed that they were clonal in origin. pbp 2X and 1A genes showed major alterations typical of cephalosporin-resistant pneumococci. The pbp 2B gene was completely unaltered, explaining the penicillin susceptibility of the isolates.
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37. |
Bast DJ,
de Azavedo JC,
Tam TY,
Kilburn L,
Duncan C,
Mandell LA,
Davidson RJ,
Low DE,
( 2001 ) Interspecies recombination contributes minimally to fluoroquinolone resistance in Streptococcus pneumoniae. PMID : 11502541 : DOI : 10.1128/aac.45.9.2631-2634.2001 PMC : PMC90704 Abstract >>
Analysis of 71 ciprofloxacin-resistant (MIC > or = 4 microg/ml) Streptococcus pneumoniae clinical isolates revealed only 1 for which the quinolone resistance-determining regions of the parC, parE, and gyrB genes were genetically related to those of viridans group streptococci. Our findings support the occurrence of interspecies recombination of type II topoisomerase genes; however, its contribution to the emergence of quinolone resistance among pneumococci appears to have been minimal.
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38. |
Weiser JN,
Bae D,
Epino H,
Gordon SB,
Kapoor M,
Zenewicz LA,
Shchepetov M,
( 2001 ) Changes in availability of oxygen accentuate differences in capsular polysaccharide expression by phenotypic variants and clinical isolates of Streptococcus pneumoniae. PMID : 11500414 : DOI : 10.1128/iai.69.9.5430-5439.2001 PMC : PMC98654 Abstract >>
Most isolates of Streptococcus pneumoniae are mixed populations of transparent (T) and opaque (O) colony phenotypes. Differences in the production of capsular polysaccharide (CPS) between O and T variants were accentuated by changes in the environmental concentration of oxygen. O variants demonstrated a 5.2- to 10.6-fold increase in amounts of CPS under anaerobic compared to atmospheric growth conditions, while CPS production remained low under all conditions for T variants. Increased amounts of CPS in O compared to T pneumococci were associated with increased expression of cps-encoded proteins. The inhibitory effect of oxygen on expression of CPS in O variants correlated with decreased tyrosine phosphorylation of CpsD, a tyrosine kinase and regulator of CPS synthesis. Modulation of CpsD expression and its activity by tyrosine phosphorylation may allow the pneumococcus to adapt to the requirements of both colonization, where decreased CPS allows for adherence, and bacteremia, where increased CPS may be required to escape from opsonic clearance. In patients with invasive infection, paired isolates from the same patient were shown to have predominantly a T colony phenotype without phosphotyrosine on CpsD when cultured from the nasopharynx, and an O phenotype that phosphorylates CpsD in response to oxygen when cultured from the blood. Differences in the availability of oxygen, therefore, may be a key factor in allowing for the selection of distinct phenotypes in these two host environments.
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39. |
Ferroni A,
Berche P,
( 2001 ) Alterations to penicillin-binding proteins 1A, 2B and 2X amongst penicillin-resistant clinical isolates of Streptococcus pneumoniae serotype 23F from the nasopharyngeal flora of children. PMID : 11549185 : DOI : 10.1099/0022-1317-50-9-828 Abstract >>
Various amino acid substitutions were identified in the three major penicillin-binding proteins (PBP1A, PBP2B and PBP2X) of eight clinical isolates of Streptococcus pneumoniae serotype 23F collected from children. The particular changes related to the level of penicillin resistance. Alterations were detected in an isolate with a penicillin MIC as low as 0.06 mg/L. These results confirm that the level of penicillin resistance in pneumococci reflects with sequential alterations of PBPs in clinical isolates.
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40. |
Margolis P,
Hackbarth C,
Lopez S,
Maniar M,
Wang W,
Yuan Z,
White R,
Trias J,
( 2001 ) Resistance of Streptococcus pneumoniae to deformylase inhibitors is due to mutations in defB. PMID : 11502510 : DOI : 10.1128/aac.45.9.2432-2435.2001 PMC : PMC90673 Abstract >>
Resistance to peptide deformylase inhibitors in Escherichia coli or Staphylococcus aureus is due to inactivation of transformylase activity. Knockout experiments in Streptococcus pneumoniae R6x indicate that the transformylase (fmt) and deformylase (defB) genes are essential and that a def paralog (defA) is not. Actinonin-resistant mutants of S. pneumoniae ATCC 49619 harbor mutations in defB but not in fmt. Reintroduction of the mutated defB gene into wild-type S. pneumoniae R6x recreates the resistance phenotype. The altered enzyme displays decreased sensitivity to actinonin.
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41. |
Echenique JR,
Trombe MC,
( 2001 ) Competence repression under oxygen limitation through the two-component MicAB signal-transducing system in Streptococcus pneumoniae and involvement of the PAS domain of MicB. PMID : 11443095 : DOI : 10.1128/JB.183.15.4599-4608.2001 PMC : PMC95355 Abstract >>
In Streptococcus pneumoniae, a fermentative aerotolerant and catalase-deficient human pathogen, oxidases with molecular oxygen as substrate are important for virulence and for competence. The signal-transducing two-component systems CiaRH and ComDE mediate the response to oxygen, culminating in competence. In this work we show that the two-component MicAB system, whose MicB kinase carries a PAS domain, is also involved in competence repression under oxygen limitation. Autophosphorylation of recombinant MicB and phosphotransfer to recombinant MicA have been demonstrated. Mutational analysis and in vitro assays showed that the C-terminal part of the protein and residue L100 in the N-terminal cap of its PAS domain are both crucial for autokinase activity in vitro. Although no insertion mutation in micA was obtained, expression of the mutated allele micA59DA did not change bacterial growth and overcame competence repression under microaerobiosis. This was related to a strong instability of MicA59DA-PO(4) in vitro. Thus, mutations which either reduced the stability of MicA-PO(4) or abolished kinase activity in MicB were related to competence derepression under microaerobiosis, suggesting that MicA-PO(4) is involved in competence repression when oxygen becomes limiting. The micAB genes are flanked by mutY and orfC. MutY is an adenine glycosylase involved in the repair of oxidized pyrimidines. OrfC shows the features of a metal binding protein. We did not obtain insertion mutation in orfC, suggesting its requirement for growth. It is proposed that MicAB, with its PAS motif, may belong to a set of functions important in the protection of the cell against oxidative stress, including the control of competence.
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42. |
Smith AM,
Klugman KP,
( 2001 ) Alterations in MurM, a cell wall muropeptide branching enzyme, increase high-level penicillin and cephalosporin resistance in Streptococcus pneumoniae. PMID : 11451707 : DOI : 10.1128/AAC.45.8.2393-2396.2001 PMC : PMC90664 Abstract >>
We report that alteration in MurM, an enzyme involved in the biosynthesis of branched-stem cell wall muropeptides, is required for maximal expression of penicillin and cefotaxime resistance in the pneumococcus. Hungarian isolate 3191 (penicillin MIC, 16 microg/ml; cefotaxime MIC, 4 microg/ml) was a source of donor DNA in transformation experiments. Penicillin-binding protein DNA was insufficient to transform recipient strain R6 to full resistance. Further transformation with altered murM DNA was required for full expression of donor penicillin and cefotaxime resistance.
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43. |
Rieux V,
Carbon C,
Azoulay-Dupuis E,
( 2001 ) Complex relationship between acquisition of beta-lactam resistance and loss of virulence in Streptococcus pneumoniae. PMID : 11398111 : DOI : 10.1086/320992 Abstract >>
In a previous study of a murine peritonitis model, no Streptococcus pneumoniae strains were found that were both clinically penicillin resistant and virulent. This study assessed the relationship between acquired resistance and virulence in single- and double-isogenic penicillin-resistant (Peni-R) mutants obtained by transformation of a virulent penicillin-susceptible recipient strain with pbp2b and pbpX polymerase chain reaction fragments from a Peni-R donor strain. Sequence analysis results of the pbp2b and pbpX alleles from these strains were in keeping with acquired penicillin resistance. The virulence of these strains was significantly reduced, which shows a relationship between beta-lactam resistance and loss of virulence. The phenotype of the 23.2x mutant remained stable after in vivo passage, which suggests that the pbpX gene is involved in growth, whereas virulent revertants of the 23.2b and 23.2b.2x mutants had no change in MIC. Compensatory mutations are implicated in the revival of virulence.
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44. |
Gay K,
Stephens DS,
( 2001 ) Structure and dissemination of a chromosomal insertion element encoding macrolide efflux in Streptococcus pneumoniae. PMID : 11398110 : DOI : 10.1086/321001 Abstract >>
Macrolide resistance associated with macrolide efflux (mef) has rapidly increased in Streptococcus pneumoniae. We defined the genetic structure and dissemination of a novel mefE-containing chromosomal insertion element. The mefE gene was found on the 5' end of a 5.5- or 5.4-kb insertion designated as the macrolide efflux genetic assembly (mega), which is found in > or =4 distinct sites of the pneumococcal genome. The element was transformable and conferred macrolide resistance to susceptible S. pneumoniae. The first 2 open-reading frames (ORFs) of the element formed an operon composed of mefE and a predicted adenosine triphosphate-binding cassette homologous to msrA. Convergent to this efflux operon were 3 ORFs with homology to stress response genes of Tn5252. Mega was related to the recently described mefA-containing element Tn1207.1 but lacked the genes necessary for transposition and had unique termini and insertion sites. In metropolitan Atlanta, macrolide resistance due to mega rapidly increased in S. pneumoniae by clonal expansion and horizontally by transformation.
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45. |
Muramatsu H,
Tachikui H,
Ushida H,
Song X,
Qiu Y,
Yamamoto S,
Muramatsu T,
( 2001 ) Molecular cloning and expression of endo-beta-N-acetylglucosaminidase D, which acts on the core structure of complex type asparagine-linked oligosaccharides. PMID : 11388907 : DOI : 10.1093/oxfordjournals.jbchem.a002938 Abstract >>
Endo-beta-N-acetylglucosaminidase D (Endo D) produced by Streptococcus pneumoniae cleaves the di-N-acetylchitobiose structure in asparagine-linked oligosaccharides. The enzyme generally acts on complex type oligosaccharides after removal of external sugars by neuraminidase, beta-galactosidase, and beta-N-acetylglucosaminidase. We cloned the gene encoding the enzyme and expressed it as a periplasm enzyme in Escherichia coli. The first 37 amino acids in the predicted sequence are removed in the mature enzyme, yielding a protein with a molecular mass of 178 kDa. The substrate specificity of the recombinant enzyme is indistinguishable from the enzyme produced by S. pneumoniae. Endo-beta-N-acetylglucosaminidase A (Endo A) from Arthrobacter protophormiae, the molecular mass of which is 72 kDa, had 32% sequence identity to Endo D, starting from the N-terminal sides of both enzymes, although Endo A hydrolyzes high-mannose-type oligosaccharides and does not hydrolyze complex type ones. Endo D is not related to endo-beta-N-acetylglucosaminidases H, F(1), F(2), or F(3), which share common structural motifs. Therefore, there are two distinct groups of endo-beta-N-acetylglucosaminidases acting on asparagine-linked oligosaccharides. The C-terminal region of Endo D shows homology to beta-galactosidase and beta-N-acetylglucosaminidase from S. pneumoniae and has an LPXTG motif typical of surface-associated proteins of Gram-positive bacteria. It is possible that Endo D is located on the surface of the bacterium and, together with other glycosidases, is involved in virulence.
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46. |
Zhang Y,
Masi AW,
Barniak V,
Mountzouros K,
Hostetter MK,
Green BA,
( 2001 ) Recombinant PhpA protein, a unique histidine motif-containing protein from Streptococcus pneumoniae, protects mice against intranasal pneumococcal challenge. PMID : 11349048 : DOI : 10.1128/IAI.69.6.3827-3836.2001 PMC : PMC98401 Abstract >>
The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the current conjugate vaccine may still cause pneumococcal disease. To address these serotypes and the remaining otitis media due to Streptococcus pneumoniae, we have been evaluating antigenically conserved proteins from S. pneumoniae as vaccine candidates. A previous report identified a 20-kDa protein with putative human complement C3-proteolytic activity. By utilizing the publicly released pneumococcal genomic sequences, we found the gene encoding the 20-kDa protein to be part of a putative open reading frame of approximately 2,400 bp. We recombinantly expressed a 79-kDa fragment (rPhpA-79) that contains a repeated HxxHxH motif and evaluated it for vaccine potential. The antibodies elicited by the purified rPhpA-79 protein were cross-reactive to proteins from multiple strains of S. pneumoniae and were against surface-exposed epitopes. Immunization with rPhpA-79 protein adjuvanted with monophosphoryl lipid A (for subcutaneous immunization) or a mutant cholera toxin, CT-E29H (for intranasal immunization), protected CBA/N mice against death and bacteremia, as well as reduced nasopharyngeal colonization, following intranasal challenge with a heterologous pneumococcal strain. In contrast, immunization with the 20-kDa portion of the PhpA protein did not protect mice. These results suggest that rPhpA-79 is a potential candidate for use as a vaccine against pneumococcal systemic disease and otitis media.
|
47. |
Maskell JP,
Sefton AM,
Hall LM,
( 2001 ) Multiple mutations modulate the function of dihydrofolate reductase in trimethoprim-resistant Streptococcus pneumoniae. PMID : 11257022 : DOI : 10.1128/AAC.45.4.1104-1108.2001 PMC : PMC90431 Abstract >>
Trimethoprim resistance in Streptococcus pneumoniae can be conferred by a single amino acid substitution (I100-L) in dihydrofolate reductase (DHFR), but resistant clinical isolates usually carry multiple DHFR mutations. DHFR genes from five trimethoprim-resistant isolates from the United Kingdom were compared to susceptible isolates and used to transform a susceptible control strain (CP1015). All trimethoprim-resistant isolates and transformants contained the I100-L mutation. The properties of DHFRs from transformants with different combinations of mutations were compared. In a transformant with only the I100-L mutation (R12/T2) and a D92-A mutation also found in the DHFRs of susceptible isolates, the enzyme was much more resistant to trimethoprim inhibition (50% inhibitory concentration [IC50], 4.2 microM) than was the DHFR from strain CP1015 (IC50, 0.09 microM). However, Km values indicated a lower affinity for the enzyme's natural substrates (Km for dihydrofolate [DHF], 3.1 microM for CP1015 and 27.5 microM for R12/T2) and a twofold decrease in the specificity constant. In transformants with additional mutations in the C-terminal portion of the enzyme, Km values for DHF were reduced (9.2 to 15.2 microM), indicating compensation for the lower affinity generated by I100-L. Additional mutations in the N-terminal portion of the enzyme were associated with up to threefold-increased resistance to trimethoprim (IC50 of up to 13.7 microM). It is postulated that carriage of the mutation M53-I-which, like I100-L, corresponds to a trimethoprim binding site in the Escherichia coli DHFR-is responsible for this increase. This study demonstrates that although the I100-L mutation alone may give rise to trimethoprim resistance, additional mutations serve to enhance resistance and modulate the effects of existing mutations on the affinity of DHFR for its natural substrates.
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48. |
Vollmer W,
Tomasz A,
( 2001 ) Identification of the teichoic acid phosphorylcholine esterase in Streptococcus pneumoniae. PMID : 11260477 : DOI : 10.1046/j.1365-2958.2001.02349.x Abstract >>
Streptococcus pneumoniae is a major human pathogen and many interactions of this bacterium with its host appear to be mediated, directly or indirectly, by components of the bacterial cell wall, specifically by the phosphorylcholine residues which serve as anchors for surface-located choline-binding proteins and are also recognized by components of the host response, such as the human C-reactive protein, a class of myeloma proteins and PAF receptors. In the present study, we describe the identification of the pneumococcal pce gene encoding for a teichoic acid phosphorylcholine esterase (Pce), an enzymatic activity capable of removing phosphorylcholine residues from the cell wall teichoic acid and lipoteichoic acid. Pce carries an N-terminal signal sequence, contains a C-terminal choline-binding domain with 10 homologous repeating units similar to those found in other pneumococcal surface proteins, and the catalytic (phosphorylcholine esterase) activity is localized on the N-terminal part of the protein. The mature protein was overexpressed in Escherichia coli and purified in a one-step procedure by choline-affinity chromatography and the enzymatic activity was followed using the chromophoric p-nitrophenyl-phosphorylcholine as a model substrate. The product of the enzymatic digestion of 3H-choline-labelled cell walls was shown to be phosphorylcholine. Inactivation of the pce gene in S. pneumoniae strains by insertion-duplication mutagenesis caused a unique change in colony morphology and a striking increase in virulence in the intraperitoneal mouse model. Pce may be a regulatory element involved with the interaction of S. pneumoniae with its human host.
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49. |
Llull D,
López R,
García E,
( 2000 ) Clonal origin of the type 37 Streptococcus pneumoniae. PMID : 11272254 : DOI : 10.1089/mdr.2000.6.269 Abstract >>
It has been recently reported that different type 37 clinical isolates of Streptococcus pneumoniae have an identical tts gene directing the formation of type 37 capsular polysaccharide. Here we show that type 37 S. pneumoniae strains isolated in two different continents (Europe and America) some 60 years apart frequently gave rise to nontypable variants upon in vitro cultivation. The tts gene from three independent nontypable mutants was PCR amplified and sequenced showing different classes of inactivating mutations. Furthermore, pulsed-field gel electrophoresis and multilocus sequence typing demonstrated that the type 37 pneumococcal isolates studied so far constitute a highly related strain cluster (a clonal complex), and strongly suggested that every type 37 pneumococcus has spread globally from a single, old clone.
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50. |
Wizemann TM,
Heinrichs JH,
Adamou JE,
Erwin AL,
Kunsch C,
Choi GH,
Barash SC,
Rosen CA,
Masure HR,
Tuomanen E,
Gayle A,
Brewah YA,
Walsh W,
Barren P,
Lathigra R,
Hanson M,
Langermann S,
Johnson S,
Koenig S,
( 2001 ) Use of a whole genome approach to identify vaccine molecules affording protection against Streptococcus pneumoniae infection. PMID : 11179332 : DOI : 10.1128/IAI.69.3.1593-1598.2001 PMC : PMC98061 Abstract >>
Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniae infection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.
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51. |
Mollerach M,
García E,
( 2000 ) The galU gene of Streptococcus pneumoniae that codes for a UDP-glucose pyrophosphorylase is highly polymorphic and suitable for molecular typing and phylogenetic studies. PMID : 11137293 : DOI : 10.1016/s0378-1119(00)00468-6 Abstract >>
The enzyme UTP-glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, UDPG:PP) is synthesized by practically all organisms, although prokaryotic UDPG:PPs are evolutionarily unrelated to the eukaryotic counterparts. The primary structure of prokaryotic UDPG:PPs is well conserved, although little information exists on the polymorphism of the genes coding for these enzymes. It has been reported that the galU gene encoding the Streptococcus pneumoniae UDPG:PP is absolutely required for the synthesis of the capsular polysaccharide, a sine qua non prerequisite for virulence. A 594 bp fragment covering 66% of the galU gene from 37 pneumococcal isolates and the type strains of Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii, Streptococcus sanguinis, Streptococcus salivarius, and Streptococcus sobrinus has been amplified by PCR and sequenced. Up to 21 different alleles were found in S. pneumoniae. They possess a mosaic-like structure and belong to, at least, two evolutionarily distinct families that show a sequence divergence of 15-20%. In spite of its marked polymorphism, phylogenetic relationships among pneumococcal strains deduced from the galU gene matched those previously established by using alternative approaches. Comparison of the pneumococcal galU alleles with those from other streptococci indicated the existence of a complex network of genetic interchange. The galU gene represents an informative marker to be used alone or in conjunction with other molecular typing methods.
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52. |
Adamou JE,
Heinrichs JH,
Erwin AL,
Walsh W,
Gayle T,
Dormitzer M,
Dagan R,
Brewah YA,
Barren P,
Lathigra R,
Langermann S,
Koenig S,
Johnson S,
( 2001 ) Identification and characterization of a novel family of pneumococcal proteins that are protective against sepsis. PMID : 11159990 : DOI : 10.1128/IAI.69.2.949-958.2001 PMC : PMC97974 Abstract >>
Four pneumococcal genes (phtA, phtB, phtD, and phtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed in Escherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.
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53. |
Haasum Y,
Ström K,
Wehelie R,
Luna V,
Roberts MC,
Maskell JP,
Hall LM,
Swedberg G,
( 2001 ) Amino acid repetitions in the dihydropteroate synthase of Streptococcus pneumoniae lead to sulfonamide resistance with limited effects on substrate K(m). PMID : 11181365 : DOI : 10.1128/AAC.45.3.805-809.2001 PMC : PMC90378 Abstract >>
Sulfonamide resistance in Streptococcus pneumoniae is due to changes in the chromosomal folP (sulA) gene coding for dihydropteroate synthase (DHPS). The first reported laboratory-selected sulfonamide-resistant S. pneumoniae isolate had a 6-bp repetition, the sul-d mutation, leading to a repetition of the amino acids Ile(66) and Glu(67) in the gene product DHPS. More recently, clinical isolates showing this and other repetitions have been reported. WA-5, a clinical isolate from Washington State, contains a 6-bp repetition in the folP gene, identical to the sul-d mutation. The repetition was deleted by site-directed mutagenesis. Enzyme kinetic measurements showed that the deletion was associated with a 35-fold difference in K(i) for sulfathiazole but changed the K(m) for p-aminobenzoic acid only 2.5-fold and did not significantly change the K(m) for 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pyrophosphate. The enzyme characteristics of the deletion variant were identical to those of DHPS from a sulfonamide-susceptible strain. DHPS from clinical isolates with repetitions of Ser(61) had very similar enzyme characteristics to the DHPS from WA-5. The results confirm that the repetitions are sufficient for development of a resistant enzyme and suggest that the fitness cost to the organism of developing resistance may be very low.
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54. |
López R,
Gindreau E,
( 2000 ) MM1, a temperate bacteriophage of the type 23F Spanish/USA multiresistant epidemic clone of Streptococcus pneumoniae: structural analysis of the site-specific integration system. PMID : 10933687 : DOI : 10.1128/jvi.74.17.7803-7813.2000 PMC : PMC112310 Abstract >>
We have characterized a temperate phage (MM1) from a clinical isolate of the multiply antibiotic-resistant Spanish/American 23F Streptococcus pneumoniae clone (Spain(23F)-1 strain). The 40-kb double-stranded genome of MM1 has been isolated as a DNA-protein complex. The use of MM1 DNA as a probe revealed that the phage genome is integrated in the host chromosome. The host and phage attachment sites, attB and attP, respectively, have been determined. Nucleotide sequencing of the attachment sites identified a 15-bp core site (5'-TTATAATTCATCCGC-3') that has not been found in any bacterial genome described so far. Sequence information revealed the presence of an integrase gene (int), which represents the first identification of an integrase in the pneumococcal system. A 1.5-kb DNA fragment embracing attP and the int gene contained all of the genetic information needed for stable integration of a nonreplicative plasmid into the attB site of a pneumococcal strain. This vector will facilitate the introduction of foreign genes into the pneumococcal chromosome. Interestingly, DNAs highly similar to that of MM1 have been detected in several clinical pneumococcal isolates of different capsular types, suggesting a widespread distribution of these phages in relevant pathogenic strains.
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55. |
Ke D,
Boissinot M,
Huletsky A,
Picard FJ,
Frenette J,
Ouellette M,
Roy PH,
Bergeron MG,
( 2000 ) Evidence for horizontal gene transfer in evolution of elongation factor Tu in enterococci. PMID : 11092850 : DOI : 10.1128/jb.182.24.6913-6920.2000 PMC : PMC94815 Abstract >>
The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus durans, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, and Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus columbae, Enterococcus faecalis, Enterococcus sulfureus, Enterococcus saccharolyticus, and Enterococcus solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes.
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56. |
Peery RB,
Meier TI,
McAllister KA,
( 2000 ) Biochemical and molecular analyses of the Streptococcus pneumoniae acyl carrier protein synthase, an enzyme essential for fatty acid biosynthesis. PMID : 10903317 : DOI : 10.1074/jbc.M004475200 Abstract >>
Acyl carrier protein synthase (AcpS) is an essential enzyme in the biosynthesis of fatty acids in all bacteria. AcpS catalyzes the transfer of 4'-phosphopantetheine from coenzyme A (CoA) to apo-ACP, thus converting apo-ACP to holo-ACP that serves as an acyl carrier for the biosynthesis of fatty acids and lipids. To further understand the physiological role of AcpS, we identified, cloned, and expressed the acpS and acpP genes of Streptococcus pneumoniae and purified both products to homogeneity. Both acpS and acpP form operons with the genes whose functions are required for other cellular metabolism. The acpS gene complements an Escherichia coli mutant defective in the production of AcpS and appears to be essential for the growth of S. pneumoniae. Gel filtration and cross-linking analyses establish that purified AcpS exists as a homotrimer. AcpS activity was significantly stimulated by apo-ACP at concentrations over 10 microm and slightly inhibited at concentrations of 5-10 microm. Double reciprocal analysis of initial velocities of AcpS at various concentrations of CoA or apo-ACP indicated a random or compulsory ordered bi bi type of reaction mechanism. Further analysis of the inhibition kinetics of the product (3',5'-ADP) suggested that it is competitive with respect to CoA but mixed (competitive and noncompetitive) with respect to apo-ACP. Finally, apo-ACP bound tightly to AcpS in the absence of CoA, but CoA failed to do so in the absence of apo-ACP. Together, these results suggest that AcpS may be allosterically regulated by apo-ACP and probably proceeds by an ordered reaction mechanism with the first formation of the AcpS-apo-ACP complex and the subsequent transfer of 4'-phosphopantetheine to the apo-ACP of the complex.
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57. |
Bongaerts RJ,
Heinz HP,
Hadding U,
Zysk G,
( 2000 ) Antigenicity, expression, and molecular characterization of surface-located pullulanase of Streptococcus pneumoniae. PMID : 11083842 : DOI : 10.1128/iai.68.12.7141-7143.2000 PMC : PMC97827 Abstract >>
A putative pullulanase-encoding gene from Streptococcus pneumoniae was identified by screening a genomic expression library with human convalescent-phase serum. The 3,864-bp gene encoded a 143-kDa protein. Surface location and pullulanase activity of the protein, designated SpuA, was demonstrated. SpuA was present in all investigated pneumococcal isolates of different serotypes. The spuA 5' end was highly conserved among clinical isolates except for a 75-bp region. The properties of SpuA reported here indicate that this novel immunogenic surface protein might have potential as a vaccine target.
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58. |
Heath RJ,
( 2000 ) A triclosan-resistant bacterial enzyme. PMID : 10910344 : DOI : 10.1038/35018162 Abstract >>
N/A
|
59. |
Diehl A,
Reichmann P,
Ehlert K,
Weber B,
( 2000 ) The fib locus in Streptococcus pneumoniae is required for peptidoglycan crosslinking and PBP-mediated beta-lactam resistance. PMID : 10867238 : DOI : 10.1111/j.1574-6968.2000.tb09172.x Abstract >>
Penicillin resistance in pneumococci is mediated by modified penicillin-binding proteins (PBPs) that have decreased affinity to beta-lactams. In high-level penicillin-resistant transformants of the laboratory strain Streptococcus pneumoniae R6 containing various combinations of low-affinity PBPs, disruption of the fib locus results in a collapse of PBP-mediated resistance. In addition, crosslinked muropeptides are highly reduced. The fib operon consists of two genes, fibA and fibB, homologous to Staphylococcus aureus femA/B which are also required for expression of methicillin resistance in this organism. FibA and FibB belong to a family of proteins of Gram-positive bacteria involved in the formation of interpeptide bridges, thus representing interesting new targets for antimicrobial compounds for this group of pathogens.
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60. |
Ingraham KA,
Iordanescu S,
So CY,
Holmes DJ,
Chalker AF,
Bryant AP,
Brown JR,
Wilding EI,
( 2000 ) Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci. PMID : 10894743 : DOI : 10.1128/jb.182.15.4319-4327.2000 PMC : PMC101949 Abstract >>
The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.
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61. |
Beall B,
Gherardi G,
Facklam RR,
Hollingshead SK,
( 2000 ) Pneumococcal pspA sequence types of prevalent multiresistant pneumococcal strains in the United States and of internationally disseminated clones. PMID : 11015380 : PMC : PMC87453 Abstract >>
In a recent genotypic survey of beta-lactam-resistant pneumococci recovered in different areas of United States during 1997, eight clonal types that each represented 3 to 40 isolates accounted for 134 of 144 isolates (G. Gherardi, C. Whitney, R. Facklam, and B. Beall, J. Infect. Dis. 181:216-229, 2000). We determined the degree of pspA gene diversity among these 134 isolates and for 11 previously characterized internationally disseminated multiresistant strains. Thirty-four different pspA restriction profiles were determined for an amplicon encompassing the variable portion of the structural gene that encodes the surface-exposed domain of PspA and a variable-length proline-rich putative cell wall-associated domain. These restriction profiles closely correlated with those of 33 different pspA sequence types of an approximately 230-residue region corresponding to residues 182 to 410 of the strain Rx1 PspA. These residues encompass a 100-residue clade-defining region known to contain cross-protective epitopes for which 17 sequence types were found. Distinct, conserved pspA sequence types were found for the majority of strains within seven of the eight U.S. clonal types assessed, while one pulsed-field gel electrophoresis type was represented by isolates of three distinct PspA clades. Sequence typing of pspA provides an added level of specificity in the subtyping of isolates and is a necessary first step in determining the components needed in a PspA vaccine which could elicit effective cross-protective coverage.
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62. |
Reichmann P,
Hakenbeck R,
( 2000 ) Allelic variation in a peptide-inducible two-component system of Streptococcus pneumoniae. PMID : 11034284 : DOI : 10.1111/j.1574-6968.2000.tb09291.x Abstract >>
The peptide SpiP of Streptococcus pneumoniae regulates the induction of a complex signal transduction system spiR1spiR2spiH. Distinct alleles of spiP and the receptor histidine protein kinase gene spiH were recognized in different pneumococcal clones. The spi system in strain KNR7/87 is adjacent to a bacteriocin gene cluster encoding putative double glycine-type bacteriocins, immunity proteins, and translocator proteins. A direct repeat element upstream of the spiR1 promoter and another three potential transcription start sites within the bacteriocin cluster indicate that SpiP functions as an inducing peptide for bacteriocin synthesis in S. pneumoniae.
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63. |
Samrakandi MM,
( 2000 ) Hyperrecombination in Streptococcus pneumoniae depends on an atypical mutY homologue. PMID : 10852864 : DOI : 10.1128/jb.182.12.3353-3360.2000 PMC : PMC101888 Abstract >>
The unusual behavior of the mutation ami36, which generates hyperrecombination in two point crosses, was previously attributed to a localized conversion process changing A/G mispairs into CG pairs. Although the mechanism was found to be dependent on the DNA polymerase I, the specific function responsible for this correction was still unknown. Analysis of the pneumococcal genome sequence has revealed the presence of an open reading frame homologous to the gene mutY of Escherichia coli. The gene mutY encodes an adenine glycosylase active on A/G and A/7,8-dihydro-8-oxoguanine (8-OxoG) mismatches, inducing their repair to CG and C/8-OxoG, respectively. Here we report that disrupting the pneumococcal mutY homologue abolishes the hyperrecombination induced by ami36 and leads to a mutator phenotype specifically enhancing AT-to-CG transversions. The deduced amino acid sequence of the pneumococcal MutY protein reveals the absence of four cysteines, highly conserved in the endonuclease III/MutY glycosylase family, which ligate a [4Fe-4S](2+) cluster. The actual function of this cluster is still intriguing, inasmuch as we show that the pneumococcal gene complements a mutY strain of E. coli.
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64. |
Cogné N,
Claverys J,
Denis F,
Martin C,
( 2000 ) A novel mutation in the alpha-helix 1 of the C subunit of the F(1)/F(0) ATPase responsible for optochin resistance of a Streptococcus pneumoniae clinical isolate. PMID : 11035244 : Abstract >>
Previously reported mutations involved in optochin resistance of Streptococcus pneumoniae clinical isolates changed residues 48, 49 or 50, in the transmembrane alpha-helix 2 of the F(1)/F(0) ATPase subunit. We report here an unusual mutation which changes the sequence of the transmembrane alpha-helix 1 of the AtpC subunit. This mutation involves a Gly to Ser substitution resulting from a G to A transition at codon 14 of the atpC gene.
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65. |
Gosink KK,
Mann ER,
Guglielmo C,
Tuomanen EI,
Masure HR,
( 2000 ) Role of novel choline binding proteins in virulence of Streptococcus pneumoniae. PMID : 10992472 : DOI : 10.1128/iai.68.10.5690-5695.2000 PMC : PMC101524 Abstract >>
The choline binding proteins (CBPs) are a family of surface proteins noncovalently bound to the phosphorylcholine moiety of the cell wall of Streptococcus pneumoniae by a conserved choline binding domain. Six new members of this family were identified, and these six plus two recently described cell wall hydrolases, LytB and LytC, were characterized for their roles in virulence. CBP-deficient mutants were constructed and tested for adherence to eukaryotic cells, colonization of the rat nasopharynx, and ability to cause sepsis. Five CBP mutants, CbpD, CbpE, CbpG, LytB, and LytC, showed significantly reduced colonization of the nasopharynx. For CbpE and -G this was attributable to a decreased ability to adhere to human cells. CbpG, a putative serine protease, also played a role in sepsis, the first observation of a pneumococcal virulence determinant strongly operative both on the mucosal surface and in the bloodstream.
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66. |
Zähner D,
Hakenbeck R,
( 2000 ) The Streptococcus pneumoniae beta-galactosidase is a surface protein. PMID : 11004197 : DOI : 10.1128/jb.182.20.5919-5921.2000 PMC : PMC94720 Abstract >>
The beta-galactosidase gene of Streptococcus pneumoniae, bgaA, encodes a putative 2,235-amino-acid protein with the two amino acid motifs characteristic of the glycosyl hydrolase family of proteins. In addition, an N-terminal signal sequence and a C-terminal LPXTG motif typical of surface-associated proteins of gram-positive bacteria are present. Trypsin treatment of cells resulted in solubilization of the enzyme, documenting that it is associated with the cell envelope. In order to obtain defined mutants suitable for lacZ reporter experiments, the bgaA gene was disrupted, resulting in a complete absence of endogenous beta-galactosidase activity. The results are consistent with beta-galactosidase being a surface protein that seems not to be involved in lactose metabolism but that may play a role during pathogenesis.
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67. |
Hollingshead SK,
Becker R,
Briles DE,
( 2000 ) Diversity of PspA: mosaic genes and evidence for past recombination in Streptococcus pneumoniae. PMID : 10992499 : DOI : 10.1128/iai.68.10.5889-5900.2000 PMC : PMC101551 Abstract >>
Pneumococcal surface protein A (PspA) is a serologically variable protein of Streptococcus pneumoniae. Twenty-four diverse alleles of the pspA gene were sequenced to investigate the genetic basis for serologic diversity and to evaluate the potential of diversity to have an impact on PspA's use in human vaccination. The 24 pspA gene sequences from unrelated strains revealed two major allelic types, termed "families," subdivided into clades. A highly mosaic gene structure was observed in which individual mosaic sequence blocks in PspAs diverged from each other by over 20% in many cases. This level of divergence exceeds that observed for blocks in the penicillin-binding proteins of S. pneumoniae or in many cross-species comparisons of gene loci. Conversely, because the mosaic pattern is so complex, each pair of pspA genes also has numerous shared blocks, but the position of conserved blocks differs from gene pair to gene pair. A central region of pspA, important for eliciting protective antibodies, was found in six clades, which each diverge from the other clades by >20%. Sequence relationships among the 24 alleles analyzed over three windows were discordant, indicating that intragenic recombination has occurred within this locus. The extensive recombination which generated the mosaic pattern seen in the pspA locus suggests that natural selection has operated in the history of this gene locus and underscores the likelihood that PspA may be important in the interaction between the pneumococcus and its human host.
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68. |
Santagati M,
Iannelli F,
Oggioni MR,
Stefani S,
Pozzi G,
( 2000 ) Characterization of a genetic element carrying the macrolide efflux gene mef(A) in Streptococcus pneumoniae. PMID : 10952626 : DOI : 10.1128/aac.44.9.2585-2587.2000 PMC : PMC90116 Abstract >>
The mef(A) gene from a clinical isolate of Streptococcus pneumoniae exhibiting the M-type resistance to macrolides was found to be part of the 7,244-bp chromosomal element Tn1207.1, which contained 8 open reading frames. orf2 encodes a resolvase/invertase, and orf5 is a homolog of the macrolide-streptogramin B resistance gene msr(SA).
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69. |
Finkel D,
Cheng Q,
( 2000 ) Novel purification scheme and functions for a C3-binding protein from Streptococcus pneumoniae. PMID : 10820017 : DOI : 10.1021/bi992157d Abstract >>
To isolate microbial proteins capable of binding the third component of complement (C3), we coupled the free sulfhydryl group of methylamine-inactivated C3 to a thiolSepharose matrix. This simple technique facilitated the purification of the first C3-binding protein isolated from a bacterium (Streptococcus pneumoniae). Both metastable (native) and thioester-disrupted C3 were recognized by this protein; binding of C3 was noncovalent, independent of thioester conformation, and preferential for the C3 alpha-chain. Sequencing of amino-terminal and internal peptides from the C3-binding protein disclosed a proline-rich region spanning approximately 20 amino acids and a signal peptide that had not been previously reported. The gene was isolated from a library of genomic DNA from laboratory strain CP1200 by screening with a 1200 bp PCR product amplified from degenerate oligonucleotides encoding the amino terminal sequence and the internal proline-rich sequence. The open reading frame spanned 1692 bp; all peptide sequences were identified in the translated gene product, which also contained at least three choline-binding repeats at the carboxy-terminus. The gene was conserved, and the translated protein was functionally active in pneumococcal clinical isolates of serotypes 1, 3, 4, 14, and 19F. Serum from a patient recovering from acute pneumococcal infection contained IgG antibodies specific for this protein by immunoblot. Wide conservation among clinical isolates, saturable binding of C3, and the ability to stimulate the human immune response have not previously been reported for this choline-binding protein. A similar biochemical approach should enable the identification of other C3-binding proteins in microorganisms able to elude complement-mediated host defense.
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70. |
Vollmer W,
( 2000 ) The pgdA gene encodes for a peptidoglycan N-acetylglucosamine deacetylase in Streptococcus pneumoniae. PMID : 10781617 : DOI : 10.1074/jbc.M910189199 Abstract >>
Analytical work on the fractionation of the glycan strands of Streptococcus pneumoniae cell wall has led to the observation that an unusually high proportion of hexosamine units (over 80% of the glucosamine and 10% of the muramic acid residues) was not N-acetylated, explaining the resistance of the peptidoglycan to the hydrolytic action of lysozyme, a muramidase that cleaves in the glycan backbone. A gene, pgdA, was identified as encoding for the peptidoglycan N-acetylglucosamine deacetylase A with amino acid sequence similarity to fungal chitin deacetylases and rhizobial NodB chitooligosaccharide deacetylases. Pneumococci in which pgdA was inactivated by insertion duplication mutagenesis produced fully N-acetylated glycan and became hypersensitive to exogenous lysozyme in the stationary phase of growth. The pgdA gene may contribute to pneumococcal virulence by providing protection against host lysozyme, which is known to accumulate in high concentrations at infection sites.
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71. |
Janulczyk R,
Iannelli F,
Sjoholm AG,
Pozzi G,
Bjorck L,
( 2000 ) Hic, a novel surface protein of Streptococcus pneumoniae that interferes with complement function. PMID : 10967103 : DOI : 10.1074/jbc.M004572200 Abstract >>
The important human pathogen Streptococcus pneumoniae was found to absorb factor H, an inhibitor of complement, from human plasma. We identified the gene encoding a novel surface protein, factor H-binding inhibitor of complement (Hic), in the pspC locus of type 3 pneumococci. Unlike PspC proteins in other serotypes, Hic is anchored to the cell wall by means of an LPXTG motif, and the overall sequence homology to various PspC proteins is low. However, the NH(2)-terminal region showed significant homology to the NH(2)-terminal region of several PspC proteins. A fragment of Hic, covering this homologous region, was expressed as a glutathione S-transferase (GST) fusion protein. GST:Hic(39-261) bound radiolabeled factor H and inhibited binding of factor H to pneumococci of different serotypes. Interaction kinetics between GST:Hic(39-261) and factor H were studied with surface plasmon resonance and showed a high affinity binding (K(A) = 5 x 10(7), K(D) = 2.3 x 10(-)(8)). Mutant pneumococci lacking Hic showed no absorption of factor H in human plasma and no binding of radiolabeled factor H, suggesting that Hic is responsible for factor H-binding in type 3 pneumococci. Factor H-dependent inhibition of the alternative pathway was not diminished by the presence of GST:Hic(39-261). In addition, an intrinsic inhibitory effect of Hic is suggested.
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72. |
Braun JS,
Park E,
Murti S,
Charpentier E,
Novak R,
( 2000 ) Extracellular targeting of choline-binding proteins in Streptococcus pneumoniae by a zinc metalloprotease. PMID : 10792723 : DOI : 10.1046/j.1365-2958.2000.01854.x Abstract >>
A genetic-based search for surface proteins of Streptococcus pneumoniae involved in adhesion identified a putative zinc metalloprotease (ZmpB). ZmpB shared high amino acid sequence similarities with IgA1 proteases of Gram-positive bacteria, but ZmpB had neither IgA1 nor IgA2 protease activity. Analysis of a family of surface-expressed proteins, the choline-binding proteins (Cbp's), in a zmpB-deficient mutant demonstrated a global loss of surface expression of CbpA, CbpE, CbpF and CbpJ. CbpA was detected within the cytoplasm. The zmpB-deficient mutant also failed to lyse with penicillin, a sign of lack of function of the Cbp LytA. Immunodetection studies revealed that the autolysin (LytA), normally located on the cell wall, was trapped in the cytoplasm colocalized with DNA and the transformation protein CinA. Trafficking of CinA and RecA to the cell membrane during genetic competence was also not observed in the zmpB-deficient mutant. These results suggest a protease dependent regulatory mechanism governing the translocation of CinA and the Cbp's LytA and CbpA of S. pneumoniae.
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73. |
Fenoll A,
Ferrándiz MJ,
( 2000 ) Horizontal transfer of parC and gyrA in fluoroquinolone-resistant clinical isolates of Streptococcus pneumoniae. PMID : 10722479 : DOI : 10.1128/aac.44.4.840-847.2000 PMC : PMC89780 Abstract >>
We have analyzed genetically three clinical isolates (3180, 3870, and 1244) of Streptococcus pneumoniae with high-level ciprofloxacin resistance. Isolates 3180 and 3870 were atypical because of their insolubility in deoxycholate. However, they hybridized specifically with pneumococcal autolysin and pneumolysin gene probes and have typical pneumococcal atpC and atpA gene sequences. Analysis of the complete sequences of the parC and gyrA genes revealed total variations of 8 and 8.7% (isolate 3180) and 7.4 and 3.6% (isolate 3870), respectively, compared to the wild-type strain R6 sequence. The variations observed between the sequences of R6 and isolate 1244 were less than 0.9%. The structure of the gyrA and parC genes from isolates 3180 and 3870 was organized in sequence blocks that show different levels of divergence, suggesting a pattern of recombination. These results are evidence for recombination at the fluoroquinolone target genes in clinical isolates of S. pneumoniae. The genetically related viridans group streptococci could act as a reservoir for fluoroquinolone resistance genes.
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74. |
Muñoz R,
López R,
García E,
( 1999 ) Construction of a new Streptococcus pneumoniae-Escherichia coli shuttle vector based on the replicon of an indigenous pneumococcal cryptic plasmid. PMID : 10943387 : Abstract >>
The nucleotide sequence of a cryptic plasmid (pRMG1) isolated from a type 1 Streptococcus pneumoniae has been determined and two recombinant plasmids, pRMGE1 and pRMGE2, bearing the pRMG1 replicon have been constructed. pRMGE2 is a shuttle vector for Escherichia coli and S. pneumoniae. The important characteristics of this cloning vector are: a size of 5.5 kb including a 1.4 kb fragment of pRMG1 (containing a double-stranded replication origin and an open reading frame encoding a putative replication initiation protein), a multicloning site, two antibiotic resistance markers for selection of plasmid containing cells, and blue-white colony screening in E. coli for identification of insert-containing plasmids.
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75. |
Ayalew S,
Lacks SA,
( 2000 ) Regulation of competence for genetic transformation in Streptococcus pneumoniae: expression of dpnA, a late competence gene encoding a DNA methyltransferase of the DpnII restriction system. PMID : 10712690 : DOI : 10.1046/j.1365-2958.2000.01777.x Abstract >>
The chromosomal DpnII gene cassette of Streptococcus pneumoniae encodes two methyltransferases and an endonuclease. One methyltransferase acts on double-stranded and the other on single-stranded DNA. Two mRNAs are transcribed from the cassette. One, a SigA promoter transcript, includes all three genes; the other includes a truncated form of the second methyltransferase gene (dpnA) and the endonuclease gene. The truncated dpnA, which is translated from the second start codon in the full gene, was shown to produce active enzyme. A promoter reporter plasmid for S. pneumoniae was devised to characterize the promoter for the second mRNA. This transcript was found to depend on a promoter that responded to the induction of competence for genetic transformation. The promoter contains the combox sequence recognized by a SigH-containing RNA polymerase. As part of the competence regulon, the dpnA gene makes a product able to methylate incoming plasmid strands to protect them from the endonuclease and allow plasmid establishment. Its function differs from most genes in the regulon, which are involved in DNA uptake. Comparison of R6 and Rx strains of S. pneumoniae showed the temperature dependence of transformation in R6 to result from temperature sensitivity of the uptake apparatus and not the development of competence.
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76. |
Paton JC,
Morona JK,
( 2000 ) Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in streptococcus pneumoniae. PMID : 10760144 : DOI : 10.1046/j.1365-2958.2000.01808.x Abstract >>
In Streptococcus pneumoniae, the first four genes of the capsule locus (cpsA to cpsD) are common to most serotypes. By analysis of various in-frame deletion and site-directed mutants, the function of their gene products in capsular polysaccharide (CPS) biosynthesis was investigated. We found that while CpsB, C and D are essential for encapsulation, CpsA is not. CpsC and CpsD have similarity to the amino-terminal and carboxy-terminal regions, respectively, of the autophosphorylating protein-tyrosine kinase Wzc from Escherichia coli. Alignment of CpsD with Wzc and other related proteins identified conserved Walker A and B sequence motifs and a tyrosine rich domain close to the carboxy-terminus. We have shown that CpsD is also an autophosphorylating protein-tyrosine kinase and that point mutations in cpsD affecting either the ATP-binding domain (Walker A motif) or the carboxy-terminal [YGX]4 repeat domain eliminated tyrosine phosphorylation of CpsD. We describe, for the first time, the phenotypic impact of these two mutations on polysaccharide production and show that they affect CPS production differently. Whereas a mutation in the Walker A motif resulted in loss of encapsulation, mutation of the tyrosines in the [YGX]4 repeat domain resulted in an apparent increase in encapsulation and a mucoid phenotype. These data suggest that autophosphorylation of CpsD at tyrosine attenuates its activity and reduces the level of encapsulation. Additionally, we demonstrated that CpsC is required for CpsD tyrosine phosphorylation and that CpsB influences dephosphorylation of CpsD. These results are consistent with CpsD tyrosine phosphorylation acting to negatively regulate CPS production. This has implications for the function of CpsC/CpsD homologues in both Gram-positive and Gram-negative bacteria and provides a mechanism to explain regulation of CPS production during pathogenesis.
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77. |
Filipe SR,
( 2000 ) Inhibition of the expression of penicillin resistance in Streptococcus pneumoniae by inactivation of cell wall muropeptide branching genes. PMID : 10759563 : DOI : 10.1073/pnas.080067697 PMC : PMC18328 Abstract >>
Penicillin-resistant strains of Streptococcus pneumoniae contain low affinity penicillin-binding proteins and often also produce abnormal indirectly crosslinked cell walls. However the relationship between cell wall abnormality and penicillin resistance has remained obscure. We now show that the genome of S. pneumoniae contains an operon composed of two genes (murM and murN) that encode enzymes involved with the biosynthesis of branched structured cell wall muropeptides. The sequences of murMN were compared in two strains: the penicillin-susceptible strain R36A producing the species-specific pneumococcal cell wall peptidoglycan in which branched stem peptides are rare, and the highly penicillin-resistant transformant strain Pen6, the cell wall of which is enriched for branched-structured stem peptides. The two strains carried different murM alleles: murM of the penicillin-resistant strain Pen6 had a "mosaic" structure encoding a protein that was only 86.5% identical to the product of murM identified in the isogenic penicillin-susceptible strain R36A. Mutants of R36A and Pen6 in which the murMN operon was interrupted by insertion-duplication mutagenesis produced peptidoglycan from which all branched muropeptide components were missing. The insertional mutant of Pen6 carried a pbp2x gene with the same "mosaic" sequence found in Pen6. On the other hand, inactivation of murMN in strain Pen6 and other resistant strains caused a virtually complete loss of penicillin resistance. Our observations indicate that the capacity to produce branched cell wall precursors plays a critical role in the expression of penicillin resistance in S. pneumoniae.
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78. |
Kim CK,
Lee H,
Peck KR,
Kim SW,
Jin JH,
Yang JW,
Song JH,
( 2000 ) Molecular characterization of multidrug-resistant Streptococcus pneumoniae isolates in Korea. The Asian Network for Surveillance of Resistant Pathogens (ANSORP) Study Group. PMID : 10747158 : PMC : PMC86510 Abstract >>
Pulsed-field gel electrophoresis, ribotyping, and fingerprinting analysis of 22 invasive isolates of multidrug-resistant (MDR) pneumococci from Korea showed that 59 to 82% were genetically related. DNA sequencing of the PBP 2B gene showed relatively uniform alterations in nucleotides (5.4 to 7.8%) and amino acids (3.0 to 4. 3%), while Asn-276-->Lys, Arg-285-->Cys and Ser-305-->Phe substitutions were unique to Korean MDR strains, suggesting the spread of a few epidemic clones of resistant pneumococci within Korea.
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79. |
Caimano MJ,
Hardy GG,
( 2000 ) Capsule biosynthesis and basic metabolism in Streptococcus pneumoniae are linked through the cellular phosphoglucomutase. PMID : 10714989 : DOI : 10.1128/jb.182.7.1854-1863.2000 PMC : PMC101867 Abstract >>
Synthesis of the type 3 capsular polysaccharide of Streptococcus pneumoniae requires UDP-glucose (UDP-Glc) and UDP-glucuronic acid (UDP-GlcUA) for production of the [3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n) polymer. The generation of UDP-Glc proceeds by conversion of Glc-6-P to Glc-1-P to UDP-Glc and is mediated by a phosphoglucomutase (PGM) and a Glc-1-P uridylyltransferase, respectively. Genes encoding both a Glc-1-P uridylyltransferase (cps3U) and a PGM homologue (cps3M) are present in the type 3 capsule locus, but these genes are not essential for capsule production. In this study, we characterized a mutant that produces fourfold less capsule than the type 3 parent. The spontaneous mutation resulting in this phenotype was not contained in the type 3 capsule locus but was instead located in a distant gene (pgm) encoding a second PGM homologue. The function of this gene product as a PGM was demonstrated through enzymatic and complementation studies. Insertional inactivation of pgm reduced capsule production to less than 10% of the parental level. The loss of PGM activity in the insertion mutants also caused growth defects and a strong selection for isolates containing second-site suppressor mutations. These results demonstrate that most of the PGM activity required for type 3 capsule biosynthesis is derived from the cellular PGM.
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80. |
Zhao W,
Black TA,
Shaw KJ,
Adrian PV,
( 2000 ) Mutations in ribosomal protein L16 conferring reduced susceptibility to evernimicin (SCH27899): implications for mechanism of action. PMID : 10681347 : DOI : 10.1128/aac.44.3.732-738.2000 PMC : PMC89755 Abstract >>
A clinical isolate of Streptococcus pneumoniae (SP#5) that showed decreased susceptibility to evernimicin (MIC, 1.5 microgram/ml) was investigated. A 4,255-bp EcoRI fragment cloned from SP#5 was identified by its ability to transform evernimicin-susceptible S. pneumoniae R6 (MIC, 0.03 microgram/ml) such that the evernimicin MIC was 1.5 microgram/ml. Nucleotide sequence analysis of this fragment revealed that it contained portions of the S10-spc ribosomal protein operons. The nucleotide sequences of resistant and susceptible isolates were compared, and a point mutation (thymine to guanine) that causes an Ile52-Ser substitution in ribosomal protein L16 was identified. The role of this mutation in decreasing susceptibility to evernimicin was confirmed by direct transformation of the altered L16 gene. The presence of the L16 mutation in the resistant strain suggests that evernimicin is an inhibitor of protein synthesis. This was confirmed by inhibition studies using radiolabeled substrates, which showed that the addition of evernimicin at sub-MIC levels resulted in a rapid decrease in the incorporation of radiolabeled isoleucine in a susceptible isolate (SP#3) but was much less effective against SP#5. The incorporation of isoleucine showed a linear response to the dose level of evernimicin. The incorporation of other classes of labeled substrates was unaffected or much delayed, indicating that these were secondary effects.
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81. |
Charpentier E,
Novak R,
( 2000 ) Signal transduction by a death signal peptide: uncovering the mechanism of bacterial killing by penicillin. PMID : 10678168 : Abstract >>
The binding of bactericidal antibiotics like penicillins, cephalosporins, and glycopeptides to their bacterial targets stops bacterial growth but does not directly cause cell death. A second process arising from the bacteria itself is necessary to trigger endogenous suicidal enzymes that dissolve the cell wall during autolysis. The signal and the trigger pathway for this event are completely unknown. Using S. pneumoniae as a model, we demonstrate that signal transduction via the two-component system VncR/S triggers multiple death pathways. We show that the signal sensed by VncR/S is a secreted peptide, Pep27, that initiates the cell death program. These data depict a novel model for the control of bacterial cell death.
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82. |
Zawadzki P,
Pickerill P,
Majewski J,
( 2000 ) Barriers to genetic exchange between bacterial species: Streptococcus pneumoniae transformation. PMID : 10648528 : DOI : 10.1128/jb.182.4.1016-1023.2000 PMC : PMC94378 Abstract >>
Interspecies genetic exchange is an important evolutionary mechanism in bacteria. It allows rapid acquisition of novel functions by transmission of adaptive genes between related species. However, the frequency of homologous recombination between bacterial species decreases sharply with the extent of DNA sequence divergence between the donor and the recipient. In Bacillus and Escherichia, this sexual isolation has been shown to be an exponential function of sequence divergence. Here we demonstrate that sexual isolation in transformation between Streptococcus pneumoniae recipient strains and donor DNA from related strains and species follows the described exponential relationship. We show that the Hex mismatch repair system poses a significant barrier to recombination over the entire range of sequence divergence (0.6 to 27%) investigated. Although mismatch repair becomes partially saturated, it is responsible for 34% of the observed sexual isolation. This is greater than the role of mismatch repair in Bacillus but less than that in Escherichia. The remaining non-Hex-mediated barrier to recombination can be provided by a variety of mechanisms. We discuss the possible additional mechanisms of sexual isolation, in view of earlier findings from Bacillus, Escherichia, and Streptococcus.
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83. |
Wittzell H,
( 1999 ) Chloroplast DNA variation and reticulate evolution in sexual and apomictic sections of dandelions. PMID : 10632854 : Abstract >>
Sequencing of the trnL-trnF intergenic spacer in chloroplast DNA (cpDNA) from 237 sexual and apomictic species of dandelions (genus Taraxacum) from Europe, Asia and arctic North America revealed 46 haplotypes, which differed mainly by a variable number of polymorphic tRNA pseudogenes next to the trnF gene. The haplotypes could be divided into 20 cpDNA lineages, but independent duplications and deletions of the pseudogene copies made it difficult to further reconstruct the phylogeny. Intraspecific cpDNA variation was found in the primitive sexual T. serotinum. However, in contrast to a recent study, no cpDNA variation was detected within 12 apomictic species representing a variety of haplotypes. The cpDNA haplotype may therefore help to define these critical apomicts. On the other hand, the genetic variation may easily be overestimated, if the clones are not correctly identified, because some morphologically similar microspecies carried very different haplotypes. In all, 36 sections of the genus were sampled. Four primitive, mainly sexual, sections only displayed a group of ancient haplotypes, whereas morphologically more advanced sections often exhibited many different haplotypes from up to seven cpDNA lineages. In the latter cases, the lineages were rarely unique to a certain section. For example, the two most widespread haplotypes, belonging to different lineages, were found together in nine sections. This suggests that significant gene flow has occurred among the advanced sections, although sexual reproduction is not currently known in several of them. The result is consistent with the reticulate distribution of morphological characters among the sections.
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84. |
Broughton K,
Woodard G,
Pickerill AP,
Efstratiou A,
Whatmore AM,
( 2000 ) Genetic relationships between clinical isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: characterization of "Atypical" pneumococci and organisms allied to S. mitis harboring S. pneumoniae virulence factor-encoding genes. PMID : 10678950 : DOI : 10.1128/iai.68.3.1374-1382.2000 PMC : PMC97291 Abstract >>
The oral streptococcal group (mitis phylogenetic group) currently consists of nine recognized species, although the group has been traditionally difficult to classify, with frequent changes in nomenclature over the years. The pneumococcus (Streptococcus pneumoniae), an important human pathogen, is traditionally distinguished from the most closely related oral streptococcal species Streptococcus mitis and Streptococcus oralis on the basis of three differentiating characteristics: optochin susceptibility, bile solubility, and agglutination with antipneumococcal polysaccharide capsule antibodies. However, there are many reports in the literature of pneumococci lacking one or more of these defining characteristics. Sometimes called "atypical" pneumococci, these isolates can be the source of considerable confusion in the clinical laboratory. Little is known to date about the genetic relationships of such organisms with classical S. pneumoniae isolates. Here we describe these relationships based on sequence analysis of housekeeping genes in comparison with previously characterized isolates of S. pneumoniae, S. mitis, and S. oralis. While most pneumococci were found to represent a closely related group these studies identified a subgroup of atypical pneumococcal isolates (bile insoluble and/or "acapsular") distinct from, though most closely related to, the "typical" pneumococcal isolates. However, a large proportion of isolates, found to be atypical on the basis of capsule reaction alone, did group with typical pneumococci, suggesting that they have either lost capsule production or represent as-yet-unrecognized capsular types. In contrast to typical S. pneumoniae, isolates phenotypically identified as S. mitis and S. oralis, which included isolates previously characterized in taxonomic studies, were genetically diverse. While most of the S. oralis isolates did fall into a well-separated group, S. mitis isolates did not cluster into a well-separated group. During the course of these studies we also identified a number of potentially important pathogenic isolates, which were frequently associated with respiratory disease, that phenotypically and genetically are most closely related to S. mitis but which harbor genes encoding the virulence determinants pneumolysin and autolysin classically associated with S. pneumoniae.
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85. |
Whitney CG,
Gherardi G,
( 2000 ) Major related sets of antibiotic-resistant Pneumococci in the United States as determined by pulsed-field gel electrophoresis and pbp1a-pbp2b-pbp2x-dhf restriction profiles. PMID : 10608770 : DOI : 10.1086/315194 Abstract >>
To assess the genetic diversity of pneumococci causing serious disease within the United States, restriction profiles of 3 penicillin-binding protein (PBP)-gene amplicons and the dhf amplicon were examined in 241 recent sterile-site isolates from 7 population centers. This analysis provided markers useful for epidemiologic studies and was generally predictive of resistances to beta-lactam antibiotics and trimethoprim-sulfamethoxazole. Eight pulsed-field gel electrophoresis (PFGE) types, each representing 3-40 isolates, accounted for 134 of the 144 beta-lactam-resistant pneumococci (MICs >/=1 microgram/mL for penicillin, cefotaxime, or both). Five of these PFGE types contained subtypes highly related to subtypes of previously characterized pneumococcal clones. Within 4 of these PFGE types, the major composite PBP gene-dhf profile was highly related to the composite profile from the previously characterized related clone. Eight capsular serotypes were found among the 144 beta-lactam-resistant pneumococci. Divergent capsular types among isolates with identical PBP gene-dhf profiles and related PFGE types indicated several instances of capsular serotype switching.
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86. |
Dos Santos D,
Ogunniyi AD,
Le Thomas I,
Le Bras G,
Chapuy-Regaud S,
Auzat I,
( 1999 ) The NADH oxidase of Streptococcus pneumoniae: its involvement in competence and virulence. PMID : 10594826 : DOI : 10.1046/j.1365-2958.1999.01663.x Abstract >>
A soluble flavoprotein that reoxidizes NADH and reduces molecular oxygen to water was purified from the facultative anaerobic human pathogen Streptococcus pneumoniae. The nucleotide sequence of nox, the gene which encodes it, has been determined and was characterized at the functional and physiological level. Several nox mutants were obtained by insertion, nonsense or missense mutation. In extracts from these strains, no NADH oxidase activity could be measured, suggesting that a single enzyme encoded by nox, having a C44 in its active site, was utilizing O2 to oxidize NADH in S. pneumoniae. The growth rate and yield of the NADH oxidase-deficient strains were not changed under aerobic or anaerobic conditions, but the efficiency of development of competence for genetic transformation during growth was markedly altered. Conditions that triggered competence induction did not affect the amount of Nox, as measured using Western blotting, indicating that nox does not belong to the competence-regulated genetic network. The decrease in competence efficiency due to the nox mutations was similar to that due to the absence of oxygen in the nox+ strain, suggesting that input of oxygen into the metabolism via NADH oxidase was important for controlling competence development throughout growth. This was not related to regulation of nox expression by O2. Interestingly, the virulence and persistence in mice of a blood isolate was attenuated by a nox insertion mutation. Global cellular responses of S. pneumoniae, such as competence for genetic exchange or virulence in a mammalian host, could thus be modulated by oxygen via the NADH oxidase activity of the bacteria, although the bacterial energetic metabolism is essentially anaerobic. The enzymatic activity of the NADH oxidase coded by nox was probably involved in transducing the external signal, corresponding to O2 availability, to the cell metabolism and physiology; thus, this enzyme may function as an oxygen sensor. This work establishes, for the first time, the role of O2 in the regulation of pneumococcal transformability and virulence.
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87. |
Doherty N,
Paul J,
Pickerill AP,
Trzcinski K,
King SJ,
Whatmore AM,
Müller-Graf CD,
( 1999 ) Population biology of Streptococcus pneumoniae isolated from oropharyngeal carriage and invasive disease. PMID : 10589738 : DOI : 10.1099/00221287-145-11-3283 Abstract >>
The population structure of Streptococcus pneumoniae in a sample of 134 carried antibiotic-susceptible isolates, and 53 resistant and susceptible invasive isolates, was examined using a DNA-based version of multilocus enzyme electrophoresis: multilocus restriction typing (MLRT). This involved RFLP analysis of PCR products generated from nine loci of housekeeping genes located around the pneumococcal chromosome. The combination of alleles at each of the nine loci gave an allelic profile or restriction type (RT). All carried (throat or nasopharyngeal) isolates from children or adults in Oxford and Manchester, UK, and from an HIV-seropositive cohort in Nairobi, Kenya, showed an epidemic population structure. Twelve carried clonal groups, each with different serotypes, were identified at both locations within the UK. Almost all of the carried clones examined (16/17) were found to possess identical RTs or sequence types (STs) to invasive isolates, indicating that frequently carried clones are also associated with cases of invasive disease. As expected from previous studies, the population of 53 invasive, mainly penicillin-resistant, isolates was also found to be at linkage equilibrium. Serotype switching was identified among 14% of RTs that possessed two or more members, or 5.7% of individual isolates within these RTs. In support of a population structure in which there is frequent recombination, there is also clear evidence that the trpA/B locus within pneumococci has evolved by horizontal gene transfer. A non-serotypable isolate from an HIV-seropositive patient in Kenya was clearly genetically distinct from other strains studied, with unique alleles at eight out of nine loci examined. However, it was initially identified as a pneumococcus by a 16S RNA gene probe (Gen-Probe), optochin susceptibility and the presence of pneumolysin and autolysin.
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88. |
Enright MC,
( 1999 ) Extensive variation in the ddl gene of penicillin-resistant Streptococcus pneumoniae results from a hitchhiking effect driven by the penicillin-binding protein 2b gene. PMID : 10605111 : DOI : 10.1093/oxfordjournals.molbev.a026082 Abstract >>
An internal fragment of the ddl gene, encoding the cytoplasmic enzyme D-alanyl-D-alanine ligase, was sequenced from 566 isolates of Streptococcus pneumoniae and single isolates of Streptococcus mitis and Streptococcus oralis. The 52 alleles found among the S. pneumoniae isolates fell into two groups. Group A alleles were very uniform in sequence and were present in both penicillin-susceptible and penicillin-resistant pneumococci. Group B alleles were much more diverse and were found only in penicillin-resistant isolates. The Streptococcus oralis and Streptococcus mitis alleles were less diverged from group A alleles than some of the group B pneumococcal alleles, suggesting that the latter alleles contain interspecies recombinational replacements. The ddl gene was located 783 bp downstream of the penicillin-binding protein 2b gene (pbp2b). Sequencing of the pbp2b-recR-ddl-murF region of three penicillin-resistant pneumococci that had diverged ddl alleles showed that the whole region from pbp2b to ddl (or beyond) was highly diverged (about 8%) compared with the sequences from three penicillin-susceptible isolates. The high levels of diversity in the group B ddl alleles from penicillin-resistant isolates were ascribed to a hitchhiking effect whereby interspecies recombinational exchanges at pbp2b, selected by penicillin usage, often extend into, or through, the ddl gene. The data allow the average size of the interspecies recombinational replacements to be estimated at about 6 kb.
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89. |
Briles DE,
Brooks-Walter A,
( 1999 ) The pspC gene of Streptococcus pneumoniae encodes a polymorphic protein, PspC, which elicits cross-reactive antibodies to PspA and provides immunity to pneumococcal bacteremia. PMID : 10569772 : PMC : PMC97064 Abstract >>
PspC is one of three designations for a pneumococcal surface protein whose gene is present in approximately 75% of all Streptococcus pneumoniae strains. Under the name SpsA, the protein has been shown to bind secretory immunoglobulin A (S. Hammerschmidt, S. R. Talay, P. Brandtzaeg, and G. S. Chhatwal, Mol. Microbiol. 25:1113-1124, 1997). Under the name CbpA, the protein has been shown to interact with human epithelial and endothelial cells (C. Rosenow et al., Mol. Microbiol. 25:819-829, 1997). The gene is paralogous to the pspA gene in S. pneumoniae and was thus called pspC (A. Brooks-Walter, R. C. Tart, D. E. Briles, and S. K. Hollingshead, Abstracts of the 97th General Meeting of the American Society for Microbiology 1997). Sequence comparisons of five published and seven new alleles reveal that this gene has a mosaic structure, and modular domains have contributed to gene diversity during evolution. Two major clades exist: clade A alleles are larger and contain an extra module that is shared with many pspA alleles; clade B alleles are smaller and lack this pspA-like domain. All alleles have a proline-rich domain and a choline-binding repeat domain that show 0% divergence from similar domains in the PspA protein. Immunization of a rabbit with a recombinant clade B PspC molecule produced antiserum that cross-reacted with both PspC and PspA from 15 pneumococcal isolates. The cross-reactive antibodies afforded cross-protection in a mouse model system. Mice immunized with PspC were protected against challenge with a strain that expressed PspA but not PspC. The PspA- and PspC-cross-reactive antibodies were directed to the proline-rich domain present in both molecules.
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90. |
Fenoll A,
Enright MC,
( 1999 ) The three major Spanish clones of penicillin-resistant Streptococcus pneumoniae are the most common clones recovered in recent cases of meningitis in Spain. PMID : 10488179 : PMC : PMC85530 Abstract >>
One hundred six isolates of Streptococcus pneumoniae recovered in Spain from patients with meningitis in 1997 and 1998 were characterized by multilocus sequence typing. A heterogeneous collection of genotypes was associated with meningitis in Spain: 65 different sequence types were resolved and, even at a genetic distance of 0.43, there were 37 distinct lineages. Thirty-eight percent of the isolates, including all isolates of serotypes 6B, 9V, 14, and 23F, were resistant to penicillin, and 24% of the isolates were members of the three major Spanish penicillin-resistant or multidrug-resistant clones of serotypes 6B, 9V, and 23F or serotype variants of these clones. These three clones (MICs, 1 to 2 microg of penicillin/ml) were the most common clones associated with pneumococcal meningitis in Spain during 1997 and 1998. Only two of the other clones associated with meningitis were penicillin resistant (MICs, 0.12 to 0.5 microg/ml). One of the two most prevalent penicillin-susceptible clones causing meningitis (serotype 3) has not been detected outside of Spain, whereas the other (serotype 18C) has been recovered from patients with meningitis in the United Kingdom, The Netherlands, and Denmark. The prevalence of meningitis caused by isolates of the three major Spanish penicillin-resistant or multiply antibiotic-resistant clones, which are now globally distributed, is disturbing and clearly establishes their ability to cause life-threatening disease.
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91. |
Molnos J,
Stieger M,
Caspers P,
Flint N,
de Saizieu A,
Wagner C,
Lange R,
( 1999 ) Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae. PMID : 10524254 : DOI : 10.1016/s0378-1119(99)00266-8 Abstract >>
In bacteria, adaptive responses to environmental stimuli are often initiated by two-component signal transduction systems (TCS). The prototypical TCS comprises two proteins: a histidine kinase (HK, hk) and a response regulator (RR rr). Recent research has suggested that compounds that inhibit two-component systems might have good antibacterial activity. In order to identify TCS that are crucial for growth or virulence of Streptococcus pneumoniae, we have examined the genomic sequence of a virulent S. pneumoniae strain for genes that are related to known histidine kinases or response regulators. Altogether 13 histidine kinases and 13 response regulators have been identified. The protein sequences encoded by these genes were compared with sequences deposited in public databases. This analysis revealed that two of the 13 pneumococcal TCSs have been described before (ciaRH and comDE) and two are homologous to the yycFG and the phoRP genes of Bacillus subtilis. All the pneumococcal response regulators contain putative DNA binding motifs within the C-terminal output domain, implying that they are involved in transcriptional control. Two of these response regulators are obviously the first representatives of a new subfamily containing an AraC-type DNA-binding effector domain. To assess the regulatory role of these transcription factors, we disrupted each of the 13 response regulator genes by insertional mutagenesis. All the viable mutant strains with disrupted response regulator genes were further characterized with regard to growth in vitro, competence, and experimental virulence. Two response regulator genes could not be inactivated, indicating that they may regulate essential cellular functions. The possibility of using these systems as targets for the development of novel antibacterials will be discussed.
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92. |
Mullen DL,
Tang J,
Zhao G,
Matsushima P,
Hoskins J,
( 1999 ) Gene disruption studies of penicillin-binding proteins 1a, 1b, and 2a in Streptococcus pneumoniae. PMID : 10515951 : PMC : PMC103796 Abstract >>
The effects of inactivation of the genes encoding penicillin-binding protein 1a (PBP1a), PBP1b, and PBP2a in Streptococcus pneumoniae were examined. Insertional mutants did not exhibit detectable changes in growth rate or morphology, although a pbp1a pbp1b double-disruption mutant grew more slowly than its parent did. Attempts to generate a pbp1a pbp2a double-disruption mutant failed. The pbp2a mutants, but not the other mutants, were more sensitive to moenomycin, a transglycosylase inhibitor. These observations suggest that individually the pbp1a, pbp1b, and pbp2a genes are dispensable but that either pbp1a or pbp2a is required for growth in vitro. These results also suggest that PBP2a is a functional transglycosylase in S. pneumoniae.
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93. |
Mollerach M,
López R,
Muñoz R,
( 1999 ) Characterization of the type 8 capsular gene cluster of Streptococcus pneumoniae. PMID : 10498742 : PMC : PMC103657 Abstract >>
The complete nucleotide sequence of the capsular gene cluster (cap8) responsible for the biosynthesis of the capsular polysaccharide of Streptococcus pneumoniae type 8 has been determined. The cap8 gene cluster, located between the genes dexB and aliA, is composed of 12 open reading frames. A 14.7-kb DNA fragment embracing the cap8 genes was sufficient to transform an unencapsulated type 3 S. pneumoniae strain to a strain with the type 8 capsule. A possible scenario for the evolution of pneumococcal types 2 and 8 is outlined.
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94. |
Padayachee T,
( 1999 ) Molecular basis of rifampin resistance in Streptococcus pneumoniae. PMID : 10508007 : PMC : PMC89483 Abstract >>
Rifampin resistance among South African clinical isolates of Streptococcus pneumoniae was shown to be due to missense mutations within the rpoB gene. Sequence analysis of 24 rifampin-resistant isolates revealed the presence of mutations within cluster I as well as novel mutations in an area designated pneumococcus cluster III. Of the 24 isolates characterized, only 1 resistant isolate did not contain any mutations in the regions sequenced. Either the cluster I or the cluster III mutations separately conferred MICs of 32 to 128 microg/ml. Clinical isolate 55, for which the MIC was 256 microg/ml, was noted to contain 9 of the 10 mutations identified, which included the cluster I and cluster III mutations. As in Escherichia coli, it is possible that cluster I (amino acids 406 to 434) and cluster III (amino acids 523 to 600) of S. pneumoniae interact to form part of the antibiotic binding site, thus accounting for the very high MIC observed for isolate 55. PCR products containing cluster I or cluster III mutations were able to transform rifampin-susceptible S. pneumoniae to resistance. Although many of the isolates studied displayed identical sequences, pulsed-field gel electrophoresis analysis revealed that the isolates were not of clonal origin.
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95. |
Morona R,
( 1999 ) Comparative genetics of capsular polysaccharide biosynthesis in Streptococcus pneumoniae types belonging to serogroup 19. PMID : 10464207 : PMC : PMC94042 Abstract >>
The genetic basis for the structural diversity of capsule polysaccharide (CPS) in Streptococcus pneumoniae serogroup 19 (consisting of types 19F, 19A, 19B, and 19C) has been determined for the first time. In this study, the genetic basis for the 19A and 19C serotypes is described, and the structures of all four serogroup 19 cps loci and their flanking sequences are compared. Transformation studies show that the structural difference between the 19A and 19F CPSs is likely to be a consequence of differences between their respective polysaccharide polymerase genes (cps19aI and cps19fI). The CPS of type 19C differs from that of type 19B by the addition of glucose. We have identified a single gene difference between the two cps loci (cps19cS), which is likely to encode a glucosyl transferase. The arrangement of the genes within the cps19 loci is highly conserved, with 13 genes (cps19A to -H and cps19K to -O) common to all four serogroup 19 members. These cps genes encode functions required for the synthesis of the shared trisaccharide component of the group 19 CPS repeat unit structures. Furthermore, the genetic differences between the group 19 cps loci identified are consistent with the CPS structures of the individual serotypes. Functions have been assigned to nearly all of the cps19 gene products, based on either gene complementation or similarity to other proteins with known functions, and putative biosynthetic pathways for production of all four group 19 CPSs have been proposed.
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96. |
Dowson CG,
( 1999 ) The autolysin-encoding gene (lytA) of Streptococcus pneumoniae displays restricted allelic variation despite localized recombination events with genes of pneumococcal bacteriophage encoding cell wall lytic enzymes. PMID : 10456899 : PMC : PMC96777 Abstract >>
The lytA-encoded autolysin (N-acetylmuramoyl-L-alanine amidase) of Streptococcus pneumoniae is believed to play an important role in the pathogenesis of pneumococcal infection and has been identified as a putative vaccine target. Allelic diversity of lytA in an extensive collection of clinical isolates was assessed by restriction fragment length polymorphism and confirmatory sequencing studies. Genetic diversity within lytA is limited, especially compared to the high levels of diversity seen in other pneumococcal virulence factor genes, although small blocks generating mosaic structure were identified. Sequence comparisons with genes encoding cell wall lytic enzymes of pneumococcal bacteriophage suggest that localized recombination events have occurred between host lytA and these bacteriophage genes. These results confirm earlier suggestions that recombination between DNA encoding bacteriophage autolytic enzymes and chromosomally encoded lytA might be important in the evolution of lytA. The implications of these findings for understanding the evolution of lytA and the potential utility of LytA as a vaccine target are discussed.
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97. |
Klugman KP,
( 1999 ) Novel expansions of the gene encoding dihydropteroate synthase in trimethoprim-sulfamethoxazole-resistant Streptococcus pneumoniae. PMID : 10471569 : PMC : PMC89451 Abstract >>
A study of eight sulfonamide-resistant clinical isolates of Streptococcus pneumoniae revealed chromosomal mutations within the gene encoding dihydropteroate synthase that play a role in conferring resistance to sulfamethoxazole. The presence of the suld mutation, found previously only in a laboratory mutant, was shown to occur in three of the wild-type clinical isolates. The duplication of Ser(61), the other previously defined mutation in the dihydropteroate synthase gene of S. pneumoniae, was observed in only one of the isolates characterized. We report two previously unidentified amino acid alterations, namely, a duplication of Arg(58) and Pro(59) and an insertion of an arginine residue between Gly(60) and Ser(61) in trimethoprim-sulfamethoxazole-resistant strains. The significance of these mutations was confirmed by site-directed mutagenesis and by the transformation of a susceptible strain of S. pneumoniae to sulfamethoxazole resistance. Two resistant isolates did not contain any mutations within the gene encoding dihydropteroate synthase. The results presented suggest the independent generation of resistant mutations among South African clinical isolates. It is also proposed that the mechanism of sulfonamide resistance in S. pneumoniae involves the expansion of a specific region within dihydropteroate synthase, which probably forms part of the sulfonamide binding site.
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98. |
López R,
Paz González M,
García E,
García JL,
( 1999 ) The molecular characterization of the first autolytic lysozyme of Streptococcus pneumoniae reveals evolutionary mobile domains. PMID : 10411730 : DOI : 10.1046/j.1365-2958.1999.01455.x Abstract >>
A biochemical approach to identify proteins with high affinity for choline-containing pneumococcal cell walls has allowed the localization, cloning and sequencing of a gene (lytC) coding for a protein that degrades the cell walls of Streptococcus pneumoniae. The lytC gene is 1506 bp long and encodes a protein (LytC) of 501 amino acid residues with a predicted M r of 58 682. LytC has a cleavable signal peptide, as demonstrated when the mature protein (about 55 kDa) was purified from S. pneumoniae. Biochemical analyses of the pure, mature protein proved that LytC is a lysozyme. Combined cell fractionation and Western blot analysis showed that the unprocessed, primary product of the lytC gene is located in the pneumococcal cytoplasm whereas the processed, active form of LytC is tightly bound to the cell envelope. In vivo experiments demonstrated that this lysozyme behaves as a pneumococcal autolytic enzyme at 30 degrees C. The DNA region encoding the 253 C-terminal amino acid residues of LytC has been cloned and expressed in Escherichia coli. The truncated protein exhibits a low, but significant, choline-independent lysozyme activity, which suggests that this polypeptide adopts an active conformation. Self-alignment of the N-terminal part of the deduced amino acid sequence of LytC revealed the presence of 11 repeated motifs. These results strongly suggest that the lysozyme reported here has changed the general building plan characteristic of the choline-binding proteins of S. pneumoniae and its bacteriophages, i.e. the choline-binding domain and the catalytic domain are located, respectively, at the N-terminal and the C-terminal moieties of LytC. This work illustrates the natural versatility exhibited by the pneumococcal genes coding for choline-binding proteins to fuse separated catalytic and substrate-binding domains and create new and functional mature proteins.
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99. |
Daniels M,
Enright MC,
Spratt BG,
( 1999 ) Serotype 14 variants of the Spanish penicillin-resistant serotype 9V clone of Streptococcus pneumoniae arose by large recombinational replacements of the cpsA-pbp1a region. PMID : 10463168 : DOI : 10.1099/13500872-145-8-2023 Abstract >>
The high prevalence of penicillin resistance among Streptococcus pneumoniae isolates from Uruguay has been associated with the emergence of a penicillin-resistant clone of serotype 14. Isolates of this clone were identical by multilocus sequence typing to members of the Spanish penicillin-resistant serotype 9V clone and possessed indistinguishable forms of the penicillin-binding protein 2b and 2x genes. Their pbp1a genes were also identical, except at the 3' end. The serotype 14 isolates were shown to be a variant of the Spanish serotype 9V clone which arose by a 22.2 kb recombinational replacement that introduced the capsular biosynthetic locus, and part of the neighbouring pbp1a gene, from a serotype 14 isolate. One end of the recombinational replacement was within the first gene of the capsular polysaccharide operon, cpsA, as the sequence of the upstream dexB gene, through most of cpsA, was identical in the penicillin-resistant serotype 9V and 14 isolates, but the sequences differed in the rest of cpsA and in cpsB. The other recombinational junction was at the end of the divergently transcribed pbp1a gene, which is approximately 11.6 kb downstream of the capsular biosynthetic locus. Isolates of this serotype variant were also detected in Spain and Denmark. Penicillin-resistant serotype 14 isolates from Poland were also closely related to the penicillin-resistant serotype 9V clone, but have emerged independently, as one end of the recombinational replacement was upstream of dexB and the other was within pbp1a, but at a different position from that in the serotype 14 variants from Uruguay, Spain and Denmark. Serotype 14 variants of the Spanish serotype 9V clone have therefore arisen on more than one occasion by large recombinational replacements that extend from the start of the cps region into the pbp1a gene.
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100. |
Morrison DA,
( 1999 ) Identification of a new regulator in Streptococcus pneumoniae linking quorum sensing to competence for genetic transformation. PMID : 10438773 : PMC : PMC93990 Abstract >>
Competence for genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system encoded by two genetic loci, comCDE and comAB. Additional competence-specific operons, cilA, cilB, cilC, cilD, cilE, cinA-recA, coiA, and cfl, involved in the DNA uptake process and recombination, share an unusual consensus sequence at -10 and -25 in the promoter, which is absent from the promoters of comAB and comCDE. This pattern suggests that a factor regulating transcription of these transformation machinery genes but not involved with comCDE and comAB expression might be an alternative sigma factor. A search for such a global transcriptional regulator was begun by purifying pneumococcal RNA polymerase holoenzyme. In preparations from competent pneumococcal cultures a protein which seemed to be responsible for cilA transcription in vitro was identified. The corresponding gene was identified and found to be present in two copies, designated comX1 and comX2, located adjacent to two of the repeated rRNA operons. Expression of transformation machinery operons, such as cilA, cilD, cilE, and cfl, but not that of the quorum-sensing operons comAB and comCDE, was shown to depend on comX, while comX expression depended on ComE but not on ComX itself. We conclude that the factor is a competence-specific global transcription modulator which links quorum-sensing information transduced to ComE to competence and propose that it acts as an alternate sigma factor. We also report that comAB and comCDE are not sufficient for shutoff of competence-stimulating peptide-induced gene expression nor for the subsequent refractory period, suggesting that these phenomena depend on one or more ComX-dependent genes.
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101. |
Tuomanen E,
Normark S,
Henriques B,
Charpentier E,
( 1999 ) Emergence of vancomycin tolerance in Streptococcus pneumoniae. PMID : 10376600 : DOI : 10.1038/21202 Abstract >>
Streptococcus pneumoniae, the pneumococcus, is the most common cause of sepsis and meningitis. Multiple-antibiotic-resistant strains are widespread, and vancomycin is the antibiotic of last resort. Emergence of vancomycin resistance in this community-acquired bacterium would be catastrophic. Antibiotic tolerance, the ability of bacteria to survive but not grow in the presence of antibiotics, is a precursor phenotype to resistance. Here we show that loss of function of the VncS histidine kinase of a two-component sensor-regulator system in S. pneumoniae produced tolerance to vancomycin and other classes of antibiotic. Bacterial two-component systems monitor environmental parameters through a sensor histidine-kinase/phosphatase, which phosphorylates/dephosphorylates a response regulator that in turn mediates changes in gene expression. These results indicate that signal transduction is critical for the bactericidal activity of antibiotics. Experimental meningitis caused by the vncS mutant failed to respond to vancomycin. Clinical isolates tolerant to vancomycin were identified and DNA sequencing revealed nucleotide alterations in vncS. We conclude that broad antibiotic tolerance of S. pneumoniae has emerged in the community by a molecular mechanism that eliminates sensitivity to the current cornerstone of therapy, vancomycin.
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102. |
Taylor DE,
( 1999 ) Characterization of gram-positive tellurite resistance encoded by the Streptococcus pneumoniae tehB gene. PMID : 10339832 : DOI : 10.1111/j.1574-6968.1999.tb13594.x Abstract >>
Streptococcus pneumoniae is a Gram-positive bacterium which is naturally resistant to tellurite. In this study, we cloned and sequenced a homologue of the Escherichia coli tellurite resistance gene tehB from S. pneumoniae. It encoded a protein of 284 amino acids which is 86 residues longer than E. coli TehB, but similar in size to Haemophilus influenzae TehB and Eikenella corrodens hemagglutinin (Hag1) as well as homologues from Actinobacillus actinomycetemcomitans, Neisseria gonorrhoeae and Neisseria meningitidis. The S. pneumoniae TehB displayed 46-58% identity (52-68% similarity) to these proteins. The results in this study showed that the S. pneumoniae tehB alone not only conferred on E. coli high level resistance to tellurite, but also caused filamentous morphology in E. coli.
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103. |
Dowson CG,
Chanter N,
Sturgeon D,
King SJ,
Doherty NC,
( 1999 ) Molecular characterization of equine isolates of Streptococcus pneumoniae: natural disruption of genes encoding the virulence factors pneumolysin and autolysin. PMID : 10338480 : PMC : PMC96581 Abstract >>
Although often considered a strict human pathogen, Streptococcus pneumoniae has been reported to infect and cause pneumonia in horses, although the pathology appears restricted compared to that of human infections. Here we report on the molecular characterization of a group of S. pneumoniae isolates obtained from horses in England and Ireland. Despite being obtained from geographically distinct locations, the isolates were found to represent a tight clonal group, virtually identical to each other but genetically distinguishable from more than 120 divergent isolates of human S. pneumoniae. A comprehensive analysis of known pneumococcal virulence determinants was undertaken in an attempt to understand the pathogenicity of equine pneumococci. Surprisingly, equine isolates appear to lack activities associated with both the hemolytic cytotoxin pneumolysin, often considered a major virulence factor of pneumococci, and the major autolysin gene lytA, also considered an important virulence factor. In support of phenotypic data, molecular studies demonstrated a deletion of parts of the coding sequences of both lytA and ply genes in equine pneumococci. The implications of these findings for the evolution and pathogenicity of equine S. pneumoniae are discussed.
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104. |
Barcus VA,
( 1999 ) Genetic diversity of the streptococcal competence (com) gene locus. PMID : 10322016 : PMC : PMC93770 Abstract >>
The com operon of naturally transformable streptococcal species contains three genes, comC, comD, and comE, involved in the regulation of competence. The comC gene encodes a competence-stimulating peptide (CSP) thought to induce competence in the bacterial population at a critical extracellular concentration. The comD and comE genes are believed to encode the transmembrane histidine kinase and response regulator proteins, respectively, of a two-component regulator, with the comD-encoded protein being a receptor for CSP. Here we report on the genetic variability of comC and comD within Streptococcus pneumoniae isolates. Comparative analysis of sequence variations of comC and comD shows that, despite evidence for horizontal gene transfer at this locus and the lack of transformability of many S. pneumoniae strains in the laboratory, there is a clear correlation between the presence of a particular comC allele and the cognate comD allele. These findings effectively rule out the possibility that the presence of noncognate comC and comD alleles may be responsible for the inability to induce competence in many isolates and indicate the importance of a functional com pathway in these isolates. In addition, we describe a number of novel CSPs from disease-associated strains of S. mitis and S. oralis. The CSPs from these isolates are much more closely related to those from S. pneumoniae than to most CSPs previously reported from S. mitis and S. oralis, suggesting that these particular organisms may be a potential source of DNA in recombination events generating the mosaic structures commonly reported in genes of S. pneumoniae that are under strong selective pressure.
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105. |
Pan XS,
( 1999 ) Streptococcus pneumoniae DNA gyrase and topoisomerase IV: overexpression, purification, and differential inhibition by fluoroquinolones. PMID : 10223925 : PMC : PMC89122 Abstract >>
Streptococcus pneumoniae gyrA and gyrB genes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones-ciprofloxacin, sparfloxacin, and clinafloxacin-which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity against S. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic pathway.
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106. |
Zhang JR,
Tuomanen EI,
Fischer W,
( 1999 ) Pneumococcal licD2 gene is involved in phosphorylcholine metabolism. PMID : 10200966 : DOI : 10.1046/j.1365-2958.1999.01291.x Abstract >>
Phosphorylcholine is an important bioactive adduct to the teichoic acid (TA) and lipoteichoic acid (LTA) of the surface of Streptococcus pneumoniae. We have identified and characterized a genetic locus lic that is required for phosphorylcholine metabolism in S. pneumoniae. The pneumococcal lic locus consists of eight genes, licA, licB, licC and licD1, licD2 and three additional open reading frames. Pneumococcal licA, licB, licC, licD1 and licD2 have significant sequence similarity to licA, licB, licC and licD of Haemophilus influenzae. Mutation of licD2 led to decreased [3H]-choline uptake, aberrant migration of LTA chains in SDS-PAGE gels, loss of several surface proteins, and a phase-locked hypertransparent colony phenotype. Moreover, the licD2- mutant falled to undergo lysis after treatment with penicillin at high cell density and showed decreased transformation competence. Finally, the licD2- mutant demonstrated decreased adherence to the human type II alveolar cells, reduced nasopharyngeal colonization in infant rats, as well as significantly impaired virulence upon intraperitoneal challenge of CF1 mice. Identification of the lic genes in the pneumococcus will facilitate further characterization of the role of surface choline in microbial physiology and pathogenesis.
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107. |
Pearce BJ,
Pozzi G,
( 1999 ) The type 2 capsule locus of Streptococcus pneumoniae. PMID : 10198036 : PMC : PMC93698 Abstract >>
The type 2 capsule locus of Streptococcus pneumoniae was characterized in Avery's strain D39, which is the parent strain of the standard transformation recipients currently used in pneumococcal research and is largely used as a virulent strain in studies on the pathogenesis of pneumococcal infections. The capsule locus was sequenced by using a 21.7-kb PCR fragment from the D39 genome as a template. Sequence data analysis showed the presence of 18 open reading frames, 17 of which have the same direction of transcription and all of which are potentially involved in capsule biosynthesis. It was also shown that R36A and R6, which are unencapsulated (rough) derivatives of D39, carry a 7,504-bp deletion involving nine capsule genes.
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108. |
Asahi Y,
Takeuchi Y,
( 1999 ) Diversity of substitutions within or adjacent to conserved amino acid motifs of penicillin-binding protein 2X in cephalosporin-resistant Streptococcus pneumoniae isolates. PMID : 10223944 : PMC : PMC89251 Abstract >>
The sequence of an approximately 1.1-kb DNA fragment of the pbp2x gene, which encodes the transpeptidase domain, was determined for 35 clinical isolates of Streptococcus pneumoniae for which the cefotaxime (CTX) MICs varied. Strains with substitutions within a conserved amino acid motif changing STMK to SAFK and a Leu-to-Val change just before the KSG motif were highly resistant to CTX (MIC, >==2 microgram/ml). Strains with substitutions adjacent to SSN or KSG motifs had low-level resistance. The amino acid substitutions were plotted on the three-dimensional crystallographic structure of the transpeptidase domain of PBP2X. Transformants containing pbp2x from strains with high-level CTX resistance increased the CTX MIC from 0. 016 microgram/ml to 0.5 to 1.0 microgram/ml.
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109. |
Munoz-Najar U,
Vijayakumar MN,
( 1999 ) An operon that confers UV resistance by evoking the SOS mutagenic response in streptococcal conjugative transposon Tn5252. PMID : 10217768 : PMC : PMC93719 Abstract >>
Streptococcus pneumoniae Rx1 is capable of repairing lesions caused by DNA-damaging agents in an error-free manner but lacks a UV-inducible error-prone repair system due to the absence of chromosomally encoded UmuDC-like proteins. We have identified an operon-like structure 8 kb from the left end of the pneumococcal conjugative transposon Tn5252 that confers SOS function in the host cells. DNA sequence analysis of this region revealed the presence of four open reading frames (ORFs). The deduced amino acid sequence of one of them, ORF13, which is capable of encoding a protein of 49.7 kDa, showed significant homology to UmuC, MucB, and other proteins involved in the SOS response. The carboxy-terminal region of another, ORF14, which is predicted to encode a 26-kDa polypeptide, shared similarity with UmuD- and MucA-like proteins that carry the amino acid residues recognized by the activated RecA* protein for proteolytic cleavage. The presence of plasmids carrying subcloned DNA from this region was found to restore UV-inducible mutagenic repair of chromosomal DNA in Escherichia coli cells defective in error-prone repair as well as in pneumococcus and Enterococcus faecalis UV202. Mutations within ORF13 abolished UV-induced mutagenesis but did not affect the conjugal transposition of the element.
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110. |
Morona R,
( 1999 ) Analysis of the 5' portion of the type 19A capsule locus identifies two classes of cpsC, cpsD, and cpsE genes in Streptococcus pneumoniae. PMID : 10348877 : PMC : PMC93832 Abstract >>
Analysis of the sequence data obtained from the 5' portion of the Streptococcus pneumoniae type 19A capsular polysaccharide biosynthesis locus (cps19a) revealed that the first seven genes are homologous to the first seven genes in the type 19F (cps19f) locus. The former genes were designated cps19aA to -G and were 70 to 90% identical to their cps19f counterparts. Southern hybridization analysis of the cps loci from various S. pneumoniae serotypes with probes specific for the cps19aC, cps19aD, and cps19aE genes indicated a hybridization pattern complementary to that previously reported for cps19fC, cps19fD, and cps19fE. That is, all serotypes tested contained high-stringency homologues of either the cps19aC to -E genes or the cps19fC to -E genes, but not both. On this basis S. pneumoniae cps loci can be divided into two distinct classes. Long-range PCR was used to amplify the cps regions between cpsB and aliA from a variety of pneumococcal serotypes. Direct sequencing of the 5' end of these PCR products, and phylogenetic analysis of the sequence data, confirmed the presence of the two distinct classes of cpsC. Whereas members within one class are greater than 95% identical to each other, the DNA sequence identity between the two classes is only approximately 70%.
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111. |
Garnier F,
Janapatla RP,
Charpentier E,
Masson G,
Grélaud C,
Stach JF,
Denis F,
Ploy MC,
( 2007 ) Insertion sequence 1515 in the ply gene of a type 1 clinical isolate of Streptococcus pneumoniae abolishes pneumolysin expression. PMID : 17494718 : DOI : 10.1128/JCM.02168-06 PMC : PMC1933007 Abstract >>
A serotype 1 Streptococcus pneumoniae strain isolated by blood culture from a woman with pneumonia was found to harbor insertion sequence (IS) 1515 in the pneumolysin gene, abolishing pneumolysin expression. To our knowledge, this is the first report of an IS in the pneumolysin gene of S. pneumoniae.
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112. |
Stanhope MJ,
Walsh SL,
Becker JA,
Miller LA,
Lefébure T,
Lang P,
Bitar PD,
Amrine-Madsen H,
( 2007 ) The relative frequency of intraspecific lateral gene transfer of penicillin binding proteins 1a, 2b, and 2x, in amoxicillin resistant Streptococcus pneumoniae. PMID : 17475572 : DOI : 10.1016/j.meegid.2007.03.004 Abstract >>
Evidence exists for both interspecific and intraspecific recombination (lateral gene transfer; LGT) involving Streptococcus pneumoniae pbp (penicillin binding protein) loci. LGT of capsular genes, or serotype switching, is also know to occur between S. pneumoniae of different serotype. It is not clear whether intraspecific pbp LGT is relatively common, whether there is a difference in the relative frequency of intraspecific LGT of different pbps, and whether serotype switching is more or less frequent than pbp LGT. The purpose of this study was to use comparative evolutionary biology analysis of 216 international clinical S. pneumoniae isolates, from the Alexander Project collection, to gain insight on these issues, as well as the possible role they might be playing in spreading amoxicillin resistance. All 216 isolates were genotyped using MLST and complete or nearly complete sequences for pbp1a, pbp2b, and pbp2x were determined. Amoxicillin MICs were available for each isolate. pbps were genotyped using phylogenetics and two or more pbp types within a MLST sequence type (ST) or clonal complex were taken as putative cases of pbp LGT; these hypotheses were statistically evaluated using the approximately unbiased (AU) test. Serotypes were determined for 171 of these isolates and the minimum number of switching events necessary to explain the serotype phenotypes for each of the STs and clonal complexes were evaluated. The majority (78%) of the amoxicillin resistant isolates were comprised in 5 clonal complexes. The relative frequency of pbp LGT was greatest for pbp2b and 2x (minimum of 10.2 and 7.8%, respectively, of the isolates consistent with the LGT hypothesis), followed by 1a (3.9%). Serotype switching was more frequent than intraspecific pbp LGT (33% of isolates consistent with serotype switching hypothesis). Although intraspecific LGT of pbps is occurring and has played a role in the spread of amoxicillin resistance in S. pneumoniae, clonal dissemination appears to be more significant.
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113. |
Cochetti I,
Tili E,
Vecchi M,
Manzin A,
Mingoia M,
Varaldo PE,
Montanari MP,
( 2007 ) New Tn916-related elements causing erm(B)-mediated erythromycin resistance in tetracycline-susceptible pneumococci. PMID : 17483548 : DOI : 10.1093/jac/dkm120 Abstract >>
To analyse the as yet unexplored genetic elements encoding erm(B)-mediated erythromycin resistance in tetracycline-susceptible pneumococci. Sixteen Streptococcus pneumoniae clinical isolates sharing erm(B)-mediated erythromycin resistance and susceptibility to tetracycline were used. Gene detection was performed by PCR using both established and specially designed primers. S. pneumoniae R6, Streptococcus pyogenes 12RF and Enterococcus faecalis JH2-2 were used as recipients in mating experiments. Of the 16 test strains, 14 bore an unexpressed tet(M) gene which in 13 strains had a genetic linkage with erm(B). Three isolates yielded a 3.2 kb and 10 an 11.9 kb erm(B)/tet(M) amplicon. The former three showed genetic organizations similar to that of the composite element Tn3872, where the erm(B)-carrying Tn917 transposon is inserted into a Tn916-like element. Of the latter 10 isolates, 9 showed genetic organizations substantially overlapping with that of Tn6002, a newly sequenced erm(B)-containing Tn916-related transposon. The tenth isolate carried a novel composite element (designated Tn6003) resulting from the insertion into a Tn6002-like transposon of a fragment [designated macrolide-aminoglycoside-streptothricin (MAS) element] containing a second erm(B) (lacking the stop codon) and a variant of the aadE-sat4-aphA-3 cluster. The two tet(M)-negative isolates had different Tn3872-related elements, one containing a complete and one a deleted MAS fragment. Conjugative transfer was obtained from donors carrying Tn6002-related elements, not from donors carrying Tn3872-related elements. In tetracycline-susceptible pneumococci with erm(B)-mediated erythromycin resistance, the erm(B) gene is carried on a variety of Tn916-related genetic elements either lacking tet(M) or, more often, carrying an unexpressed tet(M) gene.
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114. |
Jefferies JM,
Johnston CH,
Kirkham LA,
Cowan GJ,
Ross KS,
Smith A,
Clarke SC,
Brueggemann AB,
George RC,
Pichon B,
Pluschke G,
Pfluger V,
Mitchell TJ,
( 2007 ) Presence of nonhemolytic pneumolysin in serotypes of Streptococcus pneumoniae associated with disease outbreaks. PMID : 17703426 : DOI : 10.1086/520091 Abstract >>
Pneumolysin is an important virulence factor of the human pathogen Streptococcus pneumoniae. Sequence analysis of the ply gene from 121 clinical isolates of S. pneumoniae uncovered a number of alleles. Twenty-two strains were chosen for further analysis, and 14 protein alleles were discovered. Five of these had been reported previously, and the remaining 9 were novel. Cell lysates were used to determine the specific hemolytic activities of the pneumolysin proteins. Six strains showed no hemolytic activity, and the remaining 16 were hemolytic, to varying degrees. We report that the nonhemolytic allele reported previously in serotype 1, sequence type (ST) 306 isolates is also present in a number of pneumococcal isolates of serotype 8 that belong to the ST53 lineage. Serotype 1 and 8 pneumococci are known to be associated with outbreaks of invasive disease. The nonhemolytic pneumolysin allele is therefore associated with the dominant clones of outbreak-associated serotypes of S. pneumoniae.
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115. |
Del Grosso M,
Northwood JG,
Farrell DJ,
Pantosti A,
( 2007 ) The macrolide resistance genes erm(B) and mef(E) are carried by Tn2010 in dual-gene Streptococcus pneumoniae isolates belonging to clonal complex CC271. PMID : 17709465 : DOI : 10.1128/AAC.00598-07 PMC : PMC2151421 Abstract >>
The genetic elements carrying macrolide resistance genes in Streptococcus pneumoniae isolates belonging to CC271 were investigated. The international clone Taiwan(19F)-14 was found to carry Tn2009, a Tn916-like transposon containing tet(M) and mef(E). The dual erm(B) mef(E) isolates carried Tn2010, which is similar to Tn2009 with the addition of a putative new transposon, the erm(B) genetic element.
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116. |
Morand B,
Mühlemann K,
( 2007 ) Heteroresistance to penicillin in Streptococcus pneumoniae. PMID : 17704255 : DOI : 10.1073/pnas.0702377104 PMC : PMC1950099 Abstract >>
Heteroresistance to beta-lactam antibiotics has been mainly described for staphylococci, for which it complicates diagnostic procedures and therapeutic success. This study investigated whether heteroresistance to penicillin exists in Streptococcus pneumoniae. Population analysis profile (PAP) showed the presence of subpopulations with higher penicillin resistance in four of nine clinical pneumococcal strains obtained from a local surveillance program (representing the multiresistant clones ST179, ST276, and ST344) and in seven of 16 reference strains (representing the international clones Spain(23F)-1, Spain(9V)-3, Spain(14)-5, Hungary(19A)-6, South Africa(19A)-13, Taiwan(23F)-15, and Finland(6B)-12). Heteroresistant strains had penicillin minimal inhibitory concentrations (MICs) (for the majority of cells) in the intermediate- to high-level range (0.19-2.0 mug/ml). PAP curves suggested the presence of subpopulations also for the highly penicillin-resistant strains Taiwan(19F)-14, Poland(23F)-16, CSR(19A)-11, and CSR(14)-10. PAP of bacterial subpopulations with higher penicillin resistance showed a shift toward higher penicillin-resistance levels, which reverted upon multiple passages on antibiotic-free media. Convergence to a homotypic resistance phenotype did not occur. Comparison of two strains of clone ST179 showed a correlation between the heteroresistant phenotype and a higher-penicillin MIC and a greater number of altered penicillin-binding proteins (PBP1a, -2b, and -2x), respectively. Therefore, heteroresistance to penicillin occurs in international multiresistant clones of S. pneumoniae. Pneumococci may use heteroresistance to penicillin as a tool during their evolution to high penicillin resistance, because it gives the bacteria an opportunity to explore growth in the presence of antibiotics before acquisition of resistance genes.
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117. |
Lux T,
Nuhn M,
Hakenbeck R,
Reichmann P,
( 2007 ) Diversity of bacteriocins and activity spectrum in Streptococcus pneumoniae. PMID : 17704229 : DOI : 10.1128/JB.00474-07 PMC : PMC2168751 Abstract >>
The production of bacteriocins can be favorable for colonization of the host by eliminating other bacterial species that share the same environment. In Streptococcus pneumoniae, the pnc (blp) locus encoding putative bacteriocins, immunity, and export proteins is controlled by a two-component system similar to the comCDE system required for the induction of genetic competence. A detailed comparison of the pnc clusters of four genetically distinct isolates confirmed the great plasticity of this locus and documented several repeat sequences. Members of the multiple-antibiotic-resistant Spain23F-1 clone, one member of the Spain9V-3 clone, sensitive 23F strain 2306, and the TIGR4 strain produced bactericidal substances active against other gram-positive bacteria and in some cases against S. pneumoniae as well. However, other strains did not show activity against the indicator strains despite the presence of a bacteriocin cluster, indicating that other factors are required for bacteriocin activity. Analysis of strain 2306 and mutant derivatives of this strain confirmed that bacteriocin production was dependent on the two-component regulatory system and genes involved in bacteriocin transport and processing. At least one other bacteriocin gene, pncE, is located elsewhere on the chromosome and might contribute to the bacteriocin activity of this strain.
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118. |
Macheboeuf P,
Fischer DS,
Brown T,
Zervosen A,
Luxen A,
Joris B,
Dessen A,
Schofield CJ,
( 2007 ) Structural and mechanistic basis of penicillin-binding protein inhibition by lactivicins. PMID : 17676039 : DOI : 10.1038/nchembio.2007.21 Abstract >>
Beta-lactam antibiotics, including penicillins and cephalosporins, inhibit penicillin-binding proteins (PBPs), which are essential for bacterial cell wall biogenesis. Pathogenic bacteria have evolved efficient antibiotic resistance mechanisms that, in Gram-positive bacteria, include mutations to PBPs that enable them to avoid beta-lactam inhibition. Lactivicin (LTV; 1) contains separate cycloserine and gamma-lactone rings and is the only known natural PBP inhibitor that does not contain a beta-lactam. Here we show that LTV and a more potent analog, phenoxyacetyl-LTV (PLTV; 2), are active against clinically isolated, penicillin-resistant Streptococcus pneumoniae strains. Crystallographic analyses of S. pneumoniae PBP1b reveal that LTV and PLTV inhibition involves opening of both monocyclic cycloserine and gamma-lactone rings. In PBP1b complexes, the ring-derived atoms from LTV and PLTV show a notable structural convergence with those derived from a complexed cephalosporin (cefotaxime; 3). The structures imply that derivatives of LTV will be useful in the search for new antibiotics with activity against beta-lactam-resistant bacteria.
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119. |
Laponogov I,
Veselkov DA,
Sohi MK,
Pan XS,
Achari A,
Yang C,
Ferrara JD,
Fisher LM,
Sanderson MR,
( 2007 ) Breakage-reunion domain of Streptococcus pneumoniae topoisomerase IV: crystal structure of a gram-positive quinolone target. PMID : 17375187 : DOI : 10.1371/journal.pone.0000301 PMC : PMC1810434 Abstract >>
The 2.7 A crystal structure of the 55-kDa N-terminal breakage-reunion domain of topoisomerase (topo) IV subunit A (ParC) from Streptococcus pneumoniae, the first for the quinolone targets from a gram-positive bacterium, has been solved and reveals a 'closed' dimer similar in fold to Escherichia coli DNA gyrase subunit A (GyrA), but distinct from the 'open' gate structure of Escherichia coli ParC. Unlike GyrA whose DNA binding groove is largely positively charged, the DNA binding site of ParC exhibits a distinct pattern of alternating positively and negatively charged regions coincident with the predicted positions of the grooves and phosphate backbone of DNA. Based on the ParC structure, a new induced-fit model for sequence-specific recognition of the gate (G) segment by ParC has been proposed. These features may account for the unique DNA recognition and quinolone targeting properties of pneumococcal type II topoisomerases compared to their gram-negative counterparts.
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120. |
Sasková L,
Nováková L,
Basler M,
Branny P,
( 2007 ) Eukaryotic-type serine/threonine protein kinase StkP is a global regulator of gene expression in Streptococcus pneumoniae. PMID : 17416671 : DOI : 10.1128/JB.01616-06 PMC : PMC1913385 Abstract >>
Signal transduction pathways in both prokaryotes and eukaryotes utilize protein phosphorylation as a key regulatory mechanism. Recent studies have proven that eukaryotic-type serine/threonine protein kinases (Hank's type) are widespread in many bacteria, although little is known regarding the cellular processes they control. In this study, we have attempted to establish the role of a single eukaryotic-type protein kinase, StkP of Streptococcus pneumoniae, in bacterial survival. Our results indicate that the expression of StkP is important for the resistance of S. pneumoniae to various stress conditions. To investigate the impact of StkP on this phenotype, we compared the whole-genome expression profiles of the wild-type and DeltastkP mutant strains by microarray technology. This analysis revealed that StkP positively controls the transcription of a set of genes encoding functions involved in cell wall metabolism, pyrimidine biosynthesis, DNA repair, iron uptake, and oxidative stress response. Despite the reduced transformability of the stkP mutant, we found that the competence regulon was derepressed in the stkP mutant under conditions that normally repress natural competence development. Furthermore, the competence regulon was expressed independently of exogenous competence-stimulating peptide. In summary, our studies show that a eukaryotic-type serine/threonine protein kinase functions as a global regulator of gene expression in S. pneumoniae.
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121. |
Laible G,
Spratt BG,
Hakenbeck R,
( 1991 ) Interspecies recombinational events during the evolution of altered PBP 2x genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. PMID : 1766375 : DOI : 10.1111/j.1365-2958.1991.tb00821.x Abstract >>
Penicillin resistance in pneumococci is due to the appearance of high molecular-weight penicillin-binding proteins (PBPs) that have reduced affinity for the antibiotic. We have compared the PBX 2x genes (pbpX) of one penicillin-susceptible and five penicillin-resistant clinical isolates of Streptococcus pneumoniae isolated from various parts of the world. All of the resistant isolates contained a low-affinity form of PBP 2x. The 2 kb region of the two penicillin-susceptible isolates differed at only eight nucleotide sites (0.4%) and resulted in one single amino acid difference in PBP 2x. In contrast, the sequences of the PBP 2x genes from the resistant isolates differed overall from those of the susceptible isolates at between 7 and 18% of nucleotide sites and resulted in between 27 and 86 amino acid substitutions in PBP 2x. The altered PBP 2x genes consisted of regions that were similar to those of susceptible strains (less than 3% diverged), alternating with regions that were very different (18-23% diverged). The presence of highly diverged regions within the PBP 2x genes of the resistant isolates contrasts with the uniformity of the sequences of the amylomaltase genes from the same isolates, and with the uniformity of the PBP 2x genes in the two susceptible isolates. It suggests that the altered PBP 2x genes have arisen by localized interspecies recombinational events involving the PBP 2x genes of closely related streptococci, as has been suggested to occur for altered PBP 2b genes (Dowson et al., 1989b). The PBP 2x genes from the resistant isolates could transform the susceptible strain R6 to increased levels of resistance to beta-lactam antibiotics, indicating that the altered forms of PBP 2x in the resistant isolates contribute to their resistance to penicillin.
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122. |
Yother J,
Briles DE,
( 1992 ) Structural properties and evolutionary relationships of PspA, a surface protein of Streptococcus pneumoniae, as revealed by sequence analysis. PMID : 1729249 : DOI : 10.1128/jb.174.2.601-609.1992 PMC : PMC205755 Abstract >>
Analysis of the sequence for the gene encoding PspA (pneumococcal surface protein A) of Streptococcus pneumoniae revealed the presence of four distinct domains in the mature protein. The structure of the N-terminal half of PspA was highly consistent with that of an alpha-helical coiled-coil protein. The alpha-helical domain was followed by a proline-rich domain (with two regions in which 18 of 43 and 5 of 11 of the residues are prolines) and a repeat domain consisting of 10 highly conserved 20-amino-acid repeats. A fourth domain consisting of a hydrophobic region too short to serve as a membrane anchor and a poorly charged region followed the repeats and preceded the translation stop codon. The C-terminal region of PspA did not possess features conserved among numerous other surface proteins, suggesting that PspA is attached to the cell by a mechanism unique among known surface proteins of gram-positive bacteria. The repeat domain of PspA was found to have significant homology with C-terminal repeat regions of proteins from Streptococcus mutans, Streptococcus downei, Clostridium difficile, and S. pneumoniae. Comparisons of these regions with respect to functions and homologies suggested that, through evolution, the repeat regions may have lost or gained a mechanism for attachment to the bacterial cell.
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123. |
Ma Z,
Zhang JR,
( 2007 ) RR06 activates transcription of spr1996 and cbpA in Streptococcus pneumoniae. PMID : 17220227 : DOI : 10.1128/JB.01429-06 PMC : PMC1899362 Abstract >>
Streptococcus pneumoniae colonizes at the nasopharynx of humans and is able to disseminate and cause various infections. The hallmark of pneumococcal disease is rapid bacterial replication in different tissue sites leading to intense inflammation. The genetic basis of pneumococcal adaptation to different host niches remains sketchy. In this study, we investigated the regulatory effect of RR06, a response regulator protein, on gene expression of S. pneumoniae. Microarray and Northern blot analyses showed that RR06 is specifically required for transcription of spr1996 and cbpA. While the function of Spr1996 is unknown, CbpA has been well characterized as a surface-exposed protective antigen and a virulence factor of S. pneumoniae. A recombinant form of RR06 was able to bind to a 19-bp conserved sequence shared by the spr1996 and cbpA promoter regions. Furthermore, inactivation of rr06 resulted in loss of CbpA expression as detected by antibody staining and bacterial adhesion. CbpA expression was restored in trans by the intact rr06 gene. However, a mutant, RR06(D51A), with a point mutation in the aspartate residue at position 51 (a predicted major phosphorylation site) of RR06, completely abolished the CbpA expression, suggesting that RR06 phosphorylation is required for transcriptional activation of spr1996 and cbpA. Finally, inactivation of rr06 in additional pneumococcal strains also led to the loss of CbpA expression. These data implicate that RR06 activates the expression of spr1996 and cbpA in many other pneumococcal strains.
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124. |
Gould CV,
Sniegowski PD,
Shchepetov M,
Metlay JP,
Weiser JN,
( 2007 ) Identifying mutator phenotypes among fluoroquinolone-resistant strains of Streptococcus pneumoniae using fluctuation analysis. PMID : 17664329 : DOI : 10.1128/AAC.00336-07 PMC : PMC2043225 Abstract >>
The occurrence of mutator phenotypes among laboratory-generated and clinical levofloxacin-resistant strains of Streptococcus pneumoniae was determined using fluctuation analysis. The in vitro selection for levofloxacin-resistant mutants of strain D39, each with point mutations in both gyrA and parC or parE, was not associated with a significant change in the mutation rate. Two of eight clinical isolates resistant to levofloxacin (MIC, >8 microg/ml) had estimated mutation rates of 1.2 x 10(-7) and 9.4 x 10(-8) mutations per cell division, indicating potential mutator phenotypes, compared to strain D39, which had an estimated mutation rate of 1.4 x 10(-8) mutations per cell division. The levofloxacin-resistant isolates with the highest mutation rates showed evidence of dysfunctional mismatch repair and contained missense mutations in mut genes at otherwise highly conserved sites. The association of hypermutability in levofloxacin-resistant S. pneumoniae clinical isolates with mutations in DNA mismatch repair genes provides further evidence that mismatch repair mutants may have a selective advantage in the setting of antibiotic pressure, facilitating the development of further antibiotic resistance.
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125. |
Marks M,
Burns T,
Abadi M,
Seyoum B,
Thornton J,
Tuomanen E,
Pirofski LA,
( 2007 ) Influence of neutropenia on the course of serotype 8 pneumococcal pneumonia in mice. PMID : 17296760 : DOI : 10.1128/IAI.01579-06 PMC : PMC1865693 Abstract >>
Polymorphoneutrophils (PMNs) are important effector cells in host defense against pneumonia. However, PMNs can also induce inflammation and tissue damage. To investigate the contribution of PMNs to host defense against pneumococcal pneumonia, we determined the effect of the PMN-depleting rat monoclonal antibody RB6-8C5 (RB6) on survival and inflammatory and cellular response in the lungs to a lethal intranasal infection with a serotype 8 pneumococcus in BALB/c mice. Control mice received rat immunoglobulin G (rIgG). Strikingly, the survival of RB6-treated mice was significantly prolonged compared to that of rIgG-treated mice. Although the numbers of CFU in the lungs were statistically similar in both groups 4, 24, and 32 h after infection, rIgG-treated mice developed higher levels of bacteremia, and histopathological examination of the lungs of infected mice revealed marked differences between RB6- and rIgG-treated mice. RB6-treated mice had focal, perivascular lesions without accompanying parenchymal inflammation, and rIgG-treated mice had diffuse, interstitial parenchymal inflammation. Lung homogenates from the rIgG-treated mice had more leukocytes and significantly more total and apoptotic PMNs as determined by fluorescence-activated cell sorter analysis with Annexin V and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining of lung tissue samples. Studies with a pneumolysin-deficient mutant of the serotype 8 strain we used also demonstrated the prolonged survival of RB6- compared to rIgG-treated mice. Taken together, our findings suggest that PMNs enhance the likelihood of early death and alter the pathological response to pneumococcal lung infection in BALB/c mice with serotype 8 pneumonia without significantly affecting bacterial clearance or the cytokine response.
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126. |
Pallová P,
Hercík K,
Sasková L,
Nováková L,
Branny P,
( 2007 ) A eukaryotic-type serine/threonine protein kinase StkP of Streptococcus pneumoniae acts as a dimer in vivo. PMID : 17307148 : DOI : 10.1016/j.bbrc.2007.01.184 Abstract >>
Streptococcus pneumoniae carries a single Ser/Thr protein kinase gene stkP in its genome. Biochemical studies performed with recombinant StkP have revealed that this protein is a functional membrane-linked eukaryotic-type Ser/Thr protein kinase. Here, we demonstrate that the deletion of its extracellular domain negatively affects the stability of a core kinase domain. In contrast, the membrane anchored kinase domain and the full-length form of StkP were stable and capable of autophosphorylation. Furthermore, evidence is presented that StkP forms dimers through its transmembrane and extracellular domains.
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127. |
Romero P,
Llull D,
García E,
Mitchell TJ,
López R,
Moscoso M,
( 2007 ) Isolation and characterization of a new plasmid pSpnP1 from a multidrug-resistant clone of Streptococcus pneumoniae. PMID : 17275906 : DOI : 10.1016/j.plasmid.2006.12.006 Abstract >>
A novel Streptococcus pneumoniae plasmid (pSpnP1; 5413bp) has been isolated from the multidrug-resistant clone Poland(23F)-16, and its complete nucleotide sequence has been determined. Sequence analysis predicted seven co-directional open reading frames and comparative analyses revealed that plasmid pSpnP1 is different to pDP1, the only previously described pneumococcal plasmid, whereas it is highly similar to pSt08, a plasmid from Streptococcus thermophilus. A double-stranded origin for replication similar to the replication origin of the pC194/pUB110 family was located upstream of the putative rep gene (orf2). It also contained a 144-bp region with over 60% identity to the single-stranded origin type A of the Streptococcus agalactiae plasmid pMV158/pLS1. Detection of single-stranded DNA by Southern blot analysis indicated that pSpnP1 replicates via a rolling circle mechanism. Interestingly, the product of orf1 has a putative Zonular occludens toxin conserved domain present in toxigenic strains of Vibrio cholerae. Real-time PCR assays revealed that this ORF was expressed. Hybridization experiments showed that the pSpnP1 replicon was unusual among other examined antibiotic-resistant pneumococcal clones, although the recombinant plasmids based on pSpnP1 were able to replicate in Bacillus subtilis and Lactococcus lactis.
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128. |
Gherardi G,
Fallico L,
Del Grosso M,
Bonanni F,
D'Ambrosio F,
Manganelli R,
Palù G,
Dicuonzo G,
Pantosti A,
( 2007 ) Antibiotic-resistant invasive pneumococcal clones in Italy. PMID : 17122014 : DOI : 10.1128/JCM.01229-06 PMC : PMC1829026 Abstract >>
A total of 105 multiple-antibiotic-resistant invasive pneumococcal isolates recovered in Italy from 2001 to 2003 were genetically characterized. Of these, 40 were penicillin-nonsusceptible (PNSSP) and 65 were penicillin-susceptible (PSSP) Streptococcus pneumoniae strains. Among the PNSSP isolates, 8 and 11 different restriction profiles were obtained for the pbp2b and pbp2x genes, respectively. Clonal groups were established on the basis of analysis of both pulsed-field gel electrophoresis (PFGE) types and multilocus sequence typing (MLST). Several international clones, such as Spain(23F)-1/ST81, Spain(6B)-2/ST90, Spain(9V)-3/ST156, and Sweden(15A)-25/ST63 [corrected] were identified among the PNSSP isolates. Other, smaller clones, such as the minor Spanish 19F clone/ST88 and Denmark(14)-32/ST230, were also found. Among the PSSP isolates, clones related to England(14)-9/ST9, Greece(6B)-22/ST273, and Portugal(19F)-21/ST177 were found. In addition, two large clones comprised nonvaccine serotypes. One, comprising serotype 3 isolates, corresponded to the clone Netherlands(3)-31/ST180; the other, comprising serotype 15B/C isolates, ST474, was not related to any previously described clone. Two small clusters related to the newly described clones Greece(21)-30/ST193 and Netherlands(15B)-37/ST199 included isolates with unrelated PFGE profiles. An unusual finding was the inability to obtain the MLST allelic profile for an isolate of serotype 19A, belonging to the Sweden(15A)-25/ST63 [corrected] clone, due to a large deletion of the xpt gene. Capsular switching was observed among both PNSSP and PSSP isolates and involved also serotypes not included in the 7-valent pneumococcal conjugate vaccine (PCV7), such as serotypes 15B/C and 19A. Since antibiotic-resistant nonvaccine serotype clones are present in Italy, continuous monitoring of pneumococcal epidemiology should be carried out in the PCV7 era.
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129. |
Hathaway LJ,
Brugger S,
Martynova A,
Aebi S,
Mühlemann K,
( 2007 ) Use of the Agilent 2100 bioanalyzer for rapid and reproducible molecular typing of Streptococcus pneumoniae. PMID : 17202282 : DOI : 10.1128/JCM.02169-06 PMC : PMC1829109 Abstract >>
Restriction fragment length polymorphism (RFLP) analysis is an economic and fast technique for molecular typing but has the drawback of difficulties in accurately sizing DNA fragments and comparing banding patterns on agarose gels. We aimed to improve RFLP for typing of the important human pathogen Streptococcus pneumoniae and to compare the results with the commonly used typing techniques of pulsed-field gel electrophoresis and multilocus sequence typing. We designed primers to amplify a noncoding region adjacent to the pneumolysin gene. The PCR product was digested separately with six restriction endonucleases, and the DNA fragments were analyzed using an Agilent 2100 bioanalyzer for accurate sizing. The combined RFLP results for all enzymes allowed us to assign each of the 47 clinical isolates of S. pneumoniae tested to one of 33 RFLP types. RFLP analyzed using the bioanalyzer allowed discrimination between strains similar to that obtained by the more commonly used techniques of pulsed-field gel electrophoresis, which discriminated between 34 types, and multilocus sequence typing, which discriminated between 35 types, but more quickly and with less expense. RFLP of a noncoding region using the Agilent 2100 bioanalyzer could be a useful addition to the molecular typing techniques in current use for S. pneumoniae, especially as a first screen of a local population.
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130. |
Dawid S,
Roche AM,
Weiser JN,
( 2007 ) The blp bacteriocins of Streptococcus pneumoniae mediate intraspecies competition both in vitro and in vivo. PMID : 17074857 : DOI : 10.1128/IAI.01775-05 PMC : PMC1828380 Abstract >>
The introduction of the conjugate seven-valent pneumococcal vaccine has led to the replacement of vaccine serotypes with nonvaccine serotypes of Streptococcus pneumoniae. This observation implies that intraspecies competition between pneumococci occurs during nasopharyngeal colonization, allowing one strain or set of strains to predominate over others. We investigated the contribution of the blp locus, encoding putative bacteriocins and cognate immunity peptides, to intraspecies competition. We sequenced the relevant regions of the blp locus of a type 6A strain able to inhibit the growth of the type 4 strain, TIGR4, in vitro. Using deletional analysis, we confirmed that inhibitory activity is regulated by the function of the response regulator, BlpR, and requires the two putative bacteriocin genes blpM and blpN. Comparison of the TIGR4 BlpM and -N amino acid sequences demonstrated that only five amino acid differences were sufficient to target the heterologous strain. Analysis of a number of clinical isolates suggested that the BlpMN bacteriocins divide into two families. A mutant in the blpMN operon created in the clinically relevant type 19A background was deficient in both bacteriocin activity and immunity. This strain was unable to compete with both its parent strain and a serotype 4 isolate during cocolonization in the mouse nasopharynx, suggesting that the locus is functional in vivo and confirming its role in promoting intraspecies competition.
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131. |
Al-Lahham A,
Appelbaum PC,
van der Linden M,
Reinert RR,
( 2006 ) Telithromycin-nonsusceptible clinical isolates of Streptococcus pneumoniae from Europe. PMID : 17065627 : DOI : 10.1128/AAC.00057-06 PMC : PMC1635229 Abstract >>
Telithromycin-nonsusceptible pneumococcal clinical isolates (n = 17) were analyzed for their antimicrobial susceptibility, macrolide resistance mechanisms, and genetic relatedness. All strains showed the erm(B) genotype and showed a wide range of combinations of macrolide resistance mechanisms. The predominant clone (n = 7) was serotype 14, sequence type 143.
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132. |
Lovering AL,
De Castro L,
Lim D,
Strynadka NC,
( 2006 ) Structural analysis of an "open" form of PBP1B from Streptococcus pneumoniae. PMID : 16751607 : DOI : 10.1110/ps.062112106 PMC : PMC2242572 Abstract >>
The class A PBP1b from Streptococcus pneumoniae is responsible for glycosyltransferase and transpeptidase (TP) reactions, forming the peptidoglycan of the bacterial cell wall. The enzyme has been produced in a stable, soluble form and undergoes time-dependent proteolysis to leave an intact TP domain. Crystals of this TP domain were obtained, diffracting to 2.2 A resolution, and the structure was solved by using molecular replacement. Analysis of the structure revealed an "open" active site, with important conformational differences to the previously determined "closed" apoenzyme. The active-site nucleophile, Ser460, is in an orientation that allows for acylation by beta-lactams. Consistent with the productive conformation of the conserved active-site catalytic residues, adjacent loops show only minor deviation from those of known acyl-enzyme structures. These findings are discussed in the context of enzyme functionality and the possible conformational sampling of PBP1b between active and inactive states.
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133. |
Ip M,
Chi F,
Chau SS,
Hui M,
Tang J,
Chan PK,
( 2006 ) Use of the housekeeping genes, gdh (zwf) and gki, in multilocus sequence typing to differentiate Streptococcus pneumoniae from Streptococcus mitis and Streptococcus oralis. PMID : 16765553 : DOI : 10.1016/j.diagmicrobio.2006.04.013 Abstract >>
Polymerase chain reaction and sequencing of the housekeeping genes, gdh (zwf) and gki, based on the primers and alleles from multilocus sequence typing can be used to delineate and support the identity of clinical isolates of Streptococcus pneumoniae and differentiate from the closely related Streptococcus mitis and Streptococcus oralis.
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134. |
Glazunova OO,
Raoult D,
Roux V,
( 2006 ) Streptococcus massiliensis sp. nov., isolated from a patient blood culture. PMID : 16627666 : DOI : 10.1099/ijs.0.64009-0 Abstract >>
An unidentified strain of the viridans group of streptococci was isolated from a human blood sample. It was distinguished from all other recognized species of the Streptococcus sanguinis group by several biochemical characteristics. Phylogenetic analysis based on 16S rRNA gene sequence comparisons clustered this strain with Streptococcus ferus (mutans group) but phylogenetic analysis based on rpoB and sodA gene sequence comparisons included it in the S. sanguinis group. The isolate showed 95.4 and 95.2 % 16S rRNA gene sequence similarity to S. ferus and S. sanguinis, respectively, confirming it as belonging to a novel taxon, for which the name Streptococcus massiliensis sp. nov. is proposed. The type strain is 4401825T (=CIP 108498T=CCUG 49690T).
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135. |
Zhang YM,
Hurlbert J,
White SW,
Rock CO,
( 2006 ) Roles of the active site water, histidine 303, and phenylalanine 396 in the catalytic mechanism of the elongation condensing enzyme of Streptococcus pneumoniae. PMID : 16618705 : DOI : 10.1074/jbc.M513199200 Abstract >>
beta-Ketoacyl-ACP synthases catalyze the condensation steps in fatty acid and polyketide synthesis and are targets for the development of novel antibiotics and anti-obesity and anti-cancer agents. The roles of the active site residues in Streptococcus pneumoniae FabF (beta-ketoacyl-ACP synthase II; SpFabF) were investigated to clarify the mechanism for this enzyme superfamily. The nucleophilic cysteine of the active site triad was required for acyl-enzyme formation and the overall condensation activity. The two active site histidines in the elongation condensing enzyme have different electronic states and functions. His337 is essential for condensation activity, and its protonated Nepsilon stabilizes the negative charge developed on the malonyl thioester carbonyl in the transition state. The Nepsilon of His303 accelerated catalysis by deprotonating a structured active site water for nucleophilic attack on the C3 of malonate, releasing bicarbonate. Lys332 controls the electronic state of His303 and also plays a critical role in the positioning of His337. Phe396 functions as a gatekeeper that controls the order of substrate addition. These data assign specific roles for each active site residue and lead to a revised general mechanism for this important class of enzymes.
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136. |
Pimenta FC,
Ribeiro-Dias F,
Brandileone MC,
Miyaji EN,
Leite LC,
Sgambatti de Andrade AL,
( 2006 ) Genetic diversity of PspA types among nasopharyngeal isolates collected during an ongoing surveillance study of children in Brazil. PMID : 16891500 : DOI : 10.1128/JCM.00156-06 PMC : PMC1594641 Abstract >>
Pneumococcal surface protein A (PspA) has been considered a potential candidate for human vaccines because of its serotype-independent protective immunity. Nasopharyngeal (NP) pneumococcal colonization is highly prevalent in infants and precedes the invasive disease. Thus, prevention of NP colonization may reduce the burden of pneumococcal disease in children. Scarce information focusing on PspA from pneumococcal carriage in humans is available. We examined the genetic diversity of PspA from NP isolates obtained during an ongoing pneumococcal surveillance study with children. PspA families and clades of 183 community-acquired Streptococcus pneumoniae NP isolates from healthy children (n = 97) and children with respiratory tract infections (n = 48), pneumonia (n = 33), or meningitis (n = 5) were investigated. Overall, 79.8% (n = 146) of the pneumococcal isolates were classified as PspA family 1 (35.5%) and family 2 (44.3%), whereas 20.2% of the isolates could not be typed. The distribution of PspA families and clades did not differ significantly according to the clinical status of the children. A dendrogram comparing the genetic relationship between the amino acid sequences of the clade-defining region of PspA from NP strains together with 24 invasive reference strains (GenBank) closely reproduced the profile of the families and clades previously reported for pneumococcal invasive strains. These findings strengthen the idea that the use of PspA as a vaccine antigen may protect children against carriage as well as invasive pneumococcal disease.
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137. |
Rantala M,
Haanperä-Heikkinen M,
Lindgren M,
Seppälä H,
Huovinen P,
Jalava J,
( 2006 ) Streptococcus pneumoniae isolates resistant to telithromycin. PMID : 16641460 : DOI : 10.1128/AAC.50.5.1855-1858.2006 PMC : PMC1472201 Abstract >>
The telithromycin susceptibility of 210 erythromycin-resistant pneumococci was tested with the agar diffusion method. Twenty-six erm(B)-positive isolates showed heterogeneous resistance to telithromycin, which was manifested by the presence of colonies inside the inhibition zone. When these cells were cultured and tested, they showed stable, homogeneous, and high-level resistance to telithromycin.
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138. |
Llull D,
López R,
García E,
( 2006 ) Characteristic signatures of the lytA gene provide a basis for rapid and reliable diagnosis of Streptococcus pneumoniae infections. PMID : 16597847 : DOI : 10.1128/JCM.44.4.1250-1256.2006 PMC : PMC1448622 Abstract >>
The nucleotide sequences of the lytA gene from 29 pneumococcal isolates of various serotypes and 22 additional streptococci of the mitis group (including two Streptococcus pseudopneumoniae strains) have been compared and found to correspond to 19 typical (927-bp-long) and 20 atypical (921-bp-long) alleles. All the Streptococcus pneumoniae strains harbored typical lytA alleles, whereas nonpneumococcal isolates belonging to the mitis group always carried atypical alleles. A sequence alignment showed that the main difference between typical and atypical lytA alleles resided in 102 nucleotide positions (including the 6 bp absent from atypical alleles). These nucleotides were perfectly conserved in all the typical alleles studied, and the corresponding nucleotides of the atypical alleles were also perfectly conserved. The presence in these signatures of distinctive restriction sites (namely, SnaBI, XmnI, and BsaAI) allowed the development of a simple, reliable, and fast method that combines PCR amplification of the lytA gene, digestion with BsaAI, and separation of the products by agarose gel electrophoresis. This assay allows the rapid and consistent identification of true S. pneumoniae strains and represents an improved diagnostic tool for the study of pneumococcal carriage.
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139. |
Trzcinski K,
Thompson CM,
Gilbey AM,
Dowson CG,
Lipsitch M,
( 2006 ) Incremental increase in fitness cost with increased beta -lactam resistance in pneumococci evaluated by competition in an infant rat nasal colonization model. PMID : 16586368 : DOI : 10.1086/501367 Abstract >>
We evaluated the impact of resistant penicillin-binding protein (PBP) allele acquisition on the ability of penicillin-resistant (PEN-R) pneumococcal strains to compete with penicillin-susceptible (PEN-S) ancestors for upper-respiratory-tract (URT) colonization. PEN-S serotype 2, 6B, and 9V strains were transformed into derivatives expressing an increasing number of PEN-R PBP forms (2X, 2X-1A, and 2X-1A-2B for serotype 2 and 2X, 2X-2B, and 2X-2B-1A for 6B and 9V). Infant rats were inoculated intranasally with a mix of a PEN-R and PEN-S strains. For consecutive days, samples were collected for assessment of the ratio of PEN-S to PEN-R cells colonizing the URT. The selective index (SI), defined as the change in the natural logarithm of the ratio of PEN-S to PEN-R strains from the inoculum to the nasal-wash samples, quantified differences in fitness. SIs significantly > 0 (indicating a cost of resistant allele acquisition) were observed 4-5 days after colonization in all but serotype 6B pbp2x transfomants. Additional replacements with low-affinity forms of pbp2b and pbp1a genes reduced further ability to compete in all strains. The cost of penicillin-resistance acquisition for the Streptococcus pneumoniae strain competing with its susceptible ancestor to colonize the URT increases with the number of resistant pbp alleles acquired.
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140. |
Itoh Y,
Kawamura Y,
Kasai H,
Shah MM,
Nhung PH,
Yamada M,
Sun X,
Koyana T,
Hayashi M,
Ohkusu K,
Ezaki T,
( 2006 ) dnaJ and gyrB gene sequence relationship among species and strains of genus Streptococcus. PMID : 16487673 : DOI : 10.1016/j.syapm.2005.12.003 Abstract >>
The dnaJ and gyrB nucleotide sequences were determined for members of the genus Streptococcus. The average similarity between the species tested was 76.4% (69.7-100%) for dnaJ and 75.9 (70.1-98.7%) for gyrB. These data indicated that the dnaJ and gyrB genes are more divergent and more discriminatory than the 16S rDNA gene. Furthermore, the variation in the dnaJ nucleotide sequences among the mitis group was greater than that of the gyrB nucleotide sequences, especially between Streptococcus pneumoniae and Streptococcus mitis. Subsequently, the high discrimination power of dnaJ within the mitis group was confirmed. Thus, we conclude that the dnaJ and gyrB genes are efficient alternative targets for the classification of the genus Streptococcus, and that dnaJ is suitable for phylogenetic analysis of closely related Streptococcus strains.
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141. |
del Campo R,
Cafini F,
Morosini MI,
Fenoll A,
Liñares J,
Alou L,
Sevillano D,
Cantón R,
Prieto J,
Baquero F,
N/A N/A,
( 2006 ) Combinations of PBPs and MurM protein variants in early and contemporary high-level penicillin-resistant Streptococcus pneumoniae isolates in Spain. PMID : 16533824 : DOI : 10.1093/jac/dkl083 Abstract >>
High-level penicillin resistance in Streptococcus pneumoniae requires extensive re-modulation of the penicillin-binding proteins (PBPs), and murM gene function is also required for the expression of resistance. In this work, we determined whether specific changes in PBPs were associated with specific MurM variants. Two collections of highly penicillin-resistant (MIC 2-8 mg/L) isolates, including 10 early (1997-1998) and 23 contemporary (2002-2004) isolates, were studied. Most of the isolates belonged to clones Spain(6B)-2 (13 strains), Spain(23F)-1 (10 isolates) and Spain(14)-5 (20 isolates). Different protein variants of MurM (MA, MB5, MB6, MB9 and MB10), PBP1A (A-C), PBP2B (A-D) and PBP2X (A-C) were recognized, including two murM alleles not previously described. Particular [MurM-PBP1A-2B-2X] allelic combinations were predominant among the different clones, including [MA-B-B-B] for old (MIC 2 mg/L) and [MB10-C-A-B] for recent (MIC 4-8 mg/L) Spain(6B)-2 isolates, [MA-A-C-A] for Spain(23F)-1 and [MB5-A-A-A] in Spain(14)-5 isolates. Although S. pneumoniae has a basic recombinational population structure, our results indicate remarkable conservation of PBPs and MurM protein types within each clone. This suggests that particular PBPs-MurM combinations tend to be preserved and may have an independent evolutionary history in particular clones.
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142. |
Henderson-Begg SK,
Livermore DM,
Hall LM,
( 2006 ) Effect of subinhibitory concentrations of antibiotics on mutation frequency in Streptococcus pneumoniae. PMID : 16531433 : DOI : 10.1093/jac/dkl064 Abstract >>
To investigate the effect of subinhibitory concentrations of ciprofloxacin, streptomycin, trimethoprim, ampicillin and erythromycin on mutation frequency in Streptococcus pneumoniae. Frequency of mutation to rifampicin resistance was determined in three clinical isolates grown with or without antibiotic treatment. dinB was analysed using PCR and DNA sequence determination. Subinhibitory levels of ciprofloxacin and streptomycin increased the frequency of mutation to rifampicin resistance between 2- and 5-fold for all three isolates, which is comparable to the increase seen in mismatch repair mutants of this species. These increases appeared not to be dependent on the function of the error-prone DNA polymerase encoded by dinB, since one of the isolates was a naturally occurring deletion mutant for this gene. Trimethoprim increased the mutation frequency for two isolates, but not the dinB mutant; ampicillin and erythromycin had no significant effect on mutation frequencies for any isolate. Exposure to quinolones and aminoglycosides at subinhibitory concentrations may result in increased mutability in pneumococci, as well as selecting for resistance per se.
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143. |
Biçmen M,
Gülay Z,
Ramaswamy SV,
Musher DM,
Gür D,
( 2006 ) Analysis of mutations in the pbp genes of penicillin-non-susceptible pneumococci from Turkey. PMID : 16441453 : DOI : 10.1111/j.1469-0691.2005.01334.x Abstract >>
Sequence analysis of the pbp genes from 20 Streptococcus pneumoniae isolates from Turkey (eight with high-level penicillin-resistance, nine with low-level penicillin-resistance, and three that were penicillin-susceptible) was performed and phylogenetic trees were constructed. Most isolates clustered together within a single branch that was distinct from sequences deposited previously in GenBank, which suggests that these isolates have probably evolved following new recombination events. The most prominent active-site mutations, which have also been associated previously with resistance, were T371A in PBP1a, E481G followed by T451A in PBP2b, and T338A in PBP2x. All isolates also possessed a (570)SVES/TK(574) block in the PBP2b sequence, instead of the QLQPT sequence of R6, which is fairly uncommon in GenBank sequences. This is the first study to analyse alterations in the pbp sequences of pneumococci isolated in Turkey.
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144. |
Bentley SD,
Aanensen DM,
Mavroidi A,
Saunders D,
Rabbinowitsch E,
Collins M,
Donohoe K,
Harris D,
Murphy L,
Quail MA,
Samuel G,
Skovsted IC,
Kaltoft MS,
Barrell B,
Reeves PR,
Parkhill J,
Spratt BG,
( 2006 ) Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes. PMID : 16532061 : DOI : 10.1371/journal.pgen.0020031 PMC : PMC1391919 Abstract >>
Several major invasive bacterial pathogens are encapsulated. Expression of a polysaccharide capsule is essential for survival in the blood, and thus for virulence, but also is a target for host antibodies and the basis for effective vaccines. Encapsulated species typically exhibit antigenic variation and express one of a number of immunochemically distinct capsular polysaccharides that define serotypes. We provide the sequences of the capsular biosynthetic genes of all 90 serotypes of Streptococcus pneumoniae and relate these to the known polysaccharide structures and patterns of immunological reactivity of typing sera, thereby providing the most complete understanding of the genetics and origins of bacterial polysaccharide diversity, laying the foundations for molecular serotyping. This is the first time, to our knowledge, that a complete repertoire of capsular biosynthetic genes has been available, enabling a holistic analysis of a bacterial polysaccharide biosynthesis system. Remarkably, the total size of alternative coding DNA at this one locus exceeds 1.8 Mbp, almost equivalent to the entire S. pneumoniae chromosomal complement.
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145. |
Chesnel L,
Carapito R,
Croizé J,
Dideberg O,
Vernet T,
Zapun A,
( 2005 ) Identical penicillin-binding domains in penicillin-binding proteins of Streptococcus pneumoniae clinical isolates with different levels of beta-lactam resistance. PMID : 15980366 : DOI : 10.1128/AAC.49.7.2895-2902.2005 PMC : PMC1168675 Abstract >>
We have sequenced the penicillin-binding domains of the complete repertoire of penicillin-binding proteins and MurM from 22 clinical isolates of Streptococcus pneumoniae that span a wide range of beta-lactam resistance levels. Evidence of mosaicism was found in the genes encoding PBP 1a, PBP 2b, PBP 2x, MurM, and, possibly, PBP 2a. Five isolates were found to have identical PBP and MurM sequences, even though the MICs for penicillin G ranged from 0.25 to 2.0 mg/liter. When the sequences encoding PBP 1a, PBP 2b, and PBP 2x from one of these isolates were used to transform laboratory strain R6, the resulting strain had a resistance level higher than that of the less resistant isolates carrying that PBP set but lower than that of the most resistant isolates carrying that PBP set. This result demonstrates that if the R6 strain is arbitrarily defined as the standard genotype, some wild genetic backgrounds can either increase or decrease the PBP-based resistance phenotype.
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146. |
Kirkham LA,
Jefferies JM,
Kerr AR,
Jing Y,
Clarke SC,
Smith A,
Mitchell TJ,
( 2006 ) Identification of invasive serotype 1 pneumococcal isolates that express nonhemolytic pneumolysin. PMID : 16390963 : DOI : 10.1128/JCM.44.1.151-159.2006 PMC : PMC1351962 Abstract >>
Recently, there has been an increase in invasive pneumococcal disease (IPD) caused by serotype 1 Streptococcus pneumoniae throughout Europe. Serotype 1 IPD is associated with bacteremia and pneumonia in Europe and North America, especially in neonates, and is ranked among the top five most prevalent pneumococcal serotypes in at least 10 countries. The currently licensed pediatric pneumococcal vaccine does not afford protection to this serotype. Upon screening of 252 clinical isolates of S. pneumoniae, we discovered mutations in the pneumolysin gene of two out of the four serotype 1 strains present in the study group. Analysis of an additional 28 serotype 1 isolates from patients with IPD from various Scottish Health Boards, revealed that >50% had mutations in their pneumolysin genes. This resulted in the expression of nonhemolytic forms of pneumolysin. All of the strains producing nonhemolytic pneumolysin were sequence type 306 (ST306), whereas those producing "wild-type" pneumolysin were ST227. The mutations were in a region of pneumolysin involved in pore formation. These mutations can be made in vitro to give the nonhemolytic phenotype. Pneumolysin is generally conserved throughout all serotypes of S. pneumoniae and is essential for full invasive disease; however, it appears that serotype 1 ST306 does not require hemolytically active pneumolysin to cause IPD.
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147. |
Ko KS,
Oh WS,
Peck KR,
Lee JH,
Lee NY,
Song JH,
( 2005 ) Phenotypic and genotypic discrepancy of Streptococcus pneumoniae strains isolated from Asian countries. PMID : 15985224 : DOI : 10.1016/j.femsim.2005.01.012 Abstract >>
Non-typeable isolates of Streptococcus pneumoniae collected from Asian countries were characterized by optochin susceptibility test, bile solubility test, multilocus sequence typing of housekeeping genes, amplification of virulence-related genes, 16S rDNA-RsaI digestion, and 16S rDNA sequencing. Six of 54 non-typeable pneumococcal isolates showed divergence of gene sequences of recP and xpt from typical pneumococcal strains. Of these six atypical pneumococcal strains, two showed different results in optochin susceptibility or bile solubility test from typical pneumococcal strains. All six isolates showed high sequence dissimilarities of multilocus sequence typing, 16S rDNA sequences, and lytA sequences from typical S. pneumoniae strains. Data from this study suggest that classic tests such as optochin susceptibility and bile solubility tests may lead to incorrect identification of S. pneumoniae. These atypical strains may belong to different bacterial species from S. pneumoniae.
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148. |
Sperisen P,
Schmid CD,
Bucher P,
Zilian O,
( 2005 ) Stealth proteins: in silico identification of a novel protein family rendering bacterial pathogens invisible to host immune defense. PMID : 16299590 : DOI : 10.1371/journal.pcbi.0010063 PMC : PMC1285062 Abstract >>
There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC) is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.
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149. |
Maeda K,
Ida T,
Sanbongi Y,
Suzuki T,
Fukushima T,
Kurazono M,
Yonezawa M,
Ubukata K,
Inoue M,
( 2005 ) Comparison of activities of beta-lactam antibiotics against Streptococcus pneumoniae with recombinant penicillin-binding protein genes from a penicillin-resistant strain. PMID : 15856382 : DOI : 10.1007/s10156-005-0374-2 Abstract >>
We investigated the antibacterial activities of 19 beta-lactams against three recombinant bacterial strains, in which three penicillin-binding protein genes, pbp2x, pbp1a, and pbp2b, from penicillin-resistant Streptococcus pneumoniae (PRSP), were transformed to a penicillin-susceptible strain. By the acquisition of the pbp2x gene from PRSP, the minimum inhibitory concentrations (MICs) of third-generation cephalosporins were increased more than eight fold. When the strain acquired the PRSP pbp1a gene in addition to pbp2x, the MICs of all tested beta-lactams increased 2- to 16-fold. When the strain acquired the PRSP pbp2b gene in addition to pbp2x and pbp1a, the MICs of penicillins and carbapenems increased 4- to 16-fold. However, two novel carbapenems, ME1036 and L-036, showed excellent antibacterial activities against these recombinant strains, as well as against the parent PRSP.
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150. |
Ferrándiz MJ,
Ardanuy C,
Liñares J,
García-Arenzana JM,
Cercenado E,
Fleites A,
de la Campa AG,
N/A N/A,
( 2005 ) New mutations and horizontal transfer of rpoB among rifampin-resistant Streptococcus pneumoniae from four Spanish hospitals. PMID : 15917517 : DOI : 10.1128/AAC.49.6.2237-2245.2005 PMC : PMC1140543 Abstract >>
A total of 103 (0.7%) of 14,236 Streptococcus pneumoniae isolates collected in four Spanish hospitals from 1989 to 2003 were resistant to rifampin (MICs, 4 to 512 microg/ml). Only sixty-one (59.2%) of these isolates were available for molecular characterization. Resistance was mostly related to human immunodeficiency virus (HIV) infection in adult patients and to conjunctivitis in children. Thirty-six different pulsed-field gel electrophoresis patterns were identified among resistant isolates, five of which were related to international clones (Spain23F-1, Spain6B-2, Spain9V-3, Spain14-5, and clone C of serotype 19F), and accounted for 49.2% of resistant isolates. Single sense mutations at cluster N or I of the rpoB gene were found in 39 isolates, while double mutations, either at cluster I, at clusters I and II, or at clusters N and III, were found in 14 isolates. The involvement of the mutations in rifampin resistance was confirmed by genetic transformation. Single mutations at clusters N and I conferred MICs of 2 microg/ml and 4 to 32 microg/ml, respectively. Eight isolates showed high degrees of nucleotide sequence variations (2.3 to 10.8%) in rpoB, suggesting a recombinational origin for these isolates, for which viridans group streptococci are their potential gene donors. Although the majority of rifampin-resistant isolates were isolated from individual patients without temporal or geographical relationships, the clonal dissemination of rifampin-resistant isolates was observed among 12 HIV-infected patients in the two hospitals with higher rates of resistance.
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151. |
Martin B,
Humbert O,
Camara M,
Guenzi E,
Walker J,
Mitchell T,
Andrew P,
Prudhomme M,
Alloing G,
Hakenbeck R,
( 1992 ) A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae. PMID : 1630918 : DOI : 10.1093/nar/20.13.3479 PMC : PMC312505 Abstract >>
We report the discovery of a group of highly conserved DNA sequences located, in those cases studied, within intergenic regions of the chromosome of the Gram positive Streptococcus pneumoniae. The S. pneumoniae genome contains about 25 of these elements called BOX. From 5' to 3', BOX elements are composed of three subunits (boxA, boxB, and boxC) which are 59, 45 and 50 nucleotides long, respectively. BOX elements containing one, two and four copies of boxB have been observed; boxB alone was also detected in one instance. These elements are unrelated to the two most thoroughly documented families of repetitive DNA sequences present in the genomes of enterobacteria. BOX sequences have the potential to form stable stem-loop structures and one of these, at least, is transcribed. Most of these elements are located in the immediate vicinity of genes whose product has been implicated at some stage in the process of genetic transformation or in virulence of S. pneumoniae. This location raises the intriguing possibility that BOX sequences are regulatory elements shared by several coordinately controlled genes, including competence-specific and virulence-related genes.
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152. |
Smith AM,
Feldman C,
Massidda O,
McCarthy K,
Ndiweni D,
Klugman KP,
( 2005 ) Altered PBP 2A and its role in the development of penicillin, cefotaxime, and ceftriaxone resistance in a clinical isolate of Streptococcus pneumoniae. PMID : 15855525 : DOI : 10.1128/AAC.49.5.2002-2007.2005 PMC : PMC1087663 Abstract >>
We report the unusual involvement of altered PBP 2A in the development of beta-lactam resistance in Streptococcus pneumoniae. This was investigated amid three identical serotype 14 isolates (designated isolates 1, 2, and 3, respectively) of pneumococci cultured successfully from the blood of a human immunodeficiency virus-seropositive child with recurrent pneumonia. The passage of this strain through its human host induced several changes in the bacterium, which is typical of the adaptive and evolving nature of the pneumococcus. An efflux resistance mechanism, which conferred increased ciprofloxacin resistance, was induced in isolates 2 and 3. In addition, faster growth rates and larger capsules were also observed for these isolates, with respect to isolate 1. Notably, compared to isolates 1 and 2, isolate 3 showed a decrease in penicillin, cefotaxime, and ceftriaxone resistance. This change was associated with the replacement of an altered PBP 2A for an unaltered PBP 2A. In all likelihood, these events produced a strain which evolved into a fitter and more virulent type, isolate 3, that resulted in an aggravated pneumococcal infection and ultimately in the patient's death.
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153. |
Pettigrew MM,
Fennie KP,
( 2005 ) Genomic subtraction followed by dot blot screening of Streptococcus pneumoniae clinical and carriage isolates identifies genetic differences associated with strains that cause otitis media. PMID : 15845484 : DOI : 10.1128/IAI.73.5.2805-2811.2005 PMC : PMC1087362 Abstract >>
Streptococcus pneumoniae strains are the leading cause of bacterial otitis media, yet little is known about specific bacterial factors important for this disease. We utilized a molecular epidemiological approach involving genomic subtraction of the S. pneumoniae serogroup 19 middle ear strain 5093 against the laboratory strain R6. Resulting subtraction PCR (sPCR) products were used to screen a panel of 93 middle ear, 90 blood, 35 carriage, and 58 cerebrospinal fluid isolates from young children to identify genes found more frequently among middle ear isolates. Probe P41, similar to a hypothetical protein of Brucella melitensis, occurred among 41% of middle ear isolates and was found 2.8 (95% confidence interval [CI], 1.32 to 6.5), 3.3 (95% CI, 1.9 to 5.7), and 1.8 (95% CI, 1.1 to 3.0) times more frequently among middle ear strains than carriage, blood, or meningitis strains, respectively. sPCR fragment H10, similar to an unknown Streptococcus agalactiae protein, was present in 31% of middle ear isolates and occurred 3.6 (95% CI, 1.2 to 11.2), 2.8 (95% CI, 1.5 to 5.4), and 2.6 (95% CI, 1.2 to 5.5) times more often among middle ear isolates than carriage, blood, or meningitis strains, respectively. These studies have identified two genes of potential importance in otitis media virulence. Further studies are warranted to outline the precise role of these genes in otitis media pathogenesis.
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154. |
Granger D,
Boily-Larouche G,
Turgeon P,
Weiss K,
Roger M,
( 2005 ) Genetic analysis of pbp2x in clinical Streptococcus pneumoniae isolates in Quebec, Canada. PMID : 15872046 : DOI : 10.1093/jac/dki118 Abstract >>
To investigate the nature of the amino acid motifs found in penicillin-binding protein (PBP) 2x of penicillin-resistant Streptococcus pneumoniae isolates across the province of Quebec (Canada), and to obtain preliminary information regarding the prevalence of these alterations. The pbp2x genomic region encompassing codons 178-703, which includes the entire region of the transpeptidase domain, was sequenced and compared for 52 clinical isolates comprising 20 penicillin-susceptible (PSSP), 20 penicillin-intermediate (PISP) and 12 penicillin-resistant (PRSP) pneumococci. The degree of diversity within PBP2x correlated with increased resistance to beta-lactam antibiotics. There were an average of 5.0 +/- 1.8 mutations in PSSP, 37.9 +/- 4.4 in PISP, and 63.0 +/- 2.0 in PRSP isolates when compared with the control penicillin-susceptible strain R6. At least six distinct amino acid profiles were identified among PISP strains isolated in Quebec. In contrast, all PRSP isolates shared a similar pattern of altered amino acids compared with the sequence from susceptible strains. These data will be useful in future studies to monitor the genetic changes associated with the emergence and spread of beta-lactam resistance in Quebec.
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155. |
Cochetti I,
Vecchi M,
Mingoia M,
Tili E,
Catania MR,
Manzin A,
Varaldo PE,
Montanari MP,
( 2005 ) Molecular characterization of pneumococci with efflux-mediated erythromycin resistance and identification of a novel mef gene subclass, mef(I). PMID : 16304164 : DOI : 10.1128/AAC.49.12.4999-5006.2005 PMC : PMC1315940 Abstract >>
The molecular genetics of macrolide resistance were analyzed in 49 clinical pneumococci (including an "atypical" bile-insoluble strain currently assigned to the new species Streptococcus pseudopneumoniae) with efflux-mediated erythromycin resistance (M phenotype). All test strains had the mef gene, identified as mef(A) in 30 isolates and mef(E) in 19 isolates (including the S. pseudopneumoniae strain) on the basis of PCR-restriction fragment length polymorphism analysis. Twenty-eight of the 30 mef(A) isolates shared a pulsed-field gel electrophoresis (PFGE) type corresponding to the England14-9 clone. Of those isolates, 27 (20 belonging to serotype 14) yielded multilocus sequence type ST9, and one isolate yielded a new sequence type. The remaining two mef(A) isolates had different PFGE types and yielded an ST9 type and a new sequence type. Far greater heterogeneity was displayed by the 19 mef(E) isolates, which fell into 11 PFGE types, 12 serotypes (though not serotype 14), and 12 sequence types (including two new ones and an undetermined type for the S. pseudopneumoniae strain). In all mef(A) pneumococci, the mef element was a regular Tn1207.1 transposon, whereas of the mef(E) isolates, 17 carried the mega element and 2 exhibited a previously unreported organization, with no PCR evidence of the other open reading frames of mega. The mef gene of these two isolates, which did not match with the mef(E) gene of the mega element (93.6% homology) and which exhibited comparable homology (91.4%) to the mef(A) gene of the Tn1207.1 transposon, was identified as a novel mef gene variant and was designated mef(I). While penicillin-nonsusceptible isolates (three resistant isolates and one intermediate isolate) were all mef(E) strains, tetracycline resistance was also detected in three mef(A) isolates, due to the tet(M) gene carried by a Tn916-like transposon. A similar mechanism accounted for resistance in four of the five tetracycline-resistant isolates carrying mef(E), in three of which mega was inserted in the Tn916-like transposon, giving rise to the composite element Tn2009. In the fifth mef(E)-positive tetracycline-resistant isolate (the S. pseudopneumoniae strain), tetracycline resistance was due to the presence of the tet(O) gene, apparently unlinked to mef(E).
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156. |
Naser S,
Thompson FL,
Hoste B,
Gevers D,
Vandemeulebroecke K,
Cleenwerck I,
Thompson CC,
Vancanneyt M,
Swings J,
( 2005 ) Phylogeny and identification of Enterococci by atpA gene sequence analysis. PMID : 15872246 : DOI : 10.1128/JCM.43.5.2224-2230.2005 PMC : PMC1153757 Abstract >>
The relatedness among 91 Enterococcus strains representing all validly described species was investigated by comparing a 1,102-bp fragment of atpA, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However, atpA gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of atpA sequences with, at a maximum, 92% similarity. Six members of the Enterococcus faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed > 99% 16S rRNA gene sequence similarity, but the highest value of atpA gene sequence similarity was only 89.9%. The intraspecies atpA sequence similarities for all species except E. faecium strains varied from 98.6 to 100%; the E. faecium strains had a lower atpA sequence similarity of 96.3%. Our data clearly show that atpA provides an alternative tool for the phylogenetic study and identification of enterococci.
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157. |
Moscoso M,
Obregón V,
López R,
García JL,
García E,
( 2005 ) Allelic variation of polymorphic locus lytB, encoding a choline-binding protein, from streptococci of the mitis group. PMID : 16332865 : DOI : 10.1128/AEM.71.12.8706-8713.2005 PMC : PMC1317417 Abstract >>
The choline-binding protein LytB, an N-acetylglucosaminidase of Streptococcus pneumoniae, is the key enzyme for daughter cell separation and is believed to play a critical pathogenic role, facilitating bacterial spreading during infection. Because of these peculiarities LytB is a putative vaccine target. To determine the extent of LytB polymorphism, the lytB alleles from seven typical, clinical pneumococcal isolates of various serotypes and from 13 additional streptococci of the mitis group (12 atypical pneumococci and the Streptococcus mitis type strain) were sequenced. Sequence alignment showed that the main differences among alleles were differences in the number of repeats (range, 12 to 18) characteristic of choline-binding proteins. These differences were located in the region corresponding to repeats 11 to 17. Typical pneumococcal strains contained either 14, 16, or 18 repeats, whereas all of the atypical isolates except strains 1283 and 782 (which had 14 and 16 repeats, respectively) and the S. mitis type strain had only 12 repeats; atypical isolate 10546 turned out to be a DeltalytB mutant. We also found that there are two major types of alternating repeats in lytB, which encode 21 and 23 amino acids. Choline-binding proteins are linked to the choline-containing cell wall substrate through choline residues at the interface of two consecutive choline-binding repeats that create a choline-binding site. The observation that all strains contained an even number of repeats suggests that the duplication events that gave rise to the choline-binding repeats of LytB involved two repeats simultaneously, an observation that is in keeping with previous crystallographic data. Typical pneumococcal isolates usually grew as diplococci, indicating that an active LytB enzyme was present. In contrast, most atypical isolates formed long chains of cells that did not disperse after addition of purified LytB, suggesting that in these strains chains were produced through mechanisms unrelated to LytB.
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158. |
Smith AM,
Klugman KP,
( 2005 ) Amino acid mutations essential to production of an altered PBP 2X conferring high-level beta-lactam resistance in a clinical isolate of Streptococcus pneumoniae. PMID : 16251304 : DOI : 10.1128/AAC.49.11.4622-4627.2005 PMC : PMC1280142 Abstract >>
Altered penicillin-binding protein 2X (PBP 2X) is a primary beta-lactam antibiotic resistance determinant and is essential to the development of penicillin and cephalosporin resistance in the pneumococcus. We have studied the importance for resistance of 23 amino acid substitutions located in the transpeptidase domain (TD) of PBP 2X from an isolate with high-level resistance, isolate 3191 (penicillin MIC, 16 mug/ml; cefotaxime MIC, 4 microg/ml). Strain R6(2X/2B/1A/mur) (for which the MICs are as described for isolate 3191) was constructed by transforming laboratory strain R6 with all the necessary resistance determinants (altered PBPs 2X, 2B, and 1A and altered MurM) from isolate 3191. Site-directed mutagenesis was used to reverse amino acid substitutions in altered PBP 2X, followed by investigation of the impact of these reversions on resistance levels in R6(2X/2B/1A/mur). Of the 23 substitutions located in the TD of PBP 2X, reversals at six positions decreased the resistance levels in R6(2X/2B/1A/mur). Reversal of the Thr338Pro and Ile371Thr substitutions individually decreased the penicillin and cefotaxime MICs to 2 and 1 microg/ml, respectively, and individually displayed the greatest impact on resistance. To a lesser extent, reversal of the Leu364Phe, Ala369Val, Arg384Gly, and Tyr595Phe substitutions individually also decreased the penicillin and cefotaxime MICs. Reversal at all six positions collectively decreased both the penicillin and the cefotaxime MICs of R6(2X/2B/1A/mur) to 0.06 microg/ml. This study confirms the essential role of altered PBP 2X as a resistance determinant. Our data reveal that, for isolate 3191, the six amino acid substitutions described above are collectively essential to the production of an altered PBP 2X required for high-level resistance to penicillin and cefotaxime.
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159. |
Granger D,
Boily-Larouche G,
Turgeon P,
Weiss K,
Roger M,
( 2006 ) Molecular characteristics of pbp1a and pbp2b in clinical Streptococcus pneumoniae isolates in Quebec, Canada. PMID : 16282207 : DOI : 10.1093/jac/dki401 Abstract >>
To investigate the nature of the amino acid motifs found in penicillin-binding protein (PBP) 2b and PBP1a of penicillin-resistant Streptococcus pneumoniae isolates across Quebec (Canada), and to obtain preliminary information regarding the prevalence of these alterations. DNA sequences of pbp2b (codons 210-675) and pbp1a (codons 310-682) transpeptidase domains were determined and compared in 48 clinical isolates comprising 17 penicillin-susceptible (PSSP), 19 penicillin-intermediate (PISP) and 12 penicillin-resistant (PRSP) pneumococci. The degree of diversity within PBP1a and PBP2b correlated with increased resistance to beta-lactam antibiotics. There were an average of 0.6 +/- 0.4 and 2.9 +/- 0.2 mutations in PSSP, 16.8 +/- 1.4 and 36.3 +/- 5.2 in PISP, and 18.7 +/- 2.5 and 51.4 +/- 1.3 in PRSP isolates compared with control penicillin-susceptible R6-PBP2b and R6-PBP1a sequences, respectively. At least seven PBP2b and six PBP1a distinct amino acid profiles were identified among intermediate or resistant strains isolated in Quebec. The pattern of distribution of the PBPs' altered amino acids differs from that of other countries, with pneumococci isolates from Quebec showing a unique genetic signature. This study will serve as a basis for future monitoring of genetic changes associated with the emergence and spread of beta-lactam resistance in Quebec, Canada.
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160. |
Medina MJ,
Greene CM,
Gertz RE,
Facklam RR,
Jagero G,
Hamel M,
Shi YP,
Slutsker L,
Feikin DR,
Beall B,
( 2005 ) Novel antibiotic-resistant pneumococcal strains recovered from the upper respiratory tracts of HIV-infected adults and their children in Kisumu, Kenya. PMID : 15770088 : DOI : 10.1089/mdr.2005.11.9 Abstract >>
In a survey of genetic diversity within penicillin-nonsusceptible pneumococcal isolates in Kenya, we examined 162 upper respiratory isolates from 104 human immunodeficiency virus (HIV)-infected adults and 46 children in a cotrimoxazole prophylaxis study. Antibiotic resistance levels were high; 152 (94.4%) were cotrimoxazole nonsusceptible (134 fully resistant) and 124 (77%) were intermediately penicillin resistant. Isolates nonsusceptible to penicillin and cotrimoxazole (PNCNP) were found among 24 of the 29 serotypes encountered, 15 of which have rarely or never had documented nonsusceptibility to penicillin. These included serotypes 3, 4, 7C, 7F, 10A, 11A, 13, 15A, 15B, 16F, 17F, 19B, 21, 35A, and 35B. Segments of pbp2b genes from 9 PNCNP (serotypes 3, 13, 15A, 16F, 20, and 35A) were typical of resistance-conferring alleles in that they were highly divergent and contained two substitutions thought to be critical for resistance. Similarly, the dhfr genes from 3 PNCNP were divergent and contained a substitution required for cotrimoxazole resistance. Multilocus sequence typing (MLST) of 48 PNCNP revealed 33 sequence types (STs), none of which were previously recorded at http://www.mlst.net. Comparisons with all known STs revealed that 23 of these STs were unrelated to other known STs, whereas 10 STs were highly related to STs from internationally disseminated strains, including 2 of the 26 antibiotic-resistant clones recognized by the Pneumococcal Molecular Epidemiology Network. Based upon differing serotypes expressed by strains of identical or closely similar genotypes, there has been an extensive history of capsular switching within seven genetic clusters represented by these 10 STs and related STs described at http://www.mlst.net.
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161. |
Stanhope MJ,
Walsh SL,
Becker JA,
Italia MJ,
Ingraham KA,
Gwynn MN,
Mathie T,
Poupard JA,
Miller LA,
Brown JR,
Amrine-Madsen H,
( 2005 ) Molecular evolution perspectives on intraspecific lateral DNA transfer of topoisomerase and gyrase loci in Streptococcus pneumoniae, with implications for fluoroquinolone resistance development and spread. PMID : 16189113 : DOI : 10.1128/AAC.49.10.4315-4326.2005 PMC : PMC1251522 Abstract >>
Fluoroquinolones are an important class of antibiotics for the treatment of infections arising from the gram-positive respiratory pathogen Streptococcus pneumoniae. Although there is evidence supporting interspecific lateral DNA transfer of fluoroquinolone target loci, no studies have specifically been designed to assess the role of intraspecific lateral transfer of these genes in the spread of fluoroquinolone resistance. This study involves a comparative evolutionary perspective, in which the evolutionary history of a diverse set of S. pneumoniae clinical isolates is reconstructed from an expanded multilocus sequence typing data set, with putative recombinants excluded. This control history is then assessed against networks of each of the four fluoroquinolone target loci from the same isolates. The results indicate that although the majority of fluoroquinolone target loci from this set of 60 isolates are consistent with a clonal dissemination hypothesis, 3 to 10% of the sequences are consistent with an intraspecific lateral transfer hypothesis. Also evident were examples of interspecific transfer, with two isolates possessing a parE-parC gene region arising from viridans group streptococci. The Spain 23F-1 clone is the most dominant fluoroquinolone-nonsusceptible clone in this set of isolates, and the analysis suggests that its members act as frequent donors of fluoroquinolone-nonsusceptible loci. Although the majority of fluoroquinolone target gene sequences in this set of isolates can be explained on the basis of clonal dissemination, a significant number are more parsimoniously explained by intraspecific lateral DNA transfer, and in situations of high S. pneumoniae population density, such events could be an important means of resistance spread.
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162. |
Ding YF,
Zhang JH,
Mi ZH,
Qin L,
Tao YZ,
Qi X,
( 2004 ) [Study on the molecular epidemiology of beta-lactamase TEM gene in isolated Streptococcus pneumoniae]. PMID : 15769331 : Abstract >>
To investigate the beta-lactamase TEM gene of isolated Streptococcus pneumoniae (Sp) in Suzhou area. Twenty-three strains of Sp were collected from respiratory tract secretions of children with respiratory diseases in Nov 2002 to Apr 2003 at Children's Hospital of Suzhou University (reference strain ATCC49619) to build TEM polymerase chain reaction (PCR) system (reference strain E. coli. 9-j53R1 with TEM gene) TEM gene of 23 strains was detected to comparo the sequences with published TEM gene sequences in GenBank for analyzing TEM gene model. Twenty-one strains had TEM gene with a positive rate of 91.3% (21/23). TEM-129 gene were confirmed from No.17 (SR017, penicillin resistance) TEM sequence. New discovered TEM-129 sequence had a modification (ATG[M]-->ATA[I]) at No.182 code and published (GenBank: www.ncbi.nlm.nih.gov/nucleotide, AY452662). TEM-1 genes were confirmed from other TEM sequences. New discovered TEM-1 gene of isolated Sp had been published (GenBank: www.ncbi.nlm.nih.gov/nucleotide, AY392531) too. Isolated Sp had TEM gene (TEM-129, EM-1 genotype) with a positive rate of 91.3%. The result enriched the understanding of isolated Sp with penicillin resistance.
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163. |
McEllistrem MC,
Adams JM,
Visweswaran S,
Khan SA,
( 2005 ) Detection of very high-level penicillin-resistant variants of the Tennessee (23 F)-4 clone via single and serial transformations with four serotype 19 A international pneumococcal clones. PMID : 16201931 : DOI : 10.1089/mdr.2005.11.271 Abstract >>
In the United States, penicillin-resistant variants of the Tennessee (Tenn) (23 F)-4 clone account for a substantial proportion of the very-high-level penicillin-resistant (MIC 8 microg/ml) infections in the 7-valent pneumococcal protein conjugate vaccine (PCV 7) era. Serotype 19 A strains account for an increasing proportion of penicillin-nonsusceptible Streptococcus pneumoniae infections. Sequential transformations of the Tenn (23 F)-4 clone (penicillin MIC 0.1 microg/ml) were performed with four penicillin-nonsusceptible serotype 19 A international clones (penicillin MIC): S. Africa (19 A)-7 (0.5 microg/ml), Hungary (19 A)-6 (2 microg/ml), Slovakia (19 A)-11 (8 microg/ml), and South Africa (19 A)-13 (8 microg/ml). Fifty-two transformants were characterized by MICs, serogroup-specific PCR, pbp PCR restriction profile and sequence, psp A PCR restriction profile, and erm/mef PCR. A subset was analyzed with multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. Serotype 23 F transformants with penicillin MIC >or= 8 microg/ml were detected through a single transformation with the Hungary (19 A)-6 clone or serial transformations using two to three different clones. Forty-four percent (14/32) of the transformants incorporated >or=1 new MLST allele. Using encapsulated donors, very-high-level penicillin resistant variants of the Tenn (23 F)-4 clone were detected. In addition to detecting stepwise increases in penicillin MIC, a 12-fold increase in penicillin MIC was achieved through a single transformation. This large increase in MIC may explain why this clone is commonly associated with very-high-level resistance in natural populations. Recombination within the MLST housekeeping genes was commonly detected in the transformants that had acquired penicillin resistance.
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164. |
Douthwaite S,
Jalava J,
Jakobsen L,
( 2005 ) Ketolide resistance in Streptococcus pyogenes correlates with the degree of rRNA dimethylation by Erm. PMID : 16194243 : DOI : 10.1111/j.1365-2958.2005.04863.x Abstract >>
Macrolide and ketolide antibiotics inhibit protein synthesis on the bacterial ribosome. Resistance to these antibiotics is conferred by dimethylation at 23S rRNA nucleotide A2058 within the ribosomal binding site. This form of resistance is encoded by erm dimethyltransferase genes, and is found in many pathogenic bacteria. Clinical isolates of Streptococcus pneumoniae with constitutive erm(B) and Streptococcus pyogenes with constitutive erm(A) subtype (TR) are resistant to macrolides, but remain susceptible to ketolides such as telithromycin. Paradoxically, some strains of S. pyogenes that possess an identical erm(B) gene are clinically resistant to ketolides as well as macrolides. Here we explore the molecular basis for the differences in these streptococcal strains using mass spectrometry to determine the methylation status of their rRNAs. We find a correlation between the levels of A2058-dimethylation and ketolide resistance, and dimethylation is greatest in S. pyogenes strains expressing erm(B). In constitutive erm strains that are ketolide-sensitive, appreciable proportions of the rRNA remain monomethylated. Incubation of these strains with subinhibitory amounts of the macrolide erythromycin increases the proportion of dimethylated A2058 (in a manner comparable with inducible erm strains) and reduces ketolide susceptibility. The designation 'constitutive' should thus be applied with some reservation for most streptococcal erm strains. One strain worthy of the constitutive designation is S. pyogenes isolate KuoR21, which has lost part of the regulatory region upstream of erm(B). In S. pyogenes KuoR21, nucleotide A2058 is fully dimethylated under all growth conditions, and this strain displays the highest resistance to telithromycin (MIC > 64 microg ml-1).
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165. |
Faccone D,
Andres P,
Galas M,
Tokumoto M,
Rosato A,
Corso A,
( 2005 ) Emergence of a Streptococcus pneumoniae clinical isolate highly resistant to telithromycin and fluoroquinolones. PMID : 16272525 : DOI : 10.1128/JCM.43.11.5800-5803.2005 PMC : PMC1287838 Abstract >>
Streptococcus pneumoniae is a major pathogen causing community-acquired pneumonia and acute bronchitis. Macrolides, fluoroquinolones (FQs), and, recently, telithromycin (TEL) constitute primary therapeutic options, and rare cases of resistance have been reported. In this report, we describe the emergence of an S. pneumoniae clinical isolate with high-level TEL resistance (MIC, 256 microg/ml) and simultaneous resistance to FQs. Ongoing studies are oriented to elucidate the precise mechanism of resistance to TEL.
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166. |
Iannelli F,
Oggioni MR,
Pozzi G,
( 2005 ) Sensor domain of histidine kinase ComD confers competence pherotype specificity in Streptoccoccus pneumoniae. PMID : 16209911 : DOI : 10.1016/j.femsle.2005.09.008 Abstract >>
Competence for genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing mechanism involving the pheromone competence stimulating peptide (CSP) encoded by comC and a two-component signal transduction system, ComD-ComE (TCS12). In the presence of CSP, the transmembrane histidine kinase ComD receptor activates the response regulator ComE. The comC, comD and comE genes are part of an operon denoted as comCDE. In this work, the comCDE locus of 17 S. pneumoniae strains was characterized by DNA sequencing. Two major allelic combinations, comC1-comD1 and comC2-comD2 were present. Two further allelic combinations, comC1-comD3 and comC1-comD4, were also present. Comparison of the deduced amino acid sequences of the four ComD allelic variants showed that all variations are localized in the N-terminal sensor domain. In order to have the four comD alleles in the same genetic background, we constructed four different isogenic strains in which comC was deleted and the DNA encoding the sensor domain of ComD was exchanged. To formally demonstrate that the sensor domain of ComD is responsible for competence pherotype specificity, CSP-1 and CSP-2 peptides were used to induce competence in the isogenic strains: (i) strains expressing the ComD1, ComD3 and ComD4 variants were induced to competence by CSP1; (ii) the strain expressing ComD2 was induced by CSP2. Moreover, cross-induction of competence by both CSPs was observed in the ComD2 and ComD3-carrying strains in the presence of high CSP doses. This is the first formal confirmation that the ComD sensor domain is responsible for competence pherotype specificity in S. pneumoniae.
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167. |
Kong F,
Wang W,
Tao J,
Wang L,
Wang Q,
Sabananthan A,
Gilbert GL,
( 2005 ) A molecular-capsular-type prediction system for 90 Streptococcus pneumoniae serotypes using partial cpsA-cpsB sequencing and wzy- or wzx-specific PCR. PMID : 15770019 : DOI : 10.1099/jmm.0.45924-0 Abstract >>
In a previous study, a molecular capsular type (MCT) prediction system for 51 Streptococcus pneumoniae serotypes was developed based on a combination of partial cpsA-cpsB sequencing and serotype(s)/group(s)-specific PCR. In this study, another 169 S. pneumoniae isolates were added to the existing database of 427 isolates, including representatives of all 39 serotypes not previously studied. In addition to the authors' own limited sequence data for all 90 serotypes, cpsA-cpsB sequence data published by the S. pneumoniae capsular loci-sequencing group (http://www.sanger.ac.uk/Projects/S_pneumoniae/CPS/) at the Sanger Institute or available from GenBank were incorporated into the database. All serotypes, except 25A, were represented by at least two isolates. The number of sequence types identified was 138, of which 110 corresponded to single conventional serotypes (CSs); of these, 57 were represented by two or more isolates. Twenty-six sequence types were shared by between two and four CSs. To resolve these shared cpsA-cpsB sequence types and increase the discriminatory power of our system, the genes encoding the capsular polysaccharide flippase (wzx) and polymerase (wzy) were annotated and 24 new serotype(s)/group(s)-specific PCRs targeting wzy and two targeting wzx were designed. Using both cpsA-cpsB sequencing and wzx/wzy PCR, MCT correctly predicted the CSs of 516 (73 %) and the serogroup of an additional 155 (22 %) of the 708 isolates evaluated. For 5 % of isolates, MCT could not distinguish between members of five serotype pairs (37 isolates) containing members of different serogroups. Although further study of the relationship between MCT and CS is needed, this system now allows serotype or serogroup identification of 95 % of S. pneumoniae isolates.
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168. |
King SJ,
Whatmore AM,
Dowson CG,
( 2005 ) NanA, a neuraminidase from Streptococcus pneumoniae, shows high levels of sequence diversity, at least in part through recombination with Streptococcus oralis. PMID : 16030232 : DOI : 10.1128/JB.187.15.5376-5386.2005 PMC : PMC1196044 Abstract >>
Streptococcus pneumoniae, an important human pathogen, contains at least two genes, nanA and nanB, that express sialidase activity. NanA is a virulence determinant of pneumococci which is important in animal models of colonization and middle ear infections. The gene encoding NanA was detected in all 106 pneumococcal strains screened that represented 59 restriction profiles. Sequencing confirmed a high level of diversity, up to 17.2% at the nucleotide level and 14.8% at the amino acid level. NanA diversity is due to a number of mechanisms including insertions, point mutations, and recombination generating mosaic genes. The level of nucleotide divergence for each recombinant block is greater than 30% and much higher than the 20% identified within mosaic pbp genes, suggesting that a high selective pressure exists for these alterations. These data indicate that at least one of the four recombinant blocks identified originated from a Streptococcus oralis isolate, demonstrating for the first time that protein virulence determinants of pneumococci have, as identified previously for genes encoding penicillin binding proteins, evolved by recombination with oral streptococci. No amino acid alterations were identified within the aspartic boxes or predicted active site, suggesting that sequence variation may be important in evading the adaptive immune response. Furthermore, this suggests that nanA is an important target of the immune system in the interaction between the pneumococcus and host.
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169. |
Nováková L,
Sasková L,
Pallová P,
Janecek J,
Novotná J,
Ulrych A,
Echenique J,
Trombe MC,
Branny P,
( 2005 ) Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates. PMID : 15720398 : DOI : 10.1111/j.1742-4658.2005.04560.x Abstract >>
Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic-type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese-dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild-type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP-N-acetylglucosamine, an essential common precursor to cell envelope components.
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170. |
Okitsu N,
Kaieda S,
Yano H,
Nakano R,
Hosaka Y,
Okamoto R,
Kobayashi T,
Inoue M,
( 2005 ) Characterization of ermB gene transposition by Tn1545 and Tn917 in macrolide-resistant Streptococcus pneumoniae isolates. PMID : 15634967 : DOI : 10.1128/JCM.43.1.168-173.2005 PMC : PMC540176 Abstract >>
In Streptococcus pneumoniae, the ermB gene is carried by transposons, such as Tn917 and Tn1545. This study investigated the relationship between macrolide resistance and the presence of the ermB gene on Tn917 or Tn1545 in 84 Japanese pneumococcal isolates. Macrolide-resistant strains were classified into two groups as follows. Group 1 (19 strains) showed a tendency to high resistance to erythromycin (MIC at which 50% of isolates are inhibited, 4 mg/liter; MIC at which 90% of isolates are inhibited [MIC(90)], 128 mg/liter) but susceptibility to rokitamycin (MIC(90), 1 mg/liter), with the ermB gene located on Tn1545. Group 2 (65 strains) showed a tendency to high resistance to both antibiotics (MIC(90)s for both erythromycin and rokitamycin, >128 mg/liter), with the ermB gene located on Tn917. There were no strains with constitutive macrolide resistance in either group. All of the strains in group 2 had a deletion in the promoter region of ermB and an insertion of the TAAA motif in the leader peptide. The results of pulsed-field gel electrophoresis and serogrouping showed that Tn1545 spread clonally while Tn917 spread both horizontally and clonally. In conclusion, in Japanese macrolide-resistant S. pneumoniae isolates, the ermB gene is carried and spread primarily by Tn917.
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171. |
Macheboeuf P,
Di Guilmi AM,
Job V,
Vernet T,
Dideberg O,
Dessen A,
( 2005 ) Active site restructuring regulates ligand recognition in class A penicillin-binding proteins. PMID : 15637155 : DOI : 10.1073/pnas.0407186102 PMC : PMC545533 Abstract >>
Bacterial cell division is a complex, multimolecular process that requires biosynthesis of new peptidoglycan by penicillin-binding proteins (PBPs) during cell wall elongation and septum formation steps. Streptococcus pneumoniae has three bifunctional (class A) PBPs that catalyze both polymerization of glycan chains (glycosyltransfer) and cross-linking of pentapeptidic bridges (transpeptidation) during the peptidoglycan biosynthetic process. In addition to playing important roles in cell division, PBPs are also the targets for beta-lactam antibiotics and thus play key roles in drug-resistance mechanisms. The crystal structure of a soluble form of pneumococcal PBP1b (PBP1b*) has been solved to 1.9 A, thus providing previously undescribed structural information regarding a class A PBP from any organism. PBP1b* is a three-domain molecule harboring a short peptide from the glycosyltransferase domain bound to an interdomain linker region, the transpeptidase domain, and a C-terminal region. The structure of PBP1b* complexed with beta-lactam antibiotics reveals that ligand recognition requires a conformational modification involving conserved elements within the cleft. The open and closed structures of PBP1b* suggest how class A PBPs may become activated as novel peptidoglycan synthesis becomes necessary during the cell division process. In addition, this structure provides an initial framework for the understanding of the role of class A PBPs in the development of antibiotic resistance.
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172. |
Belanger AE,
Clague MJ,
Glass JI,
Leblanc DJ,
( 2004 ) Pyruvate oxidase is a determinant of Avery's rough morphology. PMID : 15576764 : DOI : 10.1128/JB.186.24.8164-8171.2004 PMC : PMC532437 Abstract >>
In pioneering studies, Avery et al. identified DNA as the hereditary material (A. T. Avery, C. M. MacLeod, and M. McCarty, J. Exp. Med. 79:137-158, 1944). They demonstrated, by means of variation in colony morphology, that this substance could transform their rough type 2 Streptococcus pneumoniae strain R36A into a smooth type 3 strain. It has become accepted as fact, from modern textbook accounts of these experiments, that smooth pneumococci make capsule, while rough strains do not. We found that rough-to-smooth morphology conversion did not occur in rough strains R36A and R6 when the ability to synthesize native type 2 capsule was restored. The continued rough morphology of these encapsulated strains was attributed to a second, since-forgotten, morphology-affecting mutation that was sustained by R36A during strain development. We used a new genome-PCR-based approach to identify spxB, the gene encoding pyruvate oxidase, as the mutated locus in R36A and R6 that, with unencapsulation, gives rise to rough colony morphology, as we know it. The variant spxB allele of R36A and R6 is associated with increased cellular pyruvate oxidase activity relative to the ancestral strain D39. Increased pyruvate oxidase activity alters colony shape by mediating cell death. R36A requires a wild-type spxB allele for the expression of smooth type 2 morphology but not for the expression of smooth type 3 morphology, the phenotype monitored by Avery et al. Thus, the mutated spxB allele did not impact their use of smooth morphology to identify the transforming principle.
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173. |
Sanbongi Y,
Ida T,
Ishikawa M,
Osaki Y,
Kataoka H,
Suzuki T,
Kondo K,
Ohsawa F,
Yonezawa M,
( 2004 ) Complete sequences of six penicillin-binding protein genes from 40 Streptococcus pneumoniae clinical isolates collected in Japan. PMID : 15155228 : DOI : 10.1128/AAC.48.6.2244-2250.2004 PMC : PMC415593 Abstract >>
All six penicillin-binding protein (PBP) genes, namely, pbp1a, pbp1b, pbp2a, pbp2b, pbp2x, and pbp3, of 40 Streptococcus pneumoniae clinical isolates, including penicillin-resistant S. pneumoniae isolates collected in Japan, were completely sequenced. The MICs of penicillin for these strains varied between 0.015 and 8 microg/ml. In PBP 2X, the Thr550Ala mutation close to the KSG motif was observed in only 1 of 40 strains, whereas the Met339Phe mutation in the STMK motif was observed in six strains. These six strains were highly resistant (MICs >/= 2 microg/ml) to cefotaxime. The MICs of cefotaxime for 27 strains bearing the Thr338Ala mutation tended to increase, but the His394Leu mutation next to the SSN motif did not exist in these strains. In PBP 2B, the Thr451Ala/Phe/Ser and Glu481Gly mutations close to the SSN motif were observed in 24 strains, which showed penicillin resistance and intermediate resistance, and the Thr624Gly mutation close to the KTG motif was observed in 2 strains for which the imipenem MIC (0.5 microg/ml) was the highest imipenem MIC detected. In PBP 1A, the Thr371Ser/Ala mutation in the STMK motif was observed in all 13 strains for which the penicillin MICs were >/=1 microg/ml. In PBP 2A, the Thr411Ala mutation in the STIK motif was observed in one strain for which with the cefotaxime MIC (8 microg/ml) was the highest cefotaxime MIC detected. On the other hand, in PBPs 1B and 3, no mutations associated with resistance were observed. The results obtained here support the concept that alterations in PBPs 2B, 2X, and 1A are mainly involved in S. pneumoniae resistance to beta-lactam antibiotics. Our findings also suggest that the Thr411Ala mutation in PBP 2A may be associated with beta-lactam resistance.
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174. |
Del Grosso M,
Scotto d'Abusco A,
Iannelli F,
Pozzi G,
Pantosti A,
( 2004 ) Tn2009, a Tn916-like element containing mef(E) in Streptococcus pneumoniae. PMID : 15155196 : DOI : 10.1128/AAC.48.6.2037-2042.2004 PMC : PMC415626 Abstract >>
The association between the macrolide efflux gene mef(E) and the tet(M) gene was studied in two clinical strains of Streptococcus pneumoniae that belonged to serotypes 19F and 6A, respectively, and that were resistant to both tetracycline and erythromycin. The mef(E)-carrying element mega (macrolide efflux genetic assembly; 5,511 bp) was found to be inserted into a Tn916-like genetic element present in the chromosomes of the two pneumococcal strains. In both strains, mega was integrated at the same site, an open reading frame identical to orf6 of Tn916. The new composite element, Tn2009, was about 23.5 kb and, with the exception of the tet(M)-coding sequence, appeared to be identical in both strains. By sequencing of the junction fragments of Tn2009 at the site of insertion into the chromosome, it was possible to show that (i) the insertion site was identical in the two clinical strains and (ii) the integration of Tn2009 caused a 9.5 kb-deletion in the pneumococcal chromosome. It was not possible to detect the conjugal transfer of Tn2009 to a recipient pneumococcal strain; however, transfer of the whole element by transformation was shown to occur. It is possible to hypothesize that Tn2009 relies on transformation for its spread among clinical strains of S. pneumoniae.
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175. |
Hathaway LJ,
Stutzmann Meier P,
Bättig P,
Aebi S,
Mühlemann K,
( 2004 ) A homologue of aliB is found in the capsule region of nonencapsulated Streptococcus pneumoniae. PMID : 15175285 : DOI : 10.1128/JB.186.12.3721-3729.2004 PMC : PMC419944 Abstract >>
The epidemiology, phylogeny, and biology of nonencapsulated Streptococcus pneumoniae are largely unknown. Increased colonization capacity and transformability are, however, intriguing features of these pneumococci and play an important role. Twenty-seven nonencapsulated pneumococci were identified in a nationwide collection of 1,980 nasopharyngeal samples and 215 blood samples obtained between 1998 and 2002. On the basis of multilocus sequence typing and capsule region analysis we divided the nonencapsulated pneumococci into two groups. Group I was closely related to encapsulated strains. Group II had a clonal population structure, including two geographically widespread clones able to cause epidemic conjunctivitis and invasive diseases. Group II strains also carried a 1,959-bp homologue of aliB (aliB-like ORF 2) in the capsule region, which was highly homologous to a sequence in the capsule region of Streptococcus mitis. In addition, strains of the two major clones in group II had an additional sequence, aliB-like ORF 1 (1,968 to 2,004 bp), upstream of aliB-like ORF 2. Expression of aliB-like ORF 1 was detected by reverse transcription-PCR, and the corresponding RNA was visualized by Northern blotting. A gene fragment homologous to capN of serotypes 33 and 37 suggests that group II strains were derived from encapsulated pneumococci some time ago. Therefore, loss of capsule expression in vivo was found to be associated with the importation of one or two aliB homologues in some nonencapsulated pneumococci.
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176. |
Hill JE,
Penny SL,
Crowell KG,
Goh SH,
Hemmingsen SM,
( 2004 ) cpnDB: a chaperonin sequence database. PMID : 15289485 : DOI : 10.1101/gr.2649204 PMC : PMC509277 Abstract >>
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.
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177. |
Baek JY,
Ko KS,
Oh WS,
Jung SI,
Kim YS,
Chang HH,
Lee H,
Kim SW,
Peck KR,
Lee NY,
Song JH,
( 2004 ) Unique variations of pbp2b sequences in penicillin-nonsusceptible Streptococcus pneumoniae isolates from Korea. PMID : 15071038 : DOI : 10.1128/jcm.42.4.1746-1750.2004 PMC : PMC387593 Abstract >>
pbp2b gene alterations were analyzed in 102 clinical isolates of Streptococcus pneumoniae (30 penicillin susceptible, 23 intermediate, and 49 resistant) from Korea. On the basis of PBP2B amino acid sequences, penicillin-nonsusceptible isolates of S. pneumoniae belonged to six groups, and 76% of the isolates in groups I to IV showed the same divergent block of amino acid alterations. Thirteen isolates (group II) also possessed a divergent block that was identical to that of Streptococcus oralis. The pbp2b genes of most Korean isolates showed novel mosaic mutations due to horizontal gene transfer. The Thr252 --> Ala substitution, previously thought to be associated only with penicillin-nonsusceptible strains, was also found in three penicillin-susceptible strains. On the basis of their pbp2b nucleotide sequences, all penicillin-nonsusceptible isolates can be detected by multiplex PCR, which can be used as a novel method for detection of antibiotic-resistant pneumococcal strains in clinical specimens.
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178. |
Hamel J,
Charland N,
Pineau I,
Ouellet C,
Rioux S,
Martin D,
Brodeur BR,
( 2004 ) Prevention of pneumococcal disease in mice immunized with conserved surface-accessible proteins. PMID : 15102774 : DOI : 10.1128/iai.72.5.2659-2670.2004 PMC : PMC387903 Abstract >>
The development of a vaccine against Streptococcus pneumoniae has been complicated by the existence of at least 90 antigenically distinct capsular serotypes. Common protein-based vaccines could represent the best strategy to prevent pneumococcal infections, regardless of serotype. In the present study, the immunoscreening of an S. pneumoniae genomic library allowed the identification of a novel immune protein target, BVH-3. We demonstrate that immunization of mice with BVH-3 elicits protective immunity against experimental sepsis and pneumonia. Sequence analysis revealed that the bvh-3 gene is highly conserved within the species. Since the BVH-3 protein shows homology at its amino-terminal end with other pneumococcal proteins, it was of interest to determine if protection was due to the homologous or to the protein-specific regions. Immunoprotection studies using recombinant BVH-3 and BVH-3-related protein fragments as antigens allowed the localization of surface-exposed and protective epitopes at the protein-specific carboxyl termini, thus establishing that BVH-3 is distinct from other previously reported protective protein antigens. Immunization with a chimeric protein comprising the carboxyl-terminal regions of BVH-3 and of a BVH-3-related protein improved the protection by targeting two surface pneumococcal components. Thus, BVH-3 and the chimeric protein hold strong promise as vaccine components to control pneumococcal disease.
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179. |
Bent CJ,
Isaacs NW,
Mitchell TJ,
Riboldi-Tunnicliffe A,
( 2004 ) Crystal structure of the response regulator 02 receiver domain, the essential YycF two-component system of Streptococcus pneumoniae in both complexed and native states. PMID : 15090529 : DOI : 10.1128/jb.186.9.2872-2879.2004 PMC : PMC387779 Abstract >>
A variety of bacterial cellular responses to environmental signals are mediated by two-component signal transduction systems comprising a membrane-associated histidine protein kinase and a cytoplasmic response regulator (RR), which interpret specific stimuli and produce a measured physiological response. In RR activation, transient phosphorylation of a highly conserved aspartic acid residue drives the conformation changes needed for full activation of the protein. Sequence homology reveals that RR02 from Streptococcus pneumoniae belongs to the OmpR subfamily of RRs. The structures of the receiver domains from four members of this family, DrrB and DrrD from Thermotoga maritima, PhoB from Escherichia coli, and PhoP from Bacillus subtilis, have been elucidated. These domains are globally very similar in that they are composed of a doubly wound alpha(5)beta(5); however, they differ remarkably in the fine detail of the beta4-alpha4 and alpha4 regions. The structures presented here reveal a further difference of the geometry in this region. RR02 is has been shown to be the essential RR in the gram-positive bacterium S. pneumoniae R. Lange, C. Wagner, A. de Saizieu, N. Flint, J. Molnos, M. Stieger, P. Caspers, M. Kamber, W. Keck, and K. E. Amrein, Gene 237:223-234, 1999; J. P. Throup, K. K. Koretke, A. P. Bryant, K. A. Ingraham, A. F. Chalker, Y. Ge, A. Marra, N. G. Wallis, J. R. Brown, D. J. Holmes, M. Rosenberg, and M. K. Burnham, Mol. Microbiol. 35:566-576, 2000). RR02 functions as part of a phosphotransfer system that ultimately controls the levels of competence within the bacteria. Here we report the native structure of the receiver domain of RR02 from serotype 4 S. pneumoniae (as well as acetate- and phosphate-bound forms) at different pH levels. Two native structures at 2.3 A, phased by single-wavelength anomalous diffraction (xenon SAD), and 1.85 A and a third structure at pH 5.9 revealed the presence of a phosphate ion outside the active site. The fourth structure revealed the presence of an acetate molecule in the active site.
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180. |
Wells J,
Gigliotti F,
Simpson-Haidaris PJ,
Haidaris CG,
( 2004 ) Epitope mapping of a protective monoclonal antibody against Pneumocystis carinii with shared reactivity to Streptococcus pneumoniae surface antigen PspA. PMID : 14977961 : DOI : 10.1128/iai.72.3.1548-1556.2004 PMC : PMC356052 Abstract >>
Pneumocystis carinii is an opportunistic fungal pathogen that causes pneumonia in the immunocompromised host. A protective monoclonal antibody (MAb) termed 4F11 generated against mouse-derived P. carinii was shown by indirect immunofluorescence assay (IFA) to bind surface antigens of P. carinii derived from multiple host species, including humans. We have identified multiple epitopes recognized by MAb 4F11 in two recombinant mouse P. carinii antigens. The epitopes mapped have similar proline content and positive charge distribution. The consensus 8-mer epitope recognized by MAb 4F11 is K/RPA/RPK/QPA/TP. Immune sera raised against intact mouse P. carinii recognized native antigens affinity purified with MAb 4F11 and a recombinant antigen reactive with MAb 4F11. Database searches for short, nearly exact matches to the mapped MAb 4F11 epitopes identified a bacterial surface antigen, Streptococcus pneumoniae PspA, with a similar proline-rich region. In an IFA, MAb 4F11 detected antigens on the S. pneumoniae surface, and Western blotting identified a protein in S. pneumoniae lysates consistent with the M(r) of PspA. A fragment of the S. pneumoniae PspA gene was cloned and sequenced, and the deduced amino acid sequence contained a region with strong similarity to the MAb 4F11 epitopes identified in P. carinii. The PspA recombinant polypeptide was recognized by MAb 4F11 in a Western blot. The ability of MAb 4F11 to recognize similar proline-rich epitopes may explain its ability to recognize P. carinii derived from multiple hosts and will permit testing of the epitopes recognized by this antibody in immunization against P. carinii.
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181. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
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182. |
Pernot L,
Chesnel L,
Le Gouellec A,
Croizé J,
Vernet T,
Dideberg O,
Dessen A,
( 2004 ) A PBP2x from a clinical isolate of Streptococcus pneumoniae exhibits an alternative mechanism for reduction of susceptibility to beta-lactam antibiotics. PMID : 14734544 : DOI : 10.1074/jbc.M313492200 Abstract >>
The human pathogen Streptococcus pneumoniae is one of the main causative agents of respiratory tract infections. At present, clinical isolates of S. pneumoniae often exhibit decreased susceptibility toward beta-lactams, a phenomenon linked to multiple mutations within the penicillin-binding proteins (PBPs). PBP2x, one of the six PBPs of S. pneumoniae, is the first target to be modified under antibiotic pressure. By comparing 89 S. pneumoniae PBP2x sequences from clinical and public data bases, we have identified one major group of sequences from drug-sensitive strains as well as two distinct groups from drug-resistant strains. The first group includes proteins that display high similarity to PBP2x from the well characterized resistant strain Sp328. The second group includes sequences in which a signature mutation, Q552E, is found adjacent to the third catalytic motif. In this work, a PBP2x from a representative strain from the latter group (S. pneumoniae 5259) was biochemically and structurally characterized. Phenotypical analyses of transformed pneumococci show that the Q552E substitution is responsible for most of the reduction of strain susceptibility toward beta-lactams. The crystal structure of 5259-PBP2x reveals a change in polarity and charge distribution around the active site cavity, as well as rearrangement of strand beta3, emulating structural changes observed for other PBPs that confer drug resistance to Gram-positive pathogens. Interestingly, the active site of 5259-PBP2x is in closed conformation, whereas that of Sp328-PBP2x is open. Consequently, S. pneumoniae has evolved to employ the same protein in two distinct mechanisms of antibiotic resistance.
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183. |
McEllistrem MC,
Noller AC,
Visweswaran S,
Adams JM,
Harrison LH,
( 2004 ) Serotype 14 variants of the France 9V(-3) clone from Baltimore, Maryland, can be differentiated by the cpsB gene. PMID : 14715761 : DOI : 10.1128/jcm.42.1.250-256.2004 PMC : PMC321660 Abstract >>
European serotype 14 variants of the France 9V(-3) clone, which have arisen through recombination events involving the penicillin binding protein 1a (pbp1a) gene, have cpsB sequences distinct from those of the 9V(-3) clone. Serotype 14 variants of the 9V(-3) clone have not been compared to genetically diverse serotype 14 strains isolated from an entire metropolitan area in the United States. All serotype 14 non-penicillin-susceptible Streptococcus pneumoniae strains causing invasive disease in Baltimore, Md., from 1995 to 1996 were compared by using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), pbp1a PCR restriction profiles, and cpsB and pbp1a sequences. The cpsB genes from strains of 13 serotypes also were analyzed to assess the correlation with serotype. Twenty-seven percent (3 of 11) of the serotype 14 strains were related by PFGE and MLST to the 9V(-3) clone. The serotype 14 variants from Baltimore, unlike the European variants, were related neither to the 9V(-3) clone nor to the R6 strain from positions 1498 to 1710 of the pbp1a gene. All serotype 14 strains had cpsB sequences that differed by
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184. |
Kong F,
Gilbert GL,
( 2003 ) Using cpsA-cpsB sequence polymorphisms and serotype-/group-specific PCR to predict 51 Streptococcus pneumoniae capsular serotypes. PMID : 14614062 : DOI : 10.1099/jmm.0.05277-0 Abstract >>
Streptococcus pneumoniae polysaccharide and protein-conjugate vaccines are available against the most commonly isolated pneumococcal serotypes. Ongoing surveillance of invasive pneumococcal disease is needed in order to monitor changes in distribution of serotypes. Based on previously published sequences of capsular polysaccharide synthesis (cps) gene clusters of 16 pneumococcal serotypes, a molecular capsular typing (MCT) system has been developed, based on a combination of partial cpsA-cpsB sequencing and serotype- or serogroup-specific PCR, targeting the genes wzy and wzx (except for serotype 3). In this study, 151 S. pneumoniae isolates of known serotype (representing 51 serotypes) and 276 recent clinical isolates were used to develop MCT and compare it with conventional serotyping (CS) (total 427 isolates). On the basis of 376 heterogeneity sites in the cpsA-cpsB region, 89 sequence types (ST) were identified, of which 76 corresponded to a single serotype and 11 contained two serotypes. The correct serotypes in two of the latter (10A-23F-g and 23F-23A) were identified using serotype 23F-specific PCR. Limited CS was required for 92 (22 %) isolates to distinguish between the two serotypes in the nine other mixed ST (6A-6B-g, 6A-6B-q, 15B-22F, 33F-33A, 17F-35B, 18B-18C, 13-20, 25F-38, 31-42). MCT is a specific, objective and practical method that can predict the serotype of most S. pneumoniae isolates; it will facilitate epidemiological studies. Further study of the relationship between MCT and CS is needed in order to improve our understanding of serotype differentiation and to improve MCT methods further.
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185. |
Calix JJ,
Oliver MB,
Sherwood LK,
Beall BW,
Hollingshead SK,
Nahm MH,
( 2011 ) Streptococcus pneumoniae serotype 9A isolates contain diverse mutations to wcjE that result in variable expression of serotype 9V-specific epitope. PMID : 21908730 : DOI : 10.1093/infdis/jir593 PMC : PMC3222105 Abstract >>
Streptococcus pneumoniae is a significant pathogen capable of expressing protective and antigenically diverse capsules. To better understand the molecular basis of capsular antigenic diversity, we investigated the hypothetical serological role of wcjE, which encodes a capsule O-acetyltransferase, in the vaccine-targeted serotype 9V and related serotype 9A. We inactivated wcjE by recombination in a serotype 9V strain and determined wcjE sequences of 11 serotype 9A clinical isolates. We determined the antigenic phenotypes of these pneumococcal strains with serogroup 9-specific antibodies and flow cytometry. Inactivation of wcjE in a serotype 9V strain resulted in expression of the 9A phenotype. Each serotype 9A clinical isolate contained a distinct mutation to wcjE. Flow cytometry showed that some 9A isolates (herein named 9A�\) expressed trace amounts of 9V-specific epitopes whereas others (named 9A�]) did not express any. Recombination with 9A�\ wcjE alleles into a 9A�] strain conferred partial expression of 9V-specific epitopes. Each serotype 9A strain independently arose from a serotype 9V strain. Furthermore, clinical isolates identified as 9A can contain mutations to wcjE that are either partially functional or completely nonfunctional, demonstrating a previously unidentified antigenic heterogeneity of serotype 9A isolates.
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186. |
Hernani Mde L,
Ferreira PC,
Ferreira DM,
Miyaji EN,
Ho PL,
Oliveira ML,
( 2011 ) Nasal immunization of mice with Lactobacillus casei expressing the pneumococcal surface protein C primes the immune system and decreases pneumococcal nasopharyngeal colonization in mice. PMID : 21492260 : DOI : 10.1111/j.1574-695X.2011.00809.x Abstract >>
Streptococcus pneumoniae colonizes the upper respiratory tract of healthy individuals, from where it can be transmitted to the community. Occasionally, bacteria invade sterile niches, causing diseases. The pneumococcal surface protein C (PspC) is a virulence factor that is important during colonization and the systemic phases of the diseases. Here, we have evaluated the effect of nasal or sublingual immunization of mice with Lactobacillus casei expressing PspC, as well as prime-boosting protocols using recombinant PspC, on nasopharyngeal pneumococcal colonization. None of the protocols tested was able to elicit significant levels of anti-PspC antibodies before challenge. However, a significant decrease in pneumococcal recovery from the nasopharynx was observed in animals immunized through the nasal route with L. casei-PspC. Immune responses evaluated after colonization challenge in this group of mice were characterized by an increase in mucosal anti-PspC immunoglobulin A (IgA) 5 days later, a time point in which the pneumococcal loads were already low. A negative correlation between the concentrations of anti-PspC IgA and pneumococcal recovery from the nasopharynx was observed, with animals with the lowest colonization levels having higher IgA concentrations. These results show that nasal immunization with L. casei-PspC primes the immune system of mice, prompting faster immune responses that result in a decrease in pneumococcal colonization.
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187. |
Harvey RM,
Ogunniyi AD,
Chen AY,
Paton JC,
( 2011 ) Pneumolysin with low hemolytic activity confers an early growth advantage to Streptococcus pneumoniae in the blood. PMID : 21788389 : DOI : 10.1128/IAI.05418-11 PMC : PMC3187248 Abstract >>
Streptococcus pneumoniae is a leading cause of human diseases such as pneumonia, bacteremia, meningitis, and otitis media. Pneumolysin (Ply) is an important virulence factor of S. pneumoniae and a promising future vaccine target. However, the expansion of clones carrying ply alleles with reduced hemolytic activity has been observed in serotypes associated with outbreaks of invasive disease and includes an allele identified in a highly virulent serotype 1 isolate (ply4496). The virulence of Ply-deficient and ply allelic-replacement derivatives of S. pneumoniae D39 was compared with that of wild-type D39. In addition, the protective immunogenicity of Ply against pneumococci with low versus high hemolytic activity was also investigated. Replacement of D39 ply with ply4496 resulted in a small but statistically significant reduction of virulence. However, both native Ply- and Ply4496-expressing strains were significantly more virulent than a Ply-deficient mutant. While the numbers of both Ply- and Ply4496-expressing isolate cells were higher in the blood than the numbers of Ply-deficient mutant cells, the growth of the Ply4496-expressing strain was superior to that of the wild type in the first 15 h postchallenge. Ply immunization provided protection regardless of the hemolytic activity of the challenge strain. In summary, we show that low-hemolytic-activity Ply alleles contribute to systemic virulence and may provide a survival advantage in the blood. Moreover, pneumococci expressing such alleles remain vulnerable to Ply-based vaccines.
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188. |
Song JH,
Baek JY,
Ko KS,
( 2011 ) Comparison of capsular genes of Streptococcus pneumoniae serotype 6A, 6B, 6C, and 6D isolates. PMID : 21411593 : DOI : 10.1128/JCM.02628-10 PMC : PMC3122669 Abstract >>
Recently, Streptococcus pneumoniae serotypes 6C and 6D have been identified. It is thought that they emerged by the replacement of wciN(�]) in the capsular loci of serotypes 6A and 6B, respectively. However, their evolution has not been unveiled yet. To investigate the evolution of four serotypes of S. pneumoniae serogroup 6, four genes of the capsular polysaccharide synthesis (cps) locus, wchA, wciN, wciO, and wciP, of isolates of S. pneumoniae serotypes 6A, 6B, 6C, and 6D were sequenced. Multilocus sequence typing (MLST) was performed to investigate their genetic backgrounds. The wchA gene of serotype 6C and 6D isolates was distinct from that of serotype 6A and 6B isolates, which may suggest cotransfer of wchA with wciN(�]). Otherwise, serotypes 6C and 6D displayed different genetic backgrounds from serotypes 6A and 6B, which was suggested by MLST analysis. In addition, serotype 6C isolates showed distinct wciP polymorphisms from other serotypes, which also indicated that serotype 6C had not recently originated from serotype 6A. Although serotype 6D shared the same amino acid polymorphisms of wciO with serotype 6B, wciP of serotype 6D differed from that of serotype 6B. The data indicate the implausibility of the scenario of a recent emergence of the cps locus of serotype 6D by genetic recombination between serotypes 6B and 6C. In addition, five serotype 6A and 6B isolates (6X group) displayed cps loci distinct from those of other isolates. The cps locus homogeneity and similar sequence types in MLST analysis suggest that most of the 6X group of isolates originated from the same ancestor and that the entire cps locus might have recently been transferred from an unknown origin. Serotype 6B isolates showed two or more cps locus subtypes, indicating a recombination-mediated mosaic structure of the cps locus of serotype 6B. The collective data favor the emergence of cps loci of serotypes 6A, 6B, 6C, and 6D by complicated recombination.
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189. |
Croucher NJ,
Harris SR,
Fraser C,
Quail MA,
Burton J,
van der Linden M,
McGee L,
von Gottberg A,
Song JH,
Ko KS,
Pichon B,
Baker S,
Parry CM,
Lambertsen LM,
Shahinas D,
Pillai DR,
Mitchell TJ,
Dougan G,
Tomasz A,
Klugman KP,
Parkhill J,
Hanage WP,
Bentley SD,
( 2011 ) Rapid pneumococcal evolution in response to clinical interventions. PMID : 21273480 : DOI : 10.1126/science.1198545 PMC : PMC3648787 Abstract >>
Epidemiological studies of the naturally transformable bacterial pathogen Streptococcus pneumoniae have previously been confounded by high rates of recombination. Sequencing 240 isolates of the PMEN1 (Spain(23F)-1) multidrug-resistant lineage enabled base substitutions to be distinguished from polymorphisms arising through horizontal sequence transfer. More than 700 recombinations were detected, with genes encoding major antigens frequently affected. Among these were 10 capsule-switching events, one of which accompanied a population shift as vaccine-escape serotype 19A isolates emerged in the USA after the introduction of the conjugate polysaccharide vaccine. The evolution of resistance to fluoroquinolones, rifampicin, and macrolides was observed to occur on multiple occasions. This study details how genomic plasticity within lineages of recombinogenic bacteria can permit adaptation to clinical interventions over remarkably short time scales.
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190. |
Totoritis R,
Duraiswami C,
Taylor AN,
Kerrigan JJ,
Campobasso N,
Smith KJ,
Ward P,
King BW,
Murrayz-Thompson M,
Jones AD,
Van Aller GS,
Aubart KM,
Zalacain M,
Thrall SH,
Meek TD,
Schwartz B,
( 2011 ) Understanding the origins of time-dependent inhibition by polypeptide deformylase inhibitors. PMID : 21711014 : DOI : 10.1021/bi200655g Abstract >>
The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes in time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss.
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191. |
Zbinden A,
Köhler N,
Bloemberg GV,
( 2011 ) recA-based PCR assay for accurate differentiation of Streptococcus pneumoniae from other viridans streptococci. PMID : 21147955 : DOI : 10.1128/JCM.01450-10 PMC : PMC3043496 Abstract >>
Proper identification of Streptococcus pneumoniae by conventional methods remains problematic. The discriminatory power of the 16S rRNA gene, which can be considered the "gold standard" for molecular identification, is too low to differentiate S. pneumoniae from closely related species such as Streptococcus pseudopneumoniae, Streptococcus mitis, and Streptococcus oralis in the routine clinical laboratory. A 313-bp part of recA was selected on the basis of variability within the S. mitis group, showing <95.8% interspecies homology. In addition, 6 signature nucleotides specific for S. pneumoniae were identified within the 313-bp recA fragment. We show that recA analysis is a useful tool for proper identification to species level within the S. mitis group, in particular, for pneumococci.
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192. |
Bonofiglio L,
Regueira M,
Pace J,
Corso A,
García E,
Mollerach M,
( 2011 ) Dissemination of an erythromycin-resistant penicillin-nonsusceptible Streptococcus pneumoniae Poland(6B)-20 clone in Argentina. PMID : 21128835 : DOI : 10.1089/mdr.2010.0027 Abstract >>
Prevalence of serotype 6B penicillin (PEN)-nonsusceptible Streptococcus pneumoniae significantly increased from 15.8% (1993-1997) to 67.3% (1998-2002) (p<0.001) in Argentina. Serogroup 6 ranks fourth among different capsular types within invasive isolates from Argentinean patients <6 years of age. To evaluate whether the increase in PEN resistance in serotype 6B pneumococci was due to the dissemination of one or more clones, the genetic diversity of 93 S. pneumoniae serotype 6B isolates was analyzed. Five BOX-polymerase chain reaction types were obtained (65.5% isolates) and a group of 15 isolates, representing 41.6% of those having a decreased susceptibility to PEN, were further characterized. The antibiotype of these isolates showed their multiresistance, with 100% of the isolates being resistant to erythromycin, 80% to tetracycline, and 73.3% to trimethoprim-sulfamethoxazole. Of the 15 isolates, 13 belonged to the same pulsed-field gel electrophoresis type and galU cluster and were members of the same clone. The identity of the clone was confirmed in four isolates by multilocus sequence typing. The sequence type found (ST315) corresponds to the Poland(6B)-20 clone. In summary, BOX-polymerase chain reaction, pulsed-field gel electrophoresis, and galU polymorphism were useful tools to detect the presence of a clone whose identity was confirmed by multilocus sequence typing. The isolates belonging to Poland(6B)-20 found in this work are described for the first time in Latin America.
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193. |
Maeda Y,
Murayama M,
Goldsmith CE,
Coulter WA,
Mason C,
Millar BC,
Dooley JS,
Lowery CJ,
Matsuda M,
Rendall JC,
Elborn JS,
Moore JE,
( 2011 ) Molecular characterization and phylogenetic analysis of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE gene loci in viridans group streptococci isolated from adult patients with cystic fibrosis. PMID : 21193474 : DOI : 10.1093/jac/dkq485 Abstract >>
Ciprofloxacin is the most frequently used member of the fluoroquinolones during initial eradication therapy of Pseudomonas aeruginosa, as well as during acute pulmonary exacerbations. However, its long-term effect on the susceptibility of the commensal flora within the cystic fibrosis (CF) airways has not yet been examined. The aim of this study was therefore to examine the consequence of oral ciprofloxacin usage on the resistance of the commensal viridans group streptococci (VGS), in terms of MICs and mutational analysis of the quinolone resistance-determining regions (QRDRs). The MICs of ciprofloxacin, efflux activities and amino acid substitutions in the QRDRs for 190 isolates of VGS, originating from the sputa of adult CF patients who had been exposed constantly to ciprofloxacin, were examined. VGS organisms included Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus infantis, Streptococcus gordonii, Streptococcus anginosus, Streptococcus cristatus, Streptococcus australis and Streptococcus mutans. Ciprofloxacin susceptibility was determined by broth microdilution and QRDRs within the gyrA, gyrB, parC and parE gene loci were explored using sequence analysis. Twenty-seven (14.2%) streptococcal isolates were resistant to ciprofloxacin (MICs ?8 mg/L) and 21 (11.1%) had reduced susceptibility (MICs 4 mg/L). As a comparator, clinically non-significant and non-invasive VGS organisms were examined in 12 consecutive non-CF patients in the community, where no resistance to ciprofloxacin was observed. Five novel QRDR PCR assays were developed to elucidate mutations within the CF VGS population, where there were six positions, which corresponded to previously reported quinolone resistance responsible mutations, and eight novel potential QRDR resistance mutations. Double mutations in gyrA and parC/parE led to MICs of 16 to >64 mg/L, while single mutations in parC or parE resulted in MICs of 8-32 mg/L and 8 mg/L, respectively. The mean homologies of each species to Streptococcus pneumoniae R6 were: gyrA, 70.3%-95%; gyrB, 69.6%-96.2%; parC, 76.1%-94.8%; and parE, 70.7%-94.7%. The close relatives of S. pneumoniae, S. mitis and S. oralis, showed high similarity for all four genes (more than 86%). Treatment of P. aeruginosa with oral ciprofloxacin in patients with CF may concurrently reduce antibiotic susceptibility in the commensal VGS flora, where these organisms may potentially act as a reservoir of fluoroquinolone resistance gene determinants for newly acquired and antibiotic-susceptible pathogens, particularly the Streptococcus milleri group.
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194. |
Maestro B,
Novaková L,
Hesek D,
Lee M,
Leyva E,
Mobashery S,
Sanz JM,
Branny P,
( 2011 ) Recognition of peptidoglycan and �]-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae. PMID : 21167155 : DOI : 10.1016/j.febslet.2010.12.016 PMC : PMC3035916 Abstract >>
The eukaryotic-type serine/threonine kinase StkP from Streptococcus pneumoniae is an important signal-transduction element that regulates the expression of numerous pneumococcal genes. We have expressed the extracellular C-terminal domain of StkP kinase (C-StkP), elaborated a three-dimensional structural model and performed a spectroscopical characterization of its structure and stability. Biophysical experiments show that C-StkP binds to synthetic samples of the cell wall peptidoglycan (PGN) and to �]-lactam antibiotics, which mimic the terminal portions of the PGN stem peptide. This is the first experimental report on the recognition of a minimal PGN unit by a PASTA-containing kinase, suggesting that non-crosslinked PGN may act as a signal for StkP function and pointing to this protein as an interesting target for �]-lactam antibiotics.
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195. |
Zhang Q,
Ma Q,
Su D,
Li Q,
Yao W,
Wang C,
( 2010 ) Identification of horizontal gene transfer and recombination of PsaA gene in streptococcus mitis group. PMID : 20536729 : DOI : 10.1111/j.1348-0421.2010.00216.x Abstract >>
Pneumococcal surface adhesin A (psaA) gene is universally confirmed as one of the Streptococcus pneumoniae adhesion genes, but it is disputed whether the psaA gene is a Streptococcus pneumoniae species-specific gene. In the present study, the presence of the psaA gene in 34 streptococcus mitis group isolates was identified by the PCR approach and a comparison of sequencing PCR products (Streptococcus pneumoniae R6 as the control strain). Also, the evolutionary scenarios of these psaA genes in these streptococcus mitis group isolates were analyzed by a phylogenetic tree based on the housekeeping genes (sodA and rnpB) and psaA genes. As a result, a high degree of conservation of open reading frame sequences in all six Streptococcus pneumoniae strains (100% similarity) and in the other species of the streptococcus mitis group (92.6-100% similarity) was revealed. Further genetics research based on housekeeping genes and psaA gene phylogenies showed that the psaA gene was of vertical inheritance only in Streptococcus pneumoniae; however, high-frequency horizontal psaA gene transfer and recombination occurred in the other species of the streptococcus mitis group. These findings confirmed that the psaA gene was not a Streptococcus pneumoniae species-specific gene, and high-frequency HGT and recombination events may explain the presence of the psaA gene in the other species of the streptococcus mitis group.
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196. |
Li Y,
Tomita H,
Lv Y,
Liu J,
Xue F,
Zheng B,
Ike Y,
( 2011 ) Molecular characterization of erm(B)- and mef(E)-mediated erythromycin-resistant Streptococcus pneumoniae in China and complete DNA sequence of Tn2010. PMID : 20961364 : DOI : 10.1111/j.1365-2672.2010.04875.x Abstract >>
To characterize the erm(B)- and mef(E)-mediated erythromycin-resistant Streptococcus pneumoniae clinical isolates obtained from ten hospitals located different cities in China. Totally 83 S. pneumoniae were collected, and eighteen representative strains of 66 strains that exhibited erythromycin resistance were used for further characterization by antibiograms, serotyping, PFGE, MLST, DNA sequencing of the macrolide-resistance elements and mapping of the elements on the chromosome. Twelve isolates showed a high-level resistance to erythromycin, and six other isolates showed a low-level resistance to erythromycin. Thirteen isolates harboured a Tn2010 transposon (26�P4 kbp) encoding the erm(B), tet(M) and mef(E) genes and were classified into three types by Tn2010 structures. The remaining five isolates harboured a Tn6002 transposon (20�P9 kbp) encoding the erm(B) and tet(M) genes and were classified into three types by Tn6002 locations on the chromosome. Three of the Tn6002 elements were located within the Tn5252-like element, implying that these composed a large mobile element. The MLST analyses showed that several clones had been disseminated and that the CC271 strains carrying the Tn2010 element expressing the high-level resistance to erythromycin were predominant in China. Four new MLST strains, which were designated as ST3262, ST3263, ST3397 and ST3398 were also identified. The erythromycin resistance determinant of S.pneumoniae that had been isolated in China was located in Tn2010 or the Tn6002 element and several clones had been disseminated, and the CC271 strains carrying the Tn2010 element expressing the high-level resistance to erythromycin were predominant in China. This is the first molecular analysis of erythromycin-resistant Streptococcus pneumoniae clinical isolates in China, and the first report of the complete nucleotide sequence of Tn2010 (26,390 bp).
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197. |
Nahm MH,
Oliver MB,
Siira L,
Kaijalainen T,
Lambertsen LM,
Virolainen A,
( 2011 ) A report of Streptococcus pneumoniae serotype 6D in Europe. PMID : 20829399 : DOI : 10.1099/jmm.0.023853-0 PMC : PMC3052471 Abstract >>
Serotype 6D of Streptococcus pneumoniae has been reported in Asia and the Fijian islands among nasopharyngeal carriage isolates. We now report a 6D isolate from a Finnish adult with invasive pneumococcal disease. Interestingly, the Finnish isolate and Asian isolate capsule gene loci are almost identical.
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198. |
Bratcher PE,
Park IH,
Oliver MB,
Hortal M,
Camilli R,
Hollingshead SK,
Camou T,
Nahm MH,
( 2011 ) Evolution of the capsular gene locus of Streptococcus pneumoniae serogroup 6. PMID : 20929956 : DOI : 10.1099/mic.0.043901-0 PMC : PMC3068628 Abstract >>
Streptococcus pneumoniae expressing serogroup 6 capsules frequently causes pneumococcal infections and the evolutionary origins of the serogroup 6 strains have been extensively studied. However, these studies were performed when serogroup 6 had only two known members (serotypes 6A and 6B) and before the two new members (serotypes 6C and 6D) expressing wciN(�]) were found. We have therefore reinvestigated the evolutionary origins of serogroup 6 by examining the profiles of the capsule gene loci and the multilocus sequence types (MLSTs) of many serogroup 6 isolates from several continents. We confirmed that there are two classes of cps locus sequences for serogroup 6 isolates. In our study, class 2 cps sequences were limited to a few serotype 6B isolates. Neighbour-joining analysis of cps sequence profiles showed a distinct clade for 6C and moderately distinct clades for class 1 6A and 6B sequences. The serotype 6D cps profile was found within the class 1 6B clade, suggesting that it was created by recombination between 6C and 6B cps loci. Interestingly, all 6C isolates also had a unique wzy allele with a 6 bp deletion. This suggests that serotype switching to 6C involves the transfer of a large (>4 kb) gene segment that includes both the wciN(�]) allele and the 'short' wzy allele. The MLST studies of serotype 6C isolates suggest that the 6C cps locus is incorporated into many different pneumococcal genomic backgrounds but that, interestingly, 6C cps may have preferentially entered strains of the same genomic backgrounds as those of serotype 6A.
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199. |
Ho PL,
Ang I,
Chow KH,
Lai EL,
Chiu SS,
( 2010 ) The prevalence and characteristics of Streptococcus pneumoniae isolates expressing serotypes 6C and 6D in Hong Kong prior to the introduction of the 7-valent pneumococcal conjugate vaccine. PMID : 20926218 : DOI : 10.1016/j.diagmicrobio.2010.07.020 Abstract >>
A study was conducted to determine the prevalence of the 2 newly described types, 6C and 6D, among pneumococcal isolates collected in Hong Kong before availability of the 7-valent pneumococcal conjugate vaccine. A total of 154 serogroup 6 isolates obtained from nasopharynx (n = 106), blood (n = 22), respiratory (n = 24), and cerebrospinal fluid (CSF) (n = 2) during 1995 to 2001 were analyzed by polymerase chain reaction typing. Five nasopharyngeal and 2 sputum isolates were found to belong to 6C and 6D, respectively. The isolates were genetically diverse, but one 6C and two 6D isolates exhibited some clonal relationship. Phylogenetic analysis of the wchA-wciN(�])-wciO nucleotide sequences showed that the Hong Kong 6C/6D isolates had 2 allelic profiles, which were more closely related to 6C/6D isolates from Fijian and Korea than were those from Brazil and the United States. However, all of the wciP gene sequences for both Hong Kong and non-Hong Kong isolates clustered together: 6C isolates with the wciP-9 allele and 6D isolates with the wciP-5 allele. In conclusion, the prevalence of the 2 newly described serotypes was low before the era of the pneumococcal conjugate vaccine. Nonetheless, results from the molecular studies indicated that the evolution of the capsular genes have involved complex pathways.
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200. |
Sheppard CL,
Pichon B,
George RC,
Hall LM,
( 2010 ) Streptococcus pneumoniae isolates expressing a capsule with epitopes of both serotypes 6A and 6B. PMID : 20876824 : DOI : 10.1128/CVI.00335-10 PMC : PMC2976087 Abstract >>
Four Streptococcus pneumoniae isolates expressing both 6A and 6B capsular serotypes were detected by a multiplex immunoassay. The sequence of WciP, a GT2-family glycosyltransferase, indicates that point mutation has compromised linkage specificity, allowing two alternative oligosaccharides to be synthesized. This finding highlights that mutation as well as recombination can mediate serotype change.
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201. |
Zhuo F,
Xiao M,
Kong F,
Oftadeh S,
Zhou F,
Zhang J,
Gilbert GL,
( 2011 ) Prevalence and genetic diversity of pneumococcal serogroup 6 in Australia. PMID : 20950338 : DOI : 10.1111/j.1469-0691.2010.03404.x Abstract >>
The prevalence of the newly discovered pneumococcal serotype 6C has increased in some countries since the introduction of seven-valent conjugate pneumococcal vaccine (PCV7). The distribution of invasive serogroup 6 serotypes, in Australia, including 6C and 6D, has not been reported previously. During the period 1999 to 2008, 6097 isolates were referred to the New South Wales Pneumococcal Reference Laboratory for serotyping. Of these, 847 were identified by Quellung reaction as belonging to serogroup 6 and 702 were available for further study. Serotypes were determined by serotype-specific PCR as follows: 6A, 197 (28.1%); 6B, 452 (64.4%); 6C, 52 (7.4%) and one 6D. The average numbers of invasive serogroup 6 isolates, per annum, fell from 62.2 before (2000-2005) to 49.7 after (2006-2008) the introduction of PCV7. The proportions of invasive 6B fell (from 72.4% to 47.3%, p 0.03), those of 6C rose (from 3.3% to 17%, p 0.02) significantly and those of 6A remained fairly constant (24.3% vs 27%, p 0.69) between the two periods. All 6C and 6D and selected 6A and 6B isolates were further characterized by multilocus sequence typing and sequence analysis of cps genes cpsA-cpsB (wzg-wzh) and wchA-wciN(beta) -wciO, wciP. Results showed considerable diversity within serotype 6C, apparently as a result of both mutation and recombination. Sequence typing indicates that, in Australia, 6C has been largely derived from 6A. The genetic diversity and rapid increase in incidence of serotype 6C causing invasive pneumococcal disease has potential implications for vaccine efficacy.
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202. |
Santoro F,
Oggioni MR,
Pozzi G,
Iannelli F,
( 2010 ) Nucleotide sequence and functional analysis of the tet (M)-carrying conjugative transposon Tn5251 of Streptococcus pneumoniae. PMID : 20487027 : DOI : 10.1111/j.1574-6968.2010.02002.x Abstract >>
The Tn916-like genetic element Tn5251 is part of the composite conjugative transposon (CTn) Tn5253 of Streptococcus pneumoniae, a 64.5-kb chromosomal element originally called Omega(cat-tet) BM6001. DNA sequence analysis showed that Tn5251 is 18 033-bp long and contains 22 ORFs, 20 of which have the same direction of transcription. Annotation was possible for 11 out of 22 ORFs, including the tet(M) tetracycline resistance gene and int and xis involved in the integration/excision process. Autonomous copies of Tn5251 were generated during matings of Tn5253-containing donors with S. pneumoniae and Enterococcus faecalis. Tn5251 was shown to integrate at different sites in the bacterial chromosome. It behaves as a fully functional CTn capable of independent conjugal transfer to a variety of bacterial species including S. pneumoniae, Streptococcus gordonii, Streptococcus pyogenes, Streptococcus agalactiae, E. faecalis and Bacillus subtilis. The excision of Tn5251 produces a circular intermediate and a deletion in Tn5253 at a level of 1.2 copies per 10(5) chromosomes.
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203. |
Calix JJ,
Nahm MH,
( 2010 ) A new pneumococcal serotype, 11E, has a variably inactivated wcjE gene. PMID : 20507232 : DOI : 10.1086/653123 PMC : PMC2880655 Abstract >>
Recently, 2 serologically and biochemically distinct subtypes-11Aalpha and 11Abeta-were discovered among serotype 11A isolates of Streptococcus pneumoniae. Sequence comparison of the capsular polysaccharide synthesis (cps) loci of the 2 subtypes identified disruption of the wcjE gene, a putative O-acetyltransferase, as the genetic hallmark of the 11Abeta phenotype. Directed disruption of wcjE in vitro in an 11Aalpha strain switched the strain to the 11Abeta phenotype, confirming the role played by the gene in the divergence between the subtypes. Furthermore, sequences from 7 11Abeta clinical strains each contained unrelated disruptive mutations in the wcjE gene, displaying an unprecedented degree of genetic heterogeneity in a pneumococcal serotype. We propose to name the 11Aalpha subtype as serotype 11A and the 11Abeta subtype as 11E, a new serotype. Our findings also suggest that the diversity of pneumococcal capsules is much greater than was previously recognized.
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204. |
Jefferies JM,
Tocheva AS,
Rubery H,
Bennett J,
Garland J,
Christodoulides M,
Faust SN,
Smith A,
Mitchell TJ,
Clarke SC,
( 2010 ) Identification of novel pneumolysin alleles from paediatric carriage isolates of Streptococcus pneumoniae. PMID : 20339017 : DOI : 10.1099/jmm.0.018663-0 Abstract >>
Pneumolysin (Ply) is a major virulence factor of Streptococcus pneumoniae and is produced by all known clinical isolates of pneumococci. Pneumolysin toxoids are being considered as vaccine candidates. We investigated the diversity of pneumolysin among 194 nasopharyngeal pneumococci characterized by serotyping and multilocus sequence typing (MLST). Eight Ply protein alleles were identified, four of which were novel. The 4 novel alleles varied at 10 different amino acid positions, from a total of 147, 3 of these substitutions have been previously reported in different combinations. The protein allele correlated closely with MLST. It is critical that the presence of pneumolysin variants is considered with regards to the potential use of Ply in future vaccine formulations, as variation in Ply amino acid sequence may influence the immunogenicity of vaccines based on the presence of an individual Ply allele.
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205. |
Kuch A,
Sadowy E,
Skoczy?ska A,
Hryniewicz W,
( 2010 ) First report of Streptococcus pneumoniae serotype 6D isolates from invasive infections. PMID : 20674875 : DOI : 10.1016/j.vaccine.2010.07.051 Abstract >>
We report the first invasive Streptococcus pneumoniae isolates of serotype 6D and the first occurrence of this serotype in Europe. Till now, the appearance of serotype 6D pneumococci in nasopharyngeal carriage has been speculated to be associated with a selective pressure from vaccination with the conjugated 7-valent antipneumococcal vaccine. Our observations indicate that this serotype is present also among unvaccinated individuals in the population where mass infant vaccination has not yet been introduced. Importantly, these strains were isolated from invasive infections, indicating the full virulence potential of pneumococci belonging to serotype 6D and its importance for future vaccine formulations.
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206. |
Nováková L,
Bezousková S,
Pompach P,
Spidlová P,
Sasková L,
Weiser J,
Branny P,
( 2010 ) Identification of multiple substrates of the StkP Ser/Thr protein kinase in Streptococcus pneumoniae. PMID : 20453092 : DOI : 10.1128/JB.01564-09 PMC : PMC2897338 Abstract >>
Monitoring the external environment and responding to its changes are essential for the survival of all living organisms. The transmission of extracellular signals in prokaryotes is mediated mainly by two-component systems. In addition, genomic analyses have revealed that many bacteria contain eukaryotic-type Ser/Thr protein kinases. The human pathogen Streptococcus pneumoniae encodes 13 two-component systems and has a single copy of a eukaryotic-like Ser/Thr protein kinase gene designated stkP. Previous studies demonstrated the pleiotropic role of the transmembrane protein kinase StkP in pneumococcal physiology. StkP regulates virulence, competence, and stress resistance and plays a role in the regulation of gene expression. To determine the intracellular signaling pathways controlled by StkP, we used a proteomic approach for identification of its substrates. We detected six proteins phosphorylated on threonine by StkP continuously during growth. We identified three new substrates of StkP: the Mn-dependent inorganic pyrophosphatase PpaC, the hypothetical protein spr0334, and the cell division protein DivIVA. Contrary to the results of a previous study, we did not confirm that the alpha-subunit of RNA polymerase is a target of StkP. We showed that StkP activation and substrate recognition depend on the presence of a peptidoglycan-binding domain comprising four extracellular penicillin-binding protein- and Ser/Thr kinase-associated domain (PASTA domain) repeats. We found that StkP is regulated in a growth-dependent manner and likely senses intracellular peptidoglycan subunits present in the cell division septa. In addition, stkP inactivation results in cell division defects. Thus, the data presented here suggest that StkP plays an important role in the regulation of cell division in pneumococcus.
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207. |
Sadowy E,
Kuch A,
Gniadkowski M,
Hryniewicz W,
( 2010 ) Expansion and evolution of the Streptococcus pneumoniae Spain9V-ST156 clonal complex in Poland. PMID : 20194703 : DOI : 10.1128/AAC.01340-09 PMC : PMC2863602 Abstract >>
In this study, we analyzed 118 penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) isolates (MICs, >or=0.12 microg/ml) recovered in Poland in 2003 to 2005 from patients with respiratory tract diseases and invasive infections. Seven different serotypes (14, 9V, 23F, 19F, 6B, 19A, and 6A, in order of descending frequency), seven alleles of the murM gene (murMA, murMB6, and the new murMB12 to -16 alleles), and 31 multilocus sequence types (STs) were observed. The vast majority of the PNSP isolates (90.7%) belonged to the international multiresistant clones, and among these, the Spain(9V)-ST156 clonal complex was the most prevalent (56 isolates) and was significantly overrepresented in invasive infections. The clone has been evolving rapidly, as demonstrated by the observed number of STs, the diversity in multiple-locus variable-number-tandem-repeat analysis (MLVA) types, and the polymorphism of pbp and pspA genes (coding for penicillin-binding proteins and the pneumococcal surface protein A, respectively). The presence and structure of the rlrA islet (encoding the pneumococcal pilus) were very well conserved. The Spain(9V)-ST156 clonal complex has been largely responsible for a decreasing susceptibility to penicillin among pneumococci in Poland in recent years, in spite of a relatively moderate antimicrobial use.
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208. |
Olson AB,
Sibley CD,
Schmidt L,
Wilcox MA,
Surette MG,
Corbett CR,
( 2010 ) Development of real-time PCR assays for detection of the Streptococcus milleri group from cystic fibrosis clinical specimens by targeting the cpn60 and 16S rRNA genes. PMID : 20164275 : DOI : 10.1128/JCM.02082-09 PMC : PMC2849594 Abstract >>
Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of the Streptococcus anginosus group (S. anginosus, S. constellatus, and S. intermedius), herein referred to as the "Streptococcus milleri group" (SMG) have been implicated as important etiological pathogens contributing to pulmonary exacerbations in CF patients. This is partly due to better microbiological detection of the SMG species through the development of a novel specific medium termed "McKay agar." McKay agar demonstrated that SMG has been an underreported respiratory pathogen contributing to lung exacerbations. Our aim was to develop a real-time PCR assay to expedite the detection of SMG within diagnostic samples. The cpn60 gene was chosen as a target, with all three members amplified using a single hybridization probe set. SMG strain analysis showed that speciation based on melting curve analysis allowed for the majority of the S. constellatus (96%), S. intermedius (94%), and S. anginosus (60%) strains to be correctly identified. To increase specificity for S. anginosus, two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific for S. anginosus (100%), while the 16s_SCI assay is specific for S. constellatus and S. intermedius (100%). These assays can detect <10 genome equivalents in pure culture and >10(4) genome equivalents in sputum samples, making this a great tool for assessment of the presence of SMG in complex polymicrobial samples. Novel molecular methods were developed providing detection ability for SMG, an emerging opportunistic pathogen.
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209. |
Cornejo OE,
McGee L,
Rozen DE,
( 2010 ) Polymorphic competence peptides do not restrict recombination in Streptococcus pneumoniae. PMID : 19942613 : DOI : 10.1093/molbev/msp287 Abstract >>
Understanding the factors that limit recombination in bacteria is critical in order to better understand and assess its effects on genetic variation and bacterial population genetic structure. Transformation in the naturally competent bacterium, Streptococcus pneumoniae, is regulated by a polymorphic competence (com) apparatus. It has been suggested that polymorphic types, called pherotypes, generate and maintain subpopulation genetic structure within this species. We test predictions stemming from this hypothesis using a cosmopolitan sample of clinical pneumococcal isolates. We sequenced the locus encoding the peptide that induces competence (comC) to assign clones to each known pherotype class and then used multilocus sequence typing to determine whether there is significant genetic differentiation between pherotypes subgroups. We find two dominant pherotypes within our sample, and both are maintained at high frequencies (CSP1 74%, CSP2 26%). Our analyses fail to detect significant genetic differentiation between pherotype groups and find strong evidence, from a coalescent analysis, for interpherotype recombination. In addition, our analyses indicate that positive selection may account for the maintenance of the fixed polymorphism in this locus (comC). Altogether, these results fail to support the prediction that the polymorphism in the competence system acts to limit recombination within S. pneumoniae populations. We discuss why this result is expected given the mechanism underlying transformation and outline a scenario to explain the evolution of polymorphism in the competence system.
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210. |
Sibold C,
Markiewicz Z,
Latorre C,
Hakenbeck R,
( 1991 ) Novel plasmids in clinical strains of Streptococcus pneumoniae. PMID : 2004700 : DOI : 10.1016/0378-1097(91)90019-7 Abstract >>
Small plasmids were found in two clinical isolates of Streptococcus pneumoniae from Spain (strains 671 and 678) and in one strain (SpR) isolated in Germany. All three strains contained one plasmid (2.75 to 3.1 kb) which is related to the only previously described pneumococcal plasmid, pDP1. Strains 678 and SpR carried a second plasmid of 2.6 kb and 2.7 kb, respectively. These two plasmids hybridized neither with each other, nor with pDP1, demonstrating that they represent new types of plasmids not having been found in pneumococci before.
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211. |
Glazunova OO,
Raoult D,
Roux V,
( 2009 ) Partial sequence comparison of the rpoB, sodA, groEL and gyrB genes within the genus Streptococcus. PMID : 19620365 : DOI : 10.1099/ijs.0.005488-0 Abstract >>
Phylogenetic analysis and species identification of members of the genus Streptococcus were carried out using partial sequence comparison of the 16S rRNA gene (1468-1478 bp), rpoB, encoding the beta subunit of RNA polymerase (659-680 bp), sodA, encoding the manganese-dependent superoxide dismutase (435-462 bp), groEL, encoding the 60 kDa heat-shock protein (757 bp), and gyrB, encoding the Beta subunit of DNA gyrase (458-461 bp). For the first time, most species within the genus Streptococcus were represented in the study (65 strains, representing 58 species and nine subspecies). Phylogenies inferred from rpoB, sodA, gyrB and groEL sequence comparisons were more discriminative than those inferred from 16S rRNA gene sequence comparison, and showed common clusters. The minimal interspecies divergence was 0.3, 2.7, 0, 2.5 and 3.4 % for the 16S rRNA gene, rpoB, sodA, gyrB and groEL, respectively. In general, groEL partial gene sequence comparison represented the best tool for identifying species and subspecies and for phylogenetic analysis.
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212. |
Bratcher PE,
Kim KH,
Kang JH,
Hong JY,
Nahm MH,
( 2010 ) Identification of natural pneumococcal isolates expressing serotype 6D by genetic, biochemical and serological characterization. PMID : 19942663 : DOI : 10.1099/mic.0.034116-0 PMC : PMC2890086 Abstract >>
The recently discovered pneumococcal serotype 6C was created when the original wciN gene in the 6A capsule gene locus was naturally replaced with a new gene. Since the capsule gene loci of 6A and 6B serotypes may differ by only one base pair in the wciP gene, it was speculated that a new serotype '6D' would be possible if the new wciN gene were inserted into the 6B capsule gene locus. Although pneumococci expressing serotype 6D could be produced in the laboratory, initial searches for natural pneumococcal isolates expressing serotype 6D were unsuccessful. However, we now report the discovery of two naturally occurring pneumococcal isolates from Korea that have the serological, genetic and biochemical features predicted for serotype 6D.
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213. |
Glazunova OO,
Raoult D,
Roux V,
( 2010 ) Partial recN gene sequencing: a new tool for identification and phylogeny within the genus Streptococcus. PMID : 19880633 : DOI : 10.1099/ijs.0.018176-0 Abstract >>
Partial sequences of the recN gene (1249 bp), which encodes a recombination and repair protein, were analysed to determine the phylogenetic relationship and identification of streptococci. The partial sequences presented interspecies nucleotide similarity of 56.4-98.2 % and intersubspecies similarity of 89.8-98 %. The mean DNA sequence similarity of recN gene sequences (66.6 %) was found to be lower than those of the 16S rRNA gene (94.1 %), rpoB (84.6 %), sodA (74.8 %), groEL (78.1 %) and gyrB (73.2 %). Phylogenetically derived trees revealed six statistically supported groups: Streptococcus salivarius, S. equinus, S. hyovaginalis/S. pluranimalium/S. thoraltensis, S. pyogenes, S. mutans and S. suis. The 'mitis' group was not supported by a significant bootstrap value, but three statistically supported subgroups were noted: Streptococcus sanguinis/S. cristatus/S. sinensis, S. anginosus/S. intermedius/S. constellatus (the 'anginosus' subgroup) and S. mitis/S. infantis/S. peroris/S. oralis/S. oligofermentans/S. pneumoniae/S. pseudopneumoniae. The partial recN gene sequence comparison highlighted a high percentage of divergence between Streptococcus dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis. This observation is confirmed by other gene sequence comparisons (groEL, gyrB, rpoB and sodA). A high percentage of similarity was found between S. intermedius and S. constellatus after sequence comparison of the recN gene. To study the genetic diversity among the 'anginosus' subgroup, recN, groEL, sodA, gyrB and rpoB sequences were determined for 36 clinical isolates. The results that were obtained confirmed the high genetic diversity within this group of streptococci.
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214. |
Domingues S,
Matos RG,
Reis FP,
Fialho AM,
Barbas A,
Arraiano CM,
( 2009 ) Biochemical characterization of the RNase II family of exoribonucleases from the human pathogens Salmonella typhimurium and Streptococcus pneumoniae. PMID : 19863111 : DOI : 10.1021/bi901105n Abstract >>
Maturation, turnover, and quality control of RNA are performed by many different classes of ribonucleases. Escherichia coli RNase II is the prototype of the RNase II family of ribonucleases, a ubiquitous family of hydrolytic, processive 3' --> 5' exonucleases crucial in RNA metabolism. RNase R is a member of this family that is modulated in response to stress and has been implicated in virulence. In this work, RNase II-like proteins were characterized in the human pathogens Salmonella typhimurium and Streptococcus pneumoniae. By sequence analysis, only one member of the RNase II family was identified in S. pneumoniae, while both RNase II and RNase R were found in Sa. typhimurium. These enzymes were cloned, expressed, purified, and characterized with regard to their biochemical features and modular architecture. The specificity of substrates and the final products generated by the enzymes were clearly demonstrated. Sa. typhimurium RNase II and RNase R behaved essentially as their respective E. coli counterparts. We have shown that the only hydrolytic RNase found in S. pneumoniae was able to degrade structured RNAs as is the case with E. coli RNase R. Our results further showed that there are differences with regard to the activity and ability to bind RNA from enzymes belonging to two distinct pneumococcal strains, and this may be related to a single amino acid substitution in the catalytic domain. Since ribonucleases have not been previously characterized in S. pneumoniae or Sa. typhimurium, this work provides an important first step in the understanding of post-transcriptional control in these pathogens.
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215. |
Tazumi A,
Maeda Y,
Goldsmith CE,
Coulter WA,
Mason C,
Millar BC,
McCalmont M,
Rendall J,
Elborn JS,
Matsuda M,
Moore JE,
( 2009 ) Molecular characterization of macrolide resistance determinants [erm(B) and mef(A)] in Streptococcus pneumoniae and viridans group streptococci (VGS) isolated from adult patients with cystic fibrosis (CF). PMID : 19584106 : DOI : 10.1093/jac/dkp213 Abstract >>
Although long-term use of azithromycin has shown a significant clinical improvement for patients with cystic fibrosis (CF), its long-term effect on the susceptibility of commensal flora within CF airways has not yet been examined. We therefore suggest that long-term use of azithromycin increases macrolide resistance in commensal streptococci. Erythromycin susceptibility in naturally colonizing viridans group streptococci (VGS) was characterized, as well as macrolide resistance gene determinants through sequence analysis, in pneumococci (n = 15) and VGS [n = 84; i.e. Streptococcus salivarius (n = 30), Streptococcus mitis (n = 17), Streptococcus sanguinis (n = 11), Streptococcus oralis (n = 10), Streptococcus parasanguinis (n = 6), Streptococcus gordonii (n = 3), Streptococcus infantis (n = 3), Streptococcus cristatus (n = 2), Streptococcus anginosus (n = 1) and Streptococcus australis (n = 1)] isolated from sputum from 24 adult CF patients, who were on oral azithromycin therapy for at least the previous 7 months. Almost three-quarters of isolates (74; 74.7%) were resistant to erythromycin, whilst a further 15 (15.2%) had reduced susceptibility, leaving only 10 (10.1%) isolates susceptible to erythromycin. The majority (89.8%) were not susceptible to erythromycin, as demonstrated by possession of the erm(B) gene in 25/99 (25.3%), the mef(A) gene in 1/99 (1.0%), the mef(E) gene in 75/99 (75.8%) and both erm(B) and mef(E) genes simultaneously in 11/99 (11.1%). These results indicate that genotypic resistance for macrolides is common in VGS in adult CF patients, with efflux being over three times more frequent. Long-term treatment with azithromycin in CF patients may reduce antibiotic susceptibility in commensal VGS, where these organisms may potentially act as a reservoir of macrolide resistance determinants for newly acquired and antibiotic-susceptible pathogens.
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216. |
Dieudonné-Vatran A,
Krentz S,
Blom AM,
Meri S,
Henriques-Normark B,
Riesbeck K,
Albiger B,
( 2009 ) Clinical isolates of Streptococcus pneumoniae bind the complement inhibitor C4b-binding protein in a PspC allele-dependent fashion. PMID : 19494311 : DOI : 10.4049/jimmunol.0802376 Abstract >>
The complement system constitutes an important component of the innate immune system. To colonize their host and/or to cause disease, many pathogens have evolved strategies to avoid complement-mediated bacterial lysis and opsonophagocytosis. In this study, using a collection of 55 clinical isolates of Streptococcus pneumoniae, we demonstrate for the first time that pneumococci bind the complement inhibitor C4b-binding protein (C4BP). C4BP binding seems to be restricted to certain serotypes such as serotype 4, 6B, 7F, and 14, of which the strains of serotype 14 are the strongest binders. We show that bacteria-bound C4BP retains its functional activity and down-regulates the activation of the classical pathway. Thus, this major respiratory pathogen may escape immune recognition and eradication by the complement system. Furthermore, we show that C4BP binding varies between strains but is dependent on the expression of pneumococcal surface protein C, PspC of group 4. The study of the distribution of group 4 pspC locus shows that most of high-binder serotype 14 isolates harbor an allelic variant of group 4 pspC. Using PspC-negative mutant strains, we identified a new allelic variant of PspC (PspC4.4) as a major ligand for C4BP, revealing a new function for this important pneumococcal virulence factor. Thus pneumococci exploit host C4BP for complement evasion in a PspC allele-dependent manner.
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217. |
Jin P,
Kong F,
Xiao M,
Oftadeh S,
Zhou F,
Liu C,
Russell F,
Gilbert GL,
( 2009 ) First report of putative Streptococcus pneumoniae serotype 6D among nasopharyngeal isolates from Fijian children. PMID : 19803727 : DOI : 10.1086/606118 Abstract >>
A putative Streptococcus pneumoniae serotype, 6D, resulting from the introduction of wciN(beta) into serotype 6B has been proposed. We studied 98 unique serogroup 6 isolates from Fijian children, two-thirds of whom had received at least 1 dose of 7-valent pneumococcal conjugate vaccine, and 51 invasive isolates from Australian children. We used a polymerase chain reaction (PCR) system that targets both wciN(beta) and the single-nucleotide polymorphism that differentiates serotypes 6A and 6B-wciP584g (6A) and wciP584a (6B). Two (9%) of 22 Australian isolates and 24 (38%) of 64 Fijian isolates previously identified as 6A by the Quellung reaction and wciP584g PCR contained wciN(beta) and were designated as 6C; 14 (41%) of 34 Fijian isolates previously identified as 6B by the Quellung reaction and wciP584a PCR contained wciN(beta) and were designated as the new putative serotype 6D. A significantly smaller proportion of children from whom serotype 6D was isolated (2/14 [14%]) had not received PCV-7, compared with the proportion of those from whom serotype 6B was isolated (11/20 [55%]) (P < .05). This is the first report of naturally occurring S. pneumoniae serotype 6D.
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218. |
Pimenta FC,
Gertz RE,
Roundtree A,
Yu J,
Nahm MH,
McDonald RR,
Carvalho Mda G,
Beall BW,
( 2009 ) Rarely occurring 19A-like cps locus from a serotype 19F pneumococcal isolate indicates continued need of serology-based quality control for PCR-based serotype determinations. PMID : 19439547 : DOI : 10.1128/JCM.00704-09 PMC : PMC2708503 Abstract >>
N/A
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219. |
Hui FM,
Morrison DA,
( 1991 ) Genetic transformation in Streptococcus pneumoniae: nucleotide sequence analysis shows comA, a gene required for competence induction, to be a member of the bacterial ATP-dependent transport protein family. PMID : 1987129 : DOI : 10.1128/jb.173.1.372-381.1991 PMC : PMC207196 Abstract >>
The complete nucleotide sequence of comA, a gene required for induction of competence for genetic transformation in Streptococcus pneumoniae, was determined by using plasmid DNA templates and synthetic oligonucleotide primers. The sequence contained a single large open reading frame, ORF1, of 2,151 bp. ORF1 was included within the comAB locus previously mapped genetically and accounted for 50% of its extent. The predicted molecular weight of the largest polypeptide encoded within ORF1, 80,290, coincided with that measured previously (77,000) for the product of in vitro transcription-translation of the cloned comA locus. A Shine-Dalgarno sequence (AAAGGAG, delta G = -14 kcal) lay immediately upstream of ORF1. A sequence (TTtAat-17 bp-TAaAAT) similar to the Escherichia coli sigma 70 promoter consensus was located 410 bp upstream of ORF1. The deduced protein sequence of ComA showed a very strong similarity to the E. coli hemolysin secretion protein, HlyB, and strong similarities to other members of the family of ATP-dependent transport proteins, including the mammalian multidrug resistance P-glycoprotein. These similarities suggest that ComA functions in the transport of some molecule, possibly pneumococcal competence factor itself.
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220. |
Osaki M,
Arcondéguy T,
Bastide A,
Touriol C,
Prats H,
Trombe MC,
( 2009 ) The StkP/PhpP signaling couple in Streptococcus pneumoniae: cellular organization and physiological characterization. PMID : 19502404 : DOI : 10.1128/JB.00196-09 PMC : PMC2715703 Abstract >>
In Streptococcus pneumoniae, stkP and phpP, encoding the eukaryotic-type serine-threonine kinase and PP2C phosphatase, respectively, form an operon. PhpP has the features of a so-called "soluble" protein, whereas StkP protein is membrane associated. Here we provide the first genetic and physiological evidence that PhpP and StkP, with antagonist enzymatic activities, constitute a signaling couple. The StkP-PhpP couple signals competence upstream of the competence-specific histidine kinase ComD, receptor for the oligopeptide pheromone "competence stimulating peptide." We show that PhpP activity is essential in a stkP(+) genetic background, suggesting tight control of StkP activity by PhpP. Proteins PhpP and StkP colocalized to the cell membrane subcellular fraction and likely belong to the same complex, as revealed by coimmunoprecipitation in cellular extracts. Specific coimmunoprecipitation of the N-kinase domain of StkP and PhpP recombinant proteins by PhpP-specific antibodies demonstrates direct interaction between these proteins. Consistently, flow cytometry analysis allowed the determination of the cytoplasmic localization of PhpP and of the N-terminal kinase domain of StkP, in contrast to the periplasmic localization of the StkP C-terminal PASTA (penicillin-binding protein and serine-threonine kinase associated) domain. A signaling route involving interplay between serine, threonine, and histidine phosphorylation is thus described for the first time in this human pathogen.
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221. |
Díaz E,
García JL,
( 1990 ) Characterization of the transcription unit encoding the major pneumococcal autolysin. PMID : 1974230 : DOI : 10.1016/0378-1119(90)90454-y Abstract >>
The pneumococcal lytA gene coding for the major autolysin (amidase) can be expressed in Streptococcus pneumoniae and Escherichia coli using unchanged promoter and termination signals. A region containing several -10, -35 and -44 promoter elements, identical to other previously described prokaryotic promoter sequences, has been found upstream from the transcription start point. A transcription terminator consisting of a hairpin structure (-20.8 kcal/mol) typical of Rho-independent prokaryotic terminators was also localized. The lytA gene has a rather long (240-bp) leader sequence with a high A + T content (70%) that contrasts with the very short (2-bp) untranslated region of the polA gene [L?pez et al., J. Biol. Chem. 264 (1989) 4255-4263], the unique pneumococcal transcription unit that had been characterized so far. Although two open reading frames have been found in the leader region it seems unlikely that these sequences can be translated due to the absence of appropriate ribosome-binding sites.
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222. |
Yamaguchi M,
Minamide Y,
Terao Y,
Isoda R,
Ogawa T,
Yokota S,
Hamada S,
Kawabata S,
( 2009 ) Nrc of Streptococcus pneumoniae suppresses capsule expression and enhances anti-phagocytosis. PMID : 19799870 : DOI : 10.1016/j.bbrc.2009.09.099 Abstract >>
Streptococcus pneumoniae is a major pathogen of community-acquired pneumonia and one of its major virulence factors is pneumolysin, which functions as a cholesterol-dependent cytolytic pore-forming toxin. In this study, we identified the ply-like gene spd0729 in a BLAST search. Unexpectedly, hemolytic and cytotoxic assays showed no significant differences between a Deltaspd0729 mutant strain and the wild-type strain, whereas the mutant strain exhibited weaker anti-phagocytic activity in human peripheral blood. In addition, real-time RT-PCR analysis revealed that four capsular biosynthesis genes in the mutant strain had expressions 7- to 432-fold greater than those of the wild type, while an enzyme-linked immunoassay showed a mean 3-fold greater amount of total capsular polysaccharide in the mutant strain. These results suggest that Spd0729 is not a cytolysin, though it plays crucial roles in anti-phagocytosis and regulation of capsule expression. Thus, we named Spd0729 as a negative regulator of capsular polysaccharide synthesis (Nrc).
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223. |
Rolo D,
Ardanuy C,
Fleites A,
Martín R,
Liñares J,
( 2009 ) Diversity of pneumococcal surface protein A (PspA) among prevalent clones in Spain. PMID : 19419534 : DOI : 10.1186/1471-2180-9-80 PMC : PMC2684541 Abstract >>
PspA is recognized as a major pneumococcal virulence factor and a possible vaccine candidate. The aim of this study was to analyze the PspA family and clade distribution among 112 Spanish pneumococci representatives of dominant clones among patients with invasive disease (n = 66) and nasopharyngeal healthy carriage in children (n = 46). PspA family 2 was predominant among invasive (63.6%) and carriage (54.3%) pneumococcal isolates. No PspA family 3 isolates were detected and only one strain was PspA negative. Although four clonal complexes contained strains of different clades, a clear association between clade and multi locus sequence typing results was found. Clades 1, 3 and 4 were associated with a wide variety of sequence types (ST) related to multiresistant and antibiotic-susceptible worldwide-disseminated clones. Clade 1 was associated with Spain 6B-ST90, Spain 14-ST18, Colombia 5-ST289, Sweden 1-ST306, Denmark 14-ST230 and Sweden 1-ST304 clones. Clade 3 was associated with Spain 23F-ST81, Spain 9V-ST156, Tennessee 14-ST67, Netherlands 3-ST180 and Netherlands 7F-ST191 clones. Clade 4 was related to Sweden 15A-ST63, Netherlands 18C-ST113 and Greece 21-ST193 clones. In contrast, PspA clade was not related to serotype, age or clinical origin of the isolates. PspA clades were associated with genotypes. PspA family 2 and family 1 were dominant among major Spanish pneumococcal clones isolated from patients with invasive disease and nasopharyngeal carriage in children.
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224. |
Neiers F,
Madhurantakam C,
Fälker S,
Manzano C,
Dessen A,
Normark S,
Henriques-Normark B,
Achour A,
( 2009 ) Two crystal structures of pneumococcal pilus sortase C provide novel insights into catalysis and substrate specificity. PMID : 19729023 : DOI : 10.1016/j.jmb.2009.08.058 Abstract >>
The respiratory tract pathogen Streptococcus pneumoniae is a primary cause of morbidity and mortality worldwide. Pili enhance initial adhesion as well as the capacity of pneumococci to cause pneumonia and bacteremia. Pilus-associated sortases (SrtB, SrtC, and SrtD) are involved in the biogenesis of pneumococcal pili, composed of repeating units of RrgB that create the stalk to which the RrgA adhesin and the preferential pilus tip subunit RrgC are covalently associated. Using single sortase-expressing strains, we demonstrate that both pilin-polymerizing sortases SrtB and SrtC can covalently link pili to the peptidoglycan cell wall, a property shared with the non-pilus-polymerizing enzyme SrtD and the housekeeping sortase SrtA. Comparative analysis of the crystal structures of S. pneumoniae SrtC and SrtB revealed structural differences explaining the incapacity of SrtC, but not of SrtB, to incorporate RrgC into the pilus. Accordingly, site-directed mutagenesis of Thr(160) in SrtB to an arginine as in SrtC (Arg(160)) partially converted its substrate specificity into that of SrtC. Solving two crystal structures for SrtC suggests that an opening of a flexible lid and a concomitant cysteine rotation are important for catalysis and the activation of the catalytic cysteine of pilus-associated sortases.
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225. |
Bratcher PE,
Park IH,
Hollingshead SK,
Nahm MH,
( 2009 ) Production of a unique pneumococcal capsule serotype belonging to serogroup 6. PMID : 19202106 : DOI : 10.1099/mic.0.024521-0 PMC : PMC3706093 Abstract >>
Serogroup 6 of Streptococcus pneumoniae contains three serotypes, named 6A, 6B and 6C, with highly homologous capsule gene loci. The 6A and 6B capsule gene loci consistently differ from each other by only one nucleotide in the wciP gene. The 6A capsule gene locus has a galactosyltransferase, which has been replaced with a glucosyltransferase in the 6C capsule gene locus. We considered that a new serotype named '6X1' would be possible if the galactosyltransferase of the 6B capsule gene locus is replaced with the glucosyltransferase of 6C. We demonstrate that this gene transfer yields a viable pneumococcal strain and that the capsular polysaccharide (PS) from this strain has the predicted chemical structure and serological similarity to the capsular PS of the 6B serotype. The new strain (i.e. serotype 6X1) is typed as 6B by the quellung reaction, but it can be distinguished from 6B strains with mAbs to 6B PS. Reexamination of 264 pneumococcal isolates that had been previously typed as 6B with classical typing methods revealed no isolates expressing serotype 6X1. Nevertheless, this study shows that this capsular PS is biochemically possible and could exist/emerge in nature.
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226. |
Contreras-Martel C,
Dahout-Gonzalez C,
Martins Ados S,
Kotnik M,
Dessen A,
( 2009 ) PBP active site flexibility as the key mechanism for beta-lactam resistance in pneumococci. PMID : 19233207 : DOI : 10.1016/j.jmb.2009.02.024 Abstract >>
Penicillin-binding proteins (PBPs), the main targets of beta-lactam antibiotics, are membrane-associated enzymes that catalyze the two last steps in the biosynthesis of peptidoglycan. In Streptococcus pneumoniae, a major human pathogen, the surge in resistance to such antibiotics is a direct consequence of the proliferation of mosaic PBP-encoding genes, which give rise to proteins containing tens of mutations. PBP2b is a major drug resistance target, and its modification is essential for the development of high levels of resistance to piperacillin. In this work, we have solved the crystal structures of PBP2b from a wild-type pneumococcal strain, as well as from a highly drug-resistant clinical isolate displaying 58 mutations. Although mutations are present throughout the entire PBP structure, those surrounding the active site influence the total charge and the polar character of the region, while those in close proximity to the catalytic nucleophile impart flexibility onto the beta3/beta4 loop area, which encapsulates the cleft. The wealth of structural data on pneumococcal PBPs now underlines the importance of high malleability in active site regions of drug-resistant strains, suggesting that active site "breathing" could be a common mechanism employed by this pathogen to prevent targeting by beta-lactams.
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227. |
Kosowska-Shick K,
Ednie LM,
McGhee P,
Appelbaum PC,
( 2009 ) Comparative antipneumococcal activities of sulopenem and other drugs. PMID : 19307366 : DOI : 10.1128/AAC.01531-08 PMC : PMC2687188 Abstract >>
For 297 penicillin-susceptible, -intermediate, and -resistant pneumococcal strains, the sulopenem MIC(50)s were 0.008, 0.06, and 0.25, respectively, and the sulopenem MIC(90)s were 0.016, 0.25, and 0.5 microg/ml, respectively. The MIC(50)s of amoxicillin for the corresponding strains were 0.03, 0.25, and 2.0 microg/ml, respectively, and the MIC(90)s were 0.03, 1.0, and 8.0 microg/ml, respectively. The combination of amoxicillin and clavulanate gave MICs similar to those obtained with amoxicillin alone. The sulopenem MICs were similar to those of imipenem and meropenem. The MICs of ss-lactams increased with those of penicillin G, and among the quinolones tested, moxifloxacin had the lowest MICs. Additionally, 45 strains of drug-resistant type 19A pneumococci were tested by agar dilution and gave sulopenem MIC(50)s and MIC(90)s of 1.0 and 2.0 microg/ml, respectively. Both sulopenem and amoxicillin (with and without clavulanate) were bactericidal against all 12 strains tested at 2x MIC after 24 h. Thirty-one strains from 10 countries with various penicillin, amoxicillin, and carbapenems MICs, including those with the highest sulopenem MICs, were selected for sequencing analysis of the pbp1a, pbp2x, and pbp2b regions encoding the transpeptidase active site and MurM. We did not find any correlations between specific penicillin-binding protein-MurM patterns and changes in the MICs.
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228. |
Kosowska-Shick K,
McGhee P,
Appelbaum PC,
( 2009 ) Binding of faropenem and other beta-lactam agents to penicillin-binding proteins of pneumococci with various beta-lactam susceptibilities. PMID : 19237649 : DOI : 10.1128/AAC.01566-08 PMC : PMC2681536 Abstract >>
Faropenem demonstrated low MICs (< or = 1 microg/ml) for all penicillin-susceptible and nonsusceptible pneumococci and exhibited very strong abilities to bind to Streptococcus pneumoniae penicillin-binding proteins (PBPs), except for PBP2X. The lower faropenem affinity for PBP2X did not affect MICs for any strains tested, and only imipenem had lower MICs, with much lower binding affinities for all PBPs tested, than faropenem.
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229. |
Manzano C,
Contreras-Martel C,
El Mortaji L,
Izoré T,
Fenel D,
Vernet T,
Schoehn G,
Di Guilmi AM,
Dessen A,
( 2008 ) Sortase-mediated pilus fiber biogenesis in Streptococcus pneumoniae. PMID : 19081060 : DOI : 10.1016/j.str.2008.10.007 Abstract >>
Streptococcus pneumoniae is a piliated pathogen whose ability to circumvent vaccination and antibiotic treatment strategies is a cause of mortality worldwide. Pili play important roles in pneumococcal infection, but little is known about their biogenesis mechanism or the relationship between components of the pilus-forming machinery, which includes the fiber pilin (RrgB), two minor pilins (RrgA, RrgC), and three sortases (SrtC-1, SrtC-2, SrtC-3). Here we show that SrtC-1 is the main pilus-polymerizing transpeptidase, and electron microscopy analyses of RrgB fibers reconstituted in vitro reveal that they structurally mimic the pneumococcal pilus backbone. Crystal structures of both SrtC-1 and SrtC-3 reveal active sites whose access is controlled by flexible lids, unlike in non-pilus sortases, and suggest that substrate specificity is dictated by surface recognition coupled to lid opening. The distinct structural features of pilus-forming sortases suggest a common pilus biogenesis mechanism that could be exploited for the development of broad-spectrum antibacterials.
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230. |
Nielsen XC,
Justesen US,
Dargis R,
Kemp M,
Christensen JJ,
( 2009 ) Identification of clinically relevant nonhemolytic Streptococci on the basis of sequence analysis of 16S-23S intergenic spacer region and partial gdh gene. PMID : 19193846 : DOI : 10.1128/JCM.01449-08 PMC : PMC2668326 Abstract >>
Nonhemolytic streptococci (NHS) cause serious infections, such as endocarditis and septicemia. Many conventional phenotypic methods are insufficient for the identification of bacteria in this group to the species level. Genetic analysis has revealed that single-gene analysis is insufficient for the identification of all species in this group of bacteria. The aim of the present study was to establish a method based on sequence analysis of the 16S-23S intergenic spacer (ITS) region and the partial gdh gene to identify clinical relevant NHS to the species level. Sequence analysis of the ITS region was performed with 57 NHS reference or clinical strains. Satisfactory identification to the species level was achieved for 14/19 NHS species included in this study on the basis of sequence analysis of the ITS region. Streptococcus salivarius and Streptococcus vestibularis obtained the expected taxon as the best taxon match, but there was a short maximum score distance to the next best match (distance, <10). Streptococcus mitis, Streptococcus oralis, and Streptococcus pneumoniae could not be unambiguously discriminated by sequence analysis of the ITS region, as was also proven by phylogenetic analysis. These five species could be identified to the group level only by ITS sequence analysis. Partial gdh sequence analysis was applied to the 11 S. oralis strains, the 11 S. mitis strains, and the 17 S. pneumoniae strains. All except one strain achieved a satisfactory identification to the species level. A phylogenetic algorithm based on the analysis of partial gdh gene sequences revealed three distinct clusters. We suggest that sequence analysis of the combination of the ITS region and the partial gdh gene can be used in the reference laboratory for the species-level identification of NHS.
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231. |
Ferreira DM,
Darrieux M,
Silva DA,
Leite LC,
Ferreira JM,
Ho PL,
Miyaji EN,
Oliveira ML,
( 2009 ) Characterization of protective mucosal and systemic immune responses elicited by pneumococcal surface protein PspA and PspC nasal vaccines against a respiratory pneumococcal challenge in mice. PMID : 19279169 : DOI : 10.1128/CVI.00395-08 PMC : PMC2681601 Abstract >>
Pneumococcal surface protein A (PspA) and PspC are virulence factors that are involved in the adhesion of Streptococcus pneumoniae to epithelial cells and/or evasion from the immune system. Here, the immune responses induced by mucosal vaccines composed of both antigens as recombinant proteins or delivered by Lactobacillus casei were evaluated. None of the PspC vaccines protected mice against an invasive challenge with pneumococcal strain ATCC 6303. On the other hand, protection was observed for immunization with vaccines composed of PspA from clade 5 (PspA5 or L. casei expressing PspA5) through the intranasal route. The protective response was distinguished by a Th1 profile with high levels of immunoglobulin G2a production, efficient complement deposition, release of proinflammatory cytokines, and infiltration of neutrophils. Intranasal immunization with PspA5 elicited the highest level of protection, characterized by increased levels of secretion of interleukin-17 and gamma interferon by lung and spleen cells, respectively, and low levels of tumor necrosis factor alpha in the respiratory tract.
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232. |
Kharat AS,
Denapaite D,
Gehre F,
Brückner R,
Vollmer W,
Hakenbeck R,
Tomasz A,
( 2008 ) Different pathways of choline metabolism in two choline-independent strains of Streptococcus pneumoniae and their impact on virulence. PMID : 18621904 : DOI : 10.1128/JB.00628-08 PMC : PMC2519527 Abstract >>
The two recently characterized Streptococcus pneumoniae strains--R6Chi and R6Cho(-)--that have lost the unique auxotrophic requirement of this bacterial species for choline differ in their mechanisms of choline independence. In strain R6Chi the mechanism is caused by a point mutation in tacF, a gene that is part of the pneumococcal lic2 operon, which is essential for growth and survival of the bacteria. Cultures of lic2 mutants of the encapsulated strain D39Chi growing in choline-containing medium formed long chains, did not autolyze, had no choline in their cell wall, and were completely avirulent in the mouse intraperitoneal model. In contrast, while the Cho(-) strain carried a complete pneumococcal lic2 operon and had no mutations in the tacF gene, deletion of the entire lic2 operon had no effect on the growth or phenotype of strain Cho(-). These observations suggest that the biochemical functions normally dependent on determinants of the pneumococcal lic2 operon may also be carried out in strain Cho(-) by a second set of genetic elements imported from Streptococcus oralis, the choline-independent streptococcal strain that served as the DNA donor in the heterologous transformation event that produced strain R6Cho(-). The identification in R6Cho(-) of a large (20-kb) S. oralis DNA insert carrying both tacF and licD genes confirms this prediction and suggests that these heterologous elements may represent a "backup" system capable of catalyzing P-choline incorporation and export of teichoic acid chains under conditions in which the native lic2 operon is not functional.
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233. |
Abbott DW,
Macauley MS,
Vocadlo DJ,
Boraston AB,
( 2009 ) Streptococcus pneumoniae endohexosaminidase D, structural and mechanistic insight into substrate-assisted catalysis in family 85 glycoside hydrolases. PMID : 19181667 : DOI : 10.1074/jbc.M809663200 PMC : PMC2670171 Abstract >>
Endo-beta-d-glucosaminidases from family 85 of glycoside hydrolases (GH85 endohexosaminidases) act to cleave the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. Endohexosaminidase D (Endo-D), produced by Streptococcus pneumoniae, is believed to contribute to the virulence of this organism by playing a role in the deglycosylation of IgG antibodies. Endohexosaminidases have received significant attention for this reason and, moreover, because they are powerful tools for chemoenzymatic synthesis of proteins having defined glycoforms. Here we describe mechanistic and structural studies of the catalytic domain (SpGH85) of Endo-D that provide compelling support for GH85 enzymes using a catalytic mechanism involving substrate-assisted catalysis. Furthermore, the structure of SpGH85 in complex with the mechanism-based competitive inhibitor NAG-thiazoline (K(d) = 28 microm) provides a coherent rationale for previous mutagenesis studies of Endo-D and other related GH85 enzymes. We also find GH85, GH56, and GH18 enzymes have a similar configuration of catalytic residues. Notably, GH85 enzymes have an asparagine in place of the aspartate residue found in these other families of glycosidases. We propose that this residue, as the imidic acid tautomer, acts analogously to the key catalytic aspartate of GH56 and GH18 enzymes. This topographically conserved arrangement of the asparagine residue and a conserved glutamic acid, coupled with previous kinetic studies, suggests these enzymes may use an unusual proton shuttle to coordinate effective general acid and base catalysis to aid cleavage of the glycosidic bond. These results collectively provide a blueprint that may be used to facilitate protein engineering of these enzymes to improve their function as biocatalysts for synthesizing glycoproteins having defined glycoforms and also may serve as a guide for generating inhibitors of GH85 enzymes.
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234. |
Del Grosso M,
Camilli R,
Libisch B,
Füzi M,
Pantosti A,
( 2009 ) New composite genetic element of the Tn916 family with dual macrolide resistance genes in a Streptococcus pneumoniae isolate belonging to clonal complex 271. PMID : 19104015 : DOI : 10.1128/AAC.01066-08 PMC : PMC2650554 Abstract >>
N/A
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235. |
Kilian M,
Poulsen K,
Blomqvist T,
Håvarstein LS,
Bek-Thomsen M,
Tettelin H,
Sørensen UB,
( 2008 ) Evolution of Streptococcus pneumoniae and its close commensal relatives. PMID : 18628950 : DOI : 10.1371/journal.pone.0002683 PMC : PMC2444020 Abstract >>
Streptococcus pneumoniae is a member of the Mitis group of streptococci which, according to 16S rRNA-sequence based phylogenetic reconstruction, includes 12 species. While other species of this group are considered prototypes of commensal bacteria, S. pneumoniae is among the most frequent microbial killers worldwide. Population genetic analysis of 118 strains, supported by demonstration of a distinct cell wall carbohydrate structure and competence pheromone sequence signature, shows that S. pneumoniae is one of several hundred evolutionary lineages forming a cluster separate from Streptococcus oralis and Streptococcus infantis. The remaining lineages of this distinct cluster are commensals previously collectively referred to as Streptococcus mitis and each represent separate species by traditional taxonomic standard. Virulence genes including the operon for capsule polysaccharide synthesis and genes encoding IgA1 protease, pneumolysin, and autolysin were randomly distributed among S. mitis lineages. Estimates of the evolutionary age of the lineages, the identical location of remnants of virulence genes in the genomes of commensal strains, the pattern of genome reductions, and the proportion of unique genes and their origin support the model that the entire cluster of S. pneumoniae, S. pseudopneumoniae, and S. mitis lineages evolved from pneumococcus-like bacteria presumably pathogenic to the common immediate ancestor of hominoids. During their adaptation to a commensal life style, most of the lineages gradually lost the majority of genes determining virulence and became genetically distinct due to sexual isolation in their respective hosts.
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236. |
Lu L,
Ma Z,
Jokiranta TS,
Whitney AR,
DeLeo FR,
Zhang JR,
( 2008 ) Species-specific interaction of Streptococcus pneumoniae with human complement factor H. PMID : 18981135 : DOI : 10.4049/jimmunol.181.10.7138 PMC : PMC2587499 Abstract >>
Streptococcus pneumoniae naturally colonizes the nasopharynx as a commensal organism and sometimes causes infections in remote tissue sites. This bacterium is highly capable of resisting host innate immunity during nasopharyngeal colonization and disseminating infections. The ability to recruit complement factor H (FH) by S. pneumoniae has been implicated as a bacterial immune evasion mechanism against complement-mediated bacterial clearance because FH is a complement alternative pathway inhibitor. S. pneumoniae recruits FH through a previously defined FH binding domain of choline-binding protein A (CbpA), a major surface protein of S. pneumoniae. In this study, we show that CbpA binds to human FH, but not to the FH proteins of mouse and other animal species tested to date. Accordingly, deleting the FH binding domain of CbpA in strain D39 did not result in obvious change in the levels of pneumococcal bacteremia or virulence in a bacteremia mouse model. Furthermore, this species-specific pneumococcal interaction with FH was shown to occur in multiple pneumococcal isolates from the blood and cerebrospinal fluid. Finally, our phagocytosis experiments with human and mouse phagocytes and complement systems provide additional evidence to support our hypothesis that CbpA acts as a bacterial determinant for pneumococcal resistance to complement-mediated host defense in humans.
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237. |
Harris KA,
Turner P,
Green EA,
Hartley JC,
( 2008 ) Duplex real-time PCR assay for detection of Streptococcus pneumoniae in clinical samples and determination of penicillin susceptibility. PMID : 18562586 : DOI : 10.1128/JCM.02462-07 PMC : PMC2519471 Abstract >>
We have developed a duplex real-time PCR for the rapid diagnosis of Streptococcus pneumoniae infection from culture-negative clinical samples with the simultaneous determination of penicillin susceptibility. The assay amplifies a lytA gene target and a penicillin binding protein 2b (pbp2b) gene target in penicillin-susceptible organisms. The assay was shown to be sensitive (detects 0.5 CFU per PCR) and specific for the detection of S. pneumoniae DNA. The assay was validated by comparing pbp2b PCR results with MIC data for 27 S. pneumoniae isolates. All 5 isolates with penicillin MICs of > 1.0 mg/liter were pbp2b real-time PCR negative, as were 9 of the 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter. One isolate with a penicillin MIC of 0.12 to 1.0 mg/liter gave an equivocal pbp2b real-time PCR result. Twelve isolates were penicillin susceptible (MICs of < or = 0.06 mg/liter) and pbp2b real-time PCR positive. These data were used to establish an algorithm for the interpretation of penicillin susceptibility from the duplex PCR result. pbp2b real-time PCR results were also compared to an established PCR-restriction fragment length polymorphism (RFLP) method previously applied to these 27 isolates and 46 culture-negative clinical samples (containing S. pneumoniae DNA by broad-range 16S rRNA gene PCR). Discordant results were seen for four isolates and six culture-negative clinical samples, as PCR-RFLP could not reliably detect penicillin MICs of 0.12 to 1.0 mg/liter. We report prospective application of the duplex PCR assay to the diagnosis of S. pneumoniae infection from 200 culture-negative clinical specimens sent to the laboratory for diagnostic broad-range 16S rRNA gene PCR. One hundred six were negative in the duplex PCR. Ninety-four were lytA PCR positive, and 70 of these were also pbp2b PCR positive and interpreted as penicillin susceptible. Fourteen were pbp2b PCR negative and interpreted as having reduced susceptibility to penicillin. For the remaining 10 samples, susceptibility to penicillin was not determined.
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238. |
Tian SF,
Chu YZ,
Chen BY,
( 2008 ) Molecular characteristics of penicillin-binding protein 2b, 2x, and 1a sequences in penicillin-nonsusceptible Streptococcus pneumoniae isolates in Shenyang, China. PMID : 18535636 : DOI : 10.1139/w08-030 Abstract >>
The aim of this study was to investigate the nature of the amino acid motifs found in penicillin-binding proteins (PBP) 2b, 2x, and 1a of penicillin-nonsusceptible Streptococcus pneumoniae isolates from Shenyang, China, and to obtain information regarding the prevalence of alterations within the motifs or in positions flanking the motifs. For 18 clinical isolates comprising 4 penicillin-susceptible S. pneumoniae, 5 penicillin-intermediate S. pneumoniae, and 9 penicillin-resistant S. pneumoniae. the DNA sequences of PBP2b, PBP2x, and PBP1a transpeptidase domains were determined and then genotyped by multilocus sequence typing. Sequence analysis revealed that most penicillin-nonsusceptible S. pneumoniae isolates (penicillin MIC > or = 1.5 microg/mL and cefotaxime MIC > or = 2 microg/mL) shared identical PBP2b, PBP2x, and PBP1a amino acid profiles. Most penicillin-resistant S. pneumoniae isolates were ST320 (4-16-19-15-6-20-1), the double-locus variant of the Taiwan19F-14 clone. This study will serve as a basis for future monitoring of genetic changes associated with the emergence and spread of beta-lactam resistance in Shenyang, China.
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239. |
Yamaguchi M,
Terao Y,
Mori Y,
Hamada S,
Kawabata S,
( 2008 ) PfbA, a novel plasmin- and fibronectin-binding protein of Streptococcus pneumoniae, contributes to fibronectin-dependent adhesion and antiphagocytosis. PMID : 18974092 : DOI : 10.1074/jbc.M807087200 PMC : PMC2662297 Abstract >>
Streptococcus pneumoniae is a major causative agent of mortality throughout the world. The initial event in invasive pneumococcal disease is the attachment of pneumococci to epithelial cells in the upper respiratory tract. Several bacterial proteins can bind to host extracellular matrix proteins and function as adhesins and invasins. To identify adhesins or invasins on the pneumococcal cell surface, we searched for several proteins with an LPXTG anchoring motif in the whole-genome sequence of Streptococcus pneumoniae and identified one, which we called PfbA (plasmin- and fibronectin-binding protein A), that bound to human serum proteins. Immunofluorescence microscopy and fluorescence-activated cell sorter analysis revealed that PfbA was expressed on the pneumococcal cell surface. A DeltapfbA mutant strain was only half as competent as the wild-type strain at adhering to and invading lung and laryngeal epithelial cells. In addition, epithelial cells infected with DeltapfbA showed morphological changes, including cell flattening and a loss of microvilli, that did not occur in cells infected with the wild-type strain. The mutant strain also exhibited a weaker antiphagocytotic activity than wild type in human peripheral blood. Moreover, the growth of wild-type bacteria in human whole blood containing anti-PfbA antibodies was reduced by approximately 50% after 3 h compared with its growth without the antibody. These results suggest that PfbA is an important factor in the development of pneumococcal infections.
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240. |
Bagnoli F,
Moschioni M,
Donati C,
Dimitrovska V,
Ferlenghi I,
Facciotti C,
Muzzi A,
Giusti F,
Emolo C,
Sinisi A,
Hilleringmann M,
Pansegrau W,
Censini S,
Rappuoli R,
Covacci A,
Masignani V,
Barocchi MA,
( 2008 ) A second pilus type in Streptococcus pneumoniae is prevalent in emerging serotypes and mediates adhesion to host cells. PMID : 18515415 : DOI : 10.1128/JB.00384-08 PMC : PMC2493256 Abstract >>
Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element containing genes typical of gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (consisting of pitA, sipA, pitB, srtG1, and srtG2) codes for a second functional pilus in pneumococcus. Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. Presence of PI-2 correlates with the genotype as defined by multilocus sequence typing and clonal complex (CC). The PI-2-positive CCs are associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging serotypes in both industrialized and developing countries. Interestingly, strains belonging to CC271 (where sequence type 271 is the predicted founder of the CC) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that play a role in the initial host cell contact to the respiratory tract and are potential antigens for inclusion in a new generation of pneumococcal vaccines.
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241. |
Dias R,
Félix D,
Caniça M,
( 2008 ) Diversity of penicillin binding proteins among clinical Streptococcus pneumoniae strains from Portugal. PMID : 18505861 : DOI : 10.1128/AAC.01655-07 PMC : PMC2443872 Abstract >>
N/A
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242. |
Cortes PR,
Orio AG,
Regueira M,
Piñas GE,
Echenique J,
( 2008 ) Characterization of in vitro-generated and clinical optochin-resistant strains of Streptococcus pneumoniae isolated from Argentina. PMID : 18417665 : DOI : 10.1128/JCM.02318-07 PMC : PMC2446823 Abstract >>
Optochin susceptibility is a key test used for pneumococcal diagnosis, but optochin-resistant (Opt(r)) pneumococci have been reported in the last 2 decades. In this work, we characterized eight Opt(r) clinical strains which presented a new mutation, G47V, a predominant A49S mutation (recently reported in Brazil) and A49T. These mutations were found in the c subunit of the F(0)F(1) ATPase encoded by the atpC gene, and W206C was found in the a subunit encoded by the atpA gene. The Opt(r) clinical isolates were analyzed by BOX PCR, multilocus sequence typing, and serotype and antimicrobial resistance profiles, and they showed no epidemiological relationship. To characterize the Opt(r) mutations that could emerge among clinical strains, we studied a pool of spontaneous Opt(r) colonies obtained in vitro from the virulent D39 strain. We compared the atpAC mutations of these Opt(r) pneumococci (with or without passage through C57BL/6 mice) with those described in the clinical isolates. This analysis revealed three new mutations, G47V and L26M in the c subunit and L184S in the a subunit. Most of the mutations identified in the laboratory-generated Opt(r) strains were also found in clinical strains, with the exception of the L26M and L184S mutations, and we suppose that both mutations could emerge among invasive strains in the future. Considering that atpAC are essential genes, we propose that all spontaneous mutations that confer in vitro optochin resistance would not present severe physiological alterations in S. pneumoniae and may be carried by circulating pneumococcal strains.
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243. |
Le Hello S,
Page S,
Garin B,
( 2008 ) Fluoroquinolone resistance in a clinical isolate of Streptococcus pneumoniae in the South Pacific. PMID : 18495439 : DOI : 10.1016/j.ijantimicag.2008.02.015 Abstract >>
N/A
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244. |
Desa MN,
Navaratnam P,
Vadivelu J,
Sekaran SD,
( 2008 ) Expression analysis of adherence-associated genes in pneumococcal clinical isolates during adherence to human respiratory epithelial cells (in vitro) by real-time PCR. PMID : 18795954 : DOI : 10.1111/j.1574-6968.2008.01345.x Abstract >>
Pneumococcal virulence determinants have been extensively studied but molecular evidence on virulence gene expression pattern is still lacking. We undertook this study to analyze the regulation pattern of adherence-associated genes; psaA, pspC, cbpG, including ply of serotypes 1, 7F, 19F and 23F clinical isolates during the bacterial adherence to human lung epithelial cells (in vitro), by real-time PCR. We were able to harvest the bacterial RNA (0.5-1 microg microL(-1)) from the infected host cell and analysis showed a consistent upregulation of psaA. Differential expressions were observed for pspC, cbpG and ply genes but the former was mostly upregulated whereas the later two frequently showed either no significant change or a downregulation. Partial nucleotide sequences of psaA, cbpG and ply were highly homologous among the isolates as well as against GenBank sequences (99%) whereas those for pspC were similar (98%) to allelic variants pspC-3 and pspC-5.
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245. |
Stanhope MJ,
Lefébure T,
Walsh SL,
Becker JA,
Lang P,
Pavinski Bitar PD,
Miller LA,
Italia MJ,
Amrine-Madsen H,
( 2008 ) Positive selection in penicillin-binding proteins 1a, 2b, and 2x from Streptococcus pneumoniae and its correlation with amoxicillin resistance development. PMID : 18394970 : DOI : 10.1016/j.meegid.2008.02.001 Abstract >>
The efficacy of beta-lactam antibiotics in Streptococcus pneumoniae has been compromised because of the development of altered penicillin-binding proteins (PBPs), however, this has been less so for amoxicillin than for penicillin. Recently, there have been a number of important methods developed to detect molecular adaptation in protein coding genes. The purpose of this study is to employ modern molecular selection approaches to predict sites under positive selection pressure in PBPs, derived from a large international S. pneumoniae collection of amoxicillin resistant and susceptible isolates, and encompassing a comparative data set of 354 pbp1a, 335 pbp2b, and 389 pbp2x gene sequences. A correspondence discriminant analysis (CDA) of positively selected pbp sites and amoxicillin MIC (minimum inhibitory concentration) values is then used to detect sites under positive selection pressure that are important in discriminating different amoxicillin MICs. Molecular adaptation was evident throughout PBP2X, with numerous positively selected sites in both the transpeptidase (TP) and C-terminal domains, strongly correlated with discriminating amoxicillin MICs. In the case of PBP1A positive selection was present in the glycosyltransfer (GT), TP and C-terminal domains. Sites within the TP domain tended to be correlated with the discrimination of low from intermediate MICs, whereas sites within the C-terminal tail, with a discrimination of intermediate from fully resistant. Most of the positively selected sites within PBP2B were in the N-terminal domain and were not correlated with amoxicillin MICs, however, several sites taken from the literature for the TP domain were strongly associated with discriminating high from intermediate level amoxicillin resistance. Many of the positively selected sites could be directly associated with functional inferences based on the crystal structures of these proteins. Our results suggest that clinical emphasis on TP domain sequences of these proteins may result in missing information relevant to antibiotic resistance development.
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246. |
Saito J,
Yamada M,
Watanabe T,
Iida M,
Kitagawa H,
Takahata S,
Ozawa T,
Takeuchi Y,
Ohsawa F,
( 2008 ) Crystal structure of enoyl-acyl carrier protein reductase (FabK) from Streptococcus pneumoniae reveals the binding mode of an inhibitor. PMID : 18305197 : DOI : 10.1110/ps.073288808 PMC : PMC2271168 Abstract >>
Enoyl-acyl carrier protein (ACP) reductases are critical for bacterial type II fatty acid biosynthesis and thus are attractive targets for developing novel antibiotics. We determined the crystal structure of enoyl-ACP reductase (FabK) from Streptococcus pneumoniae at 1.7 A resolution. There was one dimer per asymmetric unit. Each subunit formed a triose phosphate isomerase (TIM) barrel structure, and flavin mononucleotide (FMN) was bound as a cofactor in the active site. The overall structure was similar to the enoyl-ACP reductase (ER) of fungal fatty acid synthase and to 2-nitropropane dioxygenase (2-ND) from Pseudomonas aeruginosa, although there were some differences among these structures. We determined the crystal structure of FabK in complex with a phenylimidazole derivative inhibitor to envision the binding site interactions. The crystal structure reveals that the inhibitor binds to a hydrophobic pocket in the active site of FabK, and this is accompanied by induced-fit movements of two loop regions. The thiazole ring and part of the ureido moiety of the inhibitor are involved in a face-to-face pi-pi stacking interaction with the isoalloxazine ring of FMN. The side-chain conformation of the proposed catalytic residue, His144, changes upon complex formation. Lineweaver-Burk plots indicate that the inhibitor binds competitively with respect to NADH, and uncompetitively with respect to crotonoyl coenzyme A. We propose that the primary basis of the inhibitory activity is competition with NADH for binding to FabK, which is the first step of the two-step ping-pong catalytic mechanism.
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247. |
Moore MR,
Gertz RE,
Woodbury RL,
Barkocy-Gallagher GA,
Schaffner W,
Lexau C,
Gershman K,
Reingold A,
Farley M,
Harrison LH,
Hadler JL,
Bennett NM,
Thomas AR,
McGee L,
Pilishvili T,
Brueggemann AB,
Whitney CG,
Jorgensen JH,
Beall B,
( 2008 ) Population snapshot of emergent Streptococcus pneumoniae serotype 19A in the United States, 2005. PMID : 18419539 : DOI : 10.1086/528996 Abstract >>
Serotype 19A invasive pneumococcal disease (IPD) increased annually in the United States after the introduction of the 7-valent conjugate vaccine (PCV7). To understand this increase, we characterized serotype 19A isolates recovered during 2005. IPD cases during 1998-2005 were identified through population-based surveillance. We performed susceptibility testing and multilocus sequence typing on 528 (95%) of 554 serotype 19A isolates reported in 2005. The incidence of IPD due to serotype 19A increased from 0.8 to 2.5 cases per 100,000 population between 1998 and 2005 (P < .05), whereas the overall incidence of IPD decreased from 24.4 to 13.8 cases per 100,000 population (P < .05). Simultaneously, the incidence of IPD due to penicillin-resistant 19A isolates increased from 6.7% to 35% (P < .0001). Of 151 penicillin-resistant 19A isolates, 111 (73.5%) belonged to the rapidly emerging clonal complex 320, which is related to multidrug-resistant Taiwan(19F)-14. The remaining penicillin-resistant strains were highly related to other clones of PCV7 serotypes or to isolates within major 19A clonal complex 199 (CC199). In 1999, only CC199 and 3 minor clones were apparent among serotype 19A isolates. During 2005, 11 multiple-isolate clonal sets were detected, including capsular switch variants of a serotype 4 clone. PCV7 ineffectiveness against serotype 19A, antibiotic resistance, clonal expansion and emergence, and capsular switching have contributed to the genetic diversity of 19A and to its emergence as the predominant invasive pneumococcal serotype in the United States.
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248. |
Darrieux M,
Moreno AT,
Ferreira DM,
Pimenta FC,
de Andrade AL,
Lopes AP,
Leite LC,
Miyaji EN,
( 2008 ) Recognition of pneumococcal isolates by antisera raised against PspA fragments from different clades. PMID : 18287288 : DOI : 10.1099/jmm.0.47661-0 Abstract >>
Pneumococcal surface protein A (PspA) is an important vaccine candidate against pneumococcal infections, capable of inducing protection in different animal models. Based on its structural diversity, it has been suggested that a PspA-based vaccine should contain at least one fragment from each of the two major families (family 1, comprising clades 1 and 2, and family 2, comprising clades 3, 4 and 5) in order to elicit broad protection. This study analysed the recognition of a panel of 35 pneumococcal isolates bearing different PspAs by antisera raised against the N-terminal regions of PspA clades 1 to 5. The antiserum to PspA clade 4 was found to show the broadest cross-reactivity, being able to recognize pneumococcal strains containing PspAs of all clades in both families. The cross-reactivity of antibodies elicited against a PspA hybrid including the N-terminal region of clade 1 fused to a shorter and more divergent fragment (clade-defining region, or CDR) of clade 4 (PspA1-4) was also tested, and revealed a strong recognition of isolates containing clades 1, 4 and 5, and weaker reactions with clades 2 and 3. The analysis of serum reactivity against different PspA regions further revealed that the complete N-terminal region rather than just the CDR should be included in an anti-pneumococcal vaccine. A PspA-based vaccine is thus proposed to be composed of the whole N-terminal region of clades 1 and 4, which could also be expressed as a hybrid protein.
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249. |
Moschioni M,
Donati C,
Muzzi A,
Masignani V,
Censini S,
Hanage WP,
Bishop CJ,
Reis JN,
Normark S,
Henriques-Normark B,
Covacci A,
Rappuoli R,
Barocchi MA,
( 2008 ) Streptococcus pneumoniae contains 3 rlrA pilus variants that are clonally related. PMID : 18269316 : DOI : 10.1086/528375 Abstract >>
Pilus components of Streptococcus pneumoniae encoded by rlrA were recently shown to elicit protection in an animal model of infection. Limited data are available on the prevalence of the rlrA operon in pneumococci; therefore, we investigated its distribution and its antigenic variation among disease-causing strains. The prevalence of rlrA and its association with serotype and genotype were evaluated in a global panel of 424 pneumococci isolates (including the 26 drug-resistant clones described by the Pneumococcal Molecular Epidemiology Network). The rlrA islet was found in 130 isolates (30.6%) of the defined collection. Sequence alignment of 15 rlrA islets defined the presence of 3 clade types, with an overall homology of 88%-92%. The presence or absence of a pilus-encoding operon correlated with S. pneumoniae genotype (P < .001), as determined by multilocus sequence typing, and not with serotype. Further investigation identified a positive trend of rlrA occurrence among antimicrobial-resistant pneumococci. On the basis of S. pneumoniae genotype, it is possible to predict the incidence of the rlrA pilus operon in a collection of pneumococcal isolates. This will facilitate the development of a protein vaccine.
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250. |
Cochetti I,
Tili E,
Mingoia M,
Varaldo PE,
Montanari MP,
( 2008 ) erm(B)-carrying elements in tetracycline-resistant pneumococci and correspondence between Tn1545 and Tn6003. PMID : 18285489 : DOI : 10.1128/AAC.01457-07 PMC : PMC2292545 Abstract >>
This study investigated the genetic organization of erm(B)-carrying transposons of Streptococcus pneumoniae and their distribution in tetracycline-resistant clinical isolates. By comparatively analyzing reference pneumococci carrying erm(B)/tet(M) transposon Tn1545, Tn6003, Tn6002, or Tn3872, we demonstrated a substantial correspondence between Tn1545 and Tn6003, which have the same resistance gene combination [tet(M) (tetracycline), erm(B) (erythromycin), and aphA-3 (kanamycin)]; share the macrolide-aminoglycoside-streptothricin element, containing a second erm(B); and only differ by a ca. 1.2-kb insertion (containing a putative IS1239 insertion sequence) detected in Tn1545 from S. pneumoniae reference strain BM4200. These results enabled elucidation of the structure of Tn1545, the first erm(B)-carrying transposon described in S. pneumoniae. A collection of 83 erythromycin- and tetracycline-resistant clinical pneumococci, representative of recent Italian isolates carrying erm(B) as the sole erythromycin resistance gene, was used to investigate the distribution of the different transposons. All 83 organisms were positive for tet(M) and bore an erm(B)/tet(M) transposon that could be characterized by using a specific set of primer pairs; Tn3872 was detected in 18 isolates, Tn6002 in 59 isolates, and Tn6003 in 6 (the sole kanamycin-resistant) isolates. The genetic organization of transposon Tn1545, with its specific insertion, was not detected in any of the isolates tested. The erm(B)-carrying elements of tetracycline-resistant pneumococci substantially corresponded to those [bearing a silent tet(M) gene] recently detected in tetracycline-susceptible pneumococci. Overall, in erm(B)-positive pneumococci, Tn6003 was the least common erm(B)-carrying Tn916-related element and Tn6002 the most common.
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251. |
Aguiar SI,
Serrano I,
Pinto FR,
Melo-Cristino J,
Ramirez M,
( 2008 ) The presence of the pilus locus is a clonal property among pneumococcal invasive isolates. PMID : 18307767 : DOI : 10.1186/1471-2180-8-41 PMC : PMC2270847 Abstract >>
Pili were recently recognized in Streptococcus pneumoniae and implicated in the virulence of this bacterium, which led to the proposal of using these antigens in a future pneumococcal vaccine. However, pili were found to be encoded by the rlrA islet that was not universally distributed in the species. We examined the distribution of the pilus islet, using the presence of the rlrA gene as a marker for the locus, among a collection of invasive isolates recovered in Portugal and analyzed its association with capsular serotypes, clusters defined by the pulsed-field gel electrophoretic profiles (PFGE) and multilocus sequence types. Only a minority of the isolates were positive for the presence of the rlrA gene (27%). There was a high correspondence between the serotype and the presence or absence of rlrA (Wallace coefficient, W = 0.778). In particular, there was an association between the presence of rlrA and the vaccine serotypes 4, 6B, 9V and 14 whereas the gene was significantly absent from other serotypes, namely 1, 7F, 8, 12B and 23F, a group that included a vaccine serotype (23F) and serotype 1 associated with enhanced invasiveness. Even within serotypes, there was variation in the presence of the pilus islet between PFGE clones and a higher Wallace coefficient (W = 0.939) indicates that carriage of the islet is a clonal property of pneumococci. Analysis of rlrA negative isolates revealed heterogeneity in the genomic region downstream of the rfl gene, the region where the islet is found in other isolates, compatible with recent loss of the islet in some lineages. The pilus islet is present in a minority of pneumococcal isolates recovered from human invasive infections and is therefore not an essential virulence factor in these infections. Carriage of the pilus islet is a clonal property of pneumococci that may vary between isolates expressing the same serotype and loss and acquisition of the islet may be ongoing.
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252. |
Reinert RR,
Filimonova OY,
Al-Lahham A,
Grudinina SA,
Ilina EN,
Weigel LM,
Sidorenko SV,
( 2008 ) Mechanisms of macrolide resistance among Streptococcus pneumoniae isolates from Russia. PMID : 18378707 : DOI : 10.1128/AAC.01270-07 PMC : PMC2415785 Abstract >>
Among 76 macrolide-nonsusceptible Streptococcus pneumoniae isolates collected between 2003 and 2005 from Central Russia, the resistance mechanisms detected in the isolates included erm(B) alone (50%), mef alone [mef(E), mef(I), or a different mef subclass; 19.7%], or both erm(B) and mef(E) (30.3%). Isolates with dual resistance genes [erm(B) and mef(E)] belonged to clonal complex CC81 or CC271.
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253. |
Job V,
Carapito R,
Vernet T,
Dessen A,
Zapun A,
( 2008 ) Common alterations in PBP1a from resistant Streptococcus pneumoniae decrease its reactivity toward beta-lactams: structural insights. PMID : 18055459 : DOI : 10.1074/jbc.M706181200 Abstract >>
The development of high level beta-lactam resistance in the pneumococcus requires the expression of an altered form of PBP1a, in addition to modified forms of PBP2b and PBP2x, which are necessary for the appearance of low levels of resistance. Here, we present the crystal structure of a soluble form of PBP1a from the highly resistant Streptococcus pneumoniae strain 5204 (minimal inhibitory concentration of cefotaxime is 12 mg.liter(-1)). Mutations T371A, which is adjacent to the catalytic nucleophile Ser370, and TSQF(574-577)NTGY, which lie in a loop bordering the active site cleft, were investigated by site-directed mutagenesis. The consequences of these substitutions on reaction kinetics with beta-lactams were probed in vitro, and their effect on resistance was measured in vivo. The results are interpreted in the framework of the crystal structure, which displays a narrower, discontinuous active site cavity, compared with that of PBP1a from the beta-lactam susceptible strain R6, as well as a reorientation of the catalytic Ser370.
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254. |
Parthasarathy G,
Cummings R,
Becker JW,
Soisson SM,
( 2008 ) Surface-entropy reduction approaches to manipulate crystal forms of beta-ketoacyl acyl carrier protein synthase II from Streptococcus pneumoniae. PMID : 18219113 : DOI : 10.1107/S090744490705559X Abstract >>
A series of experiments with beta-ketoacyl acyl carrier protein synthase II (FabF) from Streptococcus pneumonia (spFabF) were undertaken to evaluate the capability of surface-entropy reduction (SER) to manipulate protein crystallization. Previous work has shown that this protein crystallizes in two forms. The triclinic form contains four molecules in the asymmetric unit (a.u.) and diffracts to 2.1 A resolution, while the more desirable primitive orthorhombic form contains one molecule in the a.u. and diffracts to 1.3 A. The aim was to evaluate the effect of SER mutations that were specifically engineered to avoid perturbing the crystal-packing interfaces employed by the favorable primitive orthorhombic crystal form while potentially disrupting a surface of the protein employed by the less desirable triclinic crystal form. Two mutant proteins were engineered, each of which harbored five SER mutations. Extensive crystallization screening produced crystals of the two mutants, but only under conditions that differed from those used for the native protein. One of the mutant proteins yielded crystals that were of a new form (centered orthorhombic), despite the fact that the interfaces employed by the primitive orthorhombic form of the native protein were specifically unaltered. Structure determination at 1.75 A resolution reveals that one of the mutations, E383A, appears to play a key role in disfavouring the less desirable triclinic crystal form and in generating a new surface for a packing interaction that stabilizes the new crystal form.
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255. |
Izdebski R,
Rutschmann J,
Fiett J,
Sadowy E,
Gniadkowski M,
Hryniewicz W,
Hakenbeck R,
( 2008 ) Highly variable penicillin resistance determinants PBP 2x, PBP 2b, and PBP 1a in isolates of two Streptococcus pneumoniae clonal groups, Poland 23F-16 and Poland 6B-20. PMID : 18160523 : DOI : 10.1128/AAC.01082-07 PMC : PMC2258499 Abstract >>
Penicillin-binding proteins (PBPs) in representatives of two Streptococcus pneumoniae clonal groups that are prevalent in Poland, Poland 23F-16 and Poland 6B-20, were investigated by PBP profile analysis, antibody reactivity pattern analysis, and DNA sequence analysis of the transpeptidase (TP) domain-encoding regions of the pbp2x, pbp2b, and pbp1a genes. The isolates differed in their MICs of beta-lactam antibiotics. The majority of the 6B isolates were intermediately susceptible to penicillin (penicillin MICs, 0.12 to 0.5 microg/ml), whereas all 23F isolates were penicillin resistant (MICs, >or=2 microg/ml). The 6B isolates investigated had the same sequence type (ST), determined by multilocus sequence typing, as the Poland 6B-20 reference strain (ST315), but in the 23F group, isolates with three distinct single-locus variants (SLVs) in the ddl gene (ST173, ST272, and ST1506) were included. None of the isolates showed an identical PBP profile after labeling with Bocillin FL and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and only one pair of 6B isolates and one pair of 23F isolates (ST173 and ST272) each contained an identical combination of PBP 2x, PBP 2b, and PBP 1a TP domains. Some 23F isolates contained PBP 3 with an apparently higher electrophoretic mobility, and this feature also did not correlate with their STs. The data document a highly variable pool of PBP genes as a result of multiple gene transfer and recombination events within and between different clonal groups.
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256. |
Brueggemann AB,
Pai R,
Crook DW,
Beall B,
( 2007 ) Vaccine escape recombinants emerge after pneumococcal vaccination in the United States. PMID : 18020702 : DOI : 10.1371/journal.ppat.0030168 PMC : PMC2077903 Abstract >>
The heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in the United States (US) in 2000 and has significantly reduced invasive pneumococcal disease; however, the incidence of nonvaccine serotype invasive disease, particularly due to serotype 19A, has increased. The serotype 19A increase can be explained in part by expansion of a genotype that has been circulating in the US prior to vaccine implementation (and other countries since at least 1990), but also by the emergence of a novel "vaccine escape recombinant" pneumococcal strain. This strain has a genotype that previously was only associated with vaccine serotype 4, but now expresses a nonvaccine serotype 19A capsule. Based on prior evidence for capsular switching by recombination at the capsular locus, the genetic event that resulted in this novel serotype/genotype combination might be identifiable from the DNA sequence of individual pneumococcal strains. Therefore, the aim of this study was to characterise the putative recombinational event(s) at the capsular locus that resulted in the change from a vaccine to a nonvaccine capsular type. Sequencing the capsular locus flanking regions of 51 vaccine escape (progeny), recipient, and putative donor pneumococci revealed a 39 kb recombinational fragment, which included the capsular locus, flanking regions, and two adjacent penicillin-binding proteins, and thus resulted in a capsular switch and penicillin nonsusceptibility in a single genetic event. Since 2003, 37 such vaccine escape strains have been detected, some of which had evolved further. Furthermore, two new types of serotype 19A vaccine escape strains emerged in 2005. To our knowledge, this is the first time a single recombinational event has been documented in vivo that resulted in both a change of serotype and penicillin nonsusceptibility. Vaccine escape by genetic recombination at the capsular locus has the potential to reduce PCV7 effectiveness in the longer term.
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257. |
Dias CA,
Agnes G,
Frazzon AP,
Kruger FD,
d'Azevedo PA,
Carvalho Mda G,
Facklam RR,
Teixeira LM,
( 2007 ) Diversity of mutations in the atpC gene coding for the c Subunit of F0F1 ATPase in clinical isolates of optochin-resistant Streptococcus pneumoniae from Brazil. PMID : 17626173 : DOI : 10.1128/JCM.00891-07 PMC : PMC2045260 Abstract >>
We report the characteristics of four optochin-resistant (Opt(r)) Streptococcus pneumoniae isolates from Brazil. All four Opt(r) isolates presented mutations in the nucleotide sequence coding for the c subunit of F(0)F(1) ATPase. Two isolates showed mutations in codons 23 (leading to the deduced amino acid substitution isoleucine instead of alanine) and 49 (serine instead of alanine, a novel type of mutation detected at this position), respectively. Two additional novel mutations, both located in codon 45, were detected in the other two isolates, corresponding to leucine or valine (instead of phenylalanine). The data indicate that three previously unrecognized alterations were detected in the atpC gene of S. pneumoniae and that Opt resistance among Brazilian pneumococcal isolates is not related to a specific pneumococcal serotype, antimicrobial-resistance profile, or clonal group.
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258. |
Senkovich O,
Cook WJ,
Mirza S,
Hollingshead SK,
Protasevich II,
Briles DE,
Chattopadhyay D,
( 2007 ) Structure of a complex of human lactoferrin N-lobe with pneumococcal surface protein a provides insight into microbial defense mechanism. PMID : 17543335 : DOI : 10.1016/j.jmb.2007.04.075 PMC : PMC5356469 Abstract >>
Human lactoferrin, a component of the innate immune system, kills a wide variety of microorganisms including the Gram positive bacteria Streptococcus pneumoniae. Pneumococcal surface protein A (PspA) efficiently inhibits this bactericidal action. The crystal structure of a complex of the lactoferrin-binding domain of PspA with the N-lobe of human lactoferrin reveals direct and specific interactions between the negatively charged surface of PspA helices and the highly cationic lactoferricin moiety of lactoferrin. Binding of PspA blocks surface accessibility of this bactericidal peptide preventing it from penetrating the bacterial membrane. Results of site-directed mutagenesis, in vitro protein binding assays and isothermal titration calorimetry measurements corroborate that the specific electrostatic interactions observed in the crystal structure represent major associations between PspA and lactoferrin. The structure provides a snapshot of the protective mechanism utilized by pathogens against the host's first line of defense. PspA represents a major virulence factor and a promising vaccine candidate. Insights from the structure of the complex have implications for designing therapeutic strategies for treatment and prevention of pneumococcal diseases that remain a major public health problem worldwide.
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259. |
Park IH,
Park S,
Hollingshead SK,
Nahm MH,
( 2007 ) Genetic basis for the new pneumococcal serotype, 6C. PMID : 17576753 : DOI : 10.1128/IAI.00510-07 PMC : PMC1951153 Abstract >>
We have recently reported a new pneumococcal serotype (6C), which is closely related to serotype 6A (I. H. Park et al., J. Clin. Microbiol. 45:1225-1233, 2007). To investigate the genetic basis for serotype 6C, we studied the capsule gene loci of 14 6C isolates from three different continents, including one isolated in Alabama 27 years ago. The wciN region of all 6C isolates has a 1,029-bp-long sequence that replaces the 1,222-bp-long sequence of the 6A wciN region. This recombination event has created a new 1,125-bp-long open reading frame which encodes a product that is also homologous to glycosyl transferases. Flanking this introduced gene is 300 bp upstream and 100 bp downstream with only about 90% homology with 6A and which is identical in all 6C isolates. Transfer of the wciN region converts 6A to 6C. Determination of the DNA sequence of the entire capsule gene locus of one 6C isolate showed that the 6C capsule gene locus is almost identical (>98% homologous) to that of 6A except for the wciN region. These findings indicate that the 6C capsule type originated more than 27 years ago by a single recombination event in a 6A locus in which 6A wciN was replaced by a gene of unknown origin.
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260. |
Takamatsu A,
Kawaguchiya M,
Chang B,
Ito M,
Hirano Y,
Katsuta S,
Matsuzaka S,
Serizawa Y,
Kobayashi N,
( 2017 ) First report of serotype 23B Streptococcus pneumoniae isolated from an adult patient with invasive infection in Japan. PMID : 28417006 : DOI : 10.1016/j.nmni.2017.02.008 PMC : PMC5388934 Abstract >>
Serotype 23B Streptococcus pneumoniae was isolated from a 67-year-old Japanese patient with meningitis. This isolate was susceptible to penicillin G, while genotyped as gPISP with a mutation in a penicillin-binding motif in PBP2b. The 23B isolate was assigned to ST11996 that is related to CC439, a dominant group among serotype 23B.
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261. |
Geno KA,
Saad JS,
Nahm MH,
( 2017 ) Discovery of Novel Pneumococcal Serotype 35D, a Natural WciG-Deficient Variant of Serotype 35B. PMID : 28202800 : DOI : 10.1128/JCM.00054-17 PMC : PMC5405259 Abstract >>
Pneumococcus (Streptococcus pneumoniae) remains a significant cause of morbidity and mortality, especially among those at the extremes of age. Its capsular polysaccharide is essential for systemic virulence. Over 90 serologically distinct pneumococcal capsular polysaccharides (serotypes) are recognized, but they are unequal in prevalence. Because antibodies against the capsule are protective, polysaccharide conjugate vaccines, which are constructed against the most prevalent serotypes, have caused great reductions in pneumococcal disease caused by these serotypes. In response, however, the relative prevalences of serotypes have shifted. Certain previously rare serotypes, such as serotype 35B, are increasing in prevalence. Serotype 35B is thus a likely future vaccine candidate, but due to their previous rarity, serotype 35B strains have not been scrutinized for underlying heterogeneity. We studied putative serotype 35B clinical isolates to assess the uniformity of their serological reactions. While most isolates exhibited the accepted serology of serotype 35B, one isolate failed to bind to critical serotyping reagents. We determined that the genetic basis for this aberrant serology was the presence of inactivating mutations in the O-acetyltransferase gene wciG Complementation studies in a wciG deletion strain verified that the mutant WciG was nonfunctional, and the serology of the mutant could be restored through complementation with a construct encoding a functional WciG. Nuclear magnetic resonance studies confirmed that the capsule of the WciG-deficient isolate lacked O-acetylation but was otherwise identical to serotype 35B. As this isolate expresses a unique serology with unique biochemistry and a stable genetic basis, we named its novel capsule serotype 35D.
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262. |
de la Campa AG,
Kale P,
Springhorn SS,
Lacks SA,
( 1987 ) Proteins encoded by the DpnII restriction gene cassette. Two methylases and an endonuclease. PMID : 2824782 : DOI : 10.1016/0022-2836(87)90024-6 Abstract >>
Proteins encoded by three genes in the DpnII restriction enzyme cassette of Streptococcus pneumoniae were purified and characterized. Large amounts of the proteins were produced by subcloning the cassette in an Escherichia coli expression system. All three proteins appear to be dimers composed of identical polypeptide subunits. One is the DpnII endonuclease, and the other two are DNA adenine methylase active at 5' GATC 3' sites. Inactivation of enzyme activity by insertions into the genes and comparison of the DNA sequence with the amino-terminal sequence of amino acid residues in the proteins demonstrated the following correspondence between genes and enzymes. The promoter-proximal gene in the operon, dpnM, encodes a 33 X 10(3) Mr polypeptide that gives rise to a potent DNA methylase. The next gene, dpnA, encodes the 31 x 10(3) Mr polypeptide of a weaker and less-specific methylase. The third gene, dpnB, encodes the 34 x 10(3) Mr polypeptide of the endonuclease. Although the endonuclease polypeptide is initiated from an ordinary ribosome-binding site, each of the methylase polypeptide begins at an atypical site with a consensus sequence entirely different from that of Shine & Dalgarno. This presumptive novel ribosome-binding site is well recognized in both S. pneumoniae and E. coli.
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263. |
van der Linden M,
Otten J,
Bergmann C,
Latorre C,
Liñares J,
Hakenbeck R,
( 2017 ) Insight into the Diversity of Penicillin-Binding Protein 2x Alleles and Mutations in Viridans Streptococci. PMID : 28193649 : DOI : 10.1128/AAC.02646-16 PMC : PMC5404556 Abstract >>
The identification of commensal streptococci species is an everlasting problem due to their ability to genetically transform. A new challenge in this respect is the recent description of Streptococcus pseudopneumoniae as a new species, which was distinguished from closely related pathogenic S. pneumoniae and commensal S. mitis by a variety of physiological and molecular biological tests. Forty-one atypical S. pneumoniae isolates have been collected at the German National Reference Center for Streptococci (GNRCS). Multilocus sequence typing (MLST) confirmed 35 isolates as the species S. pseudopneumoniae A comparison with the pbp2x sequences from 120 commensal streptococci isolated from different continents revealed that pbp2x is distinct among penicillin-susceptible S. pseudopneumoniae isolates. Four penicillin-binding protein x (PBPx) alleles of penicillin-sensitive S. mitis account for most of the diverse sequence blocks in resistant S. pseudopneumoniae, S. pneumoniae, and S. mitis, and S. infantis and S. oralis sequences were found in S. pneumoniae from Japan. PBP2x genes of the family of mosaic genes related to pbp2x in the S. pneumoniae clone Spain23F-1 were observed in S. oralis and S. infantis as well, confirming its global distribution. Thirty-eight sites were altered within the PBP2x transpeptidase domains of penicillin-resistant strains, excluding another 37 sites present in the reference genes of sensitive strains. Specific mutational patterns were detected depending on the parental sequence blocks, in agreement with distinct mutational pathways during the development of beta-lactam resistance. The majority of the mutations clustered around the active site, whereas others are likely to affect stability or interactions with the C-terminal domain or partner proteins.
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264. |
de la Campa AG,
Springhorn SS,
Kale P,
Lacks SA,
( 1988 ) Proteins encoded by the DpnI restriction gene cassette. Hyperproduction and characterization of the DpnI endonuclease. PMID : 2844782 : Abstract >>
Insertion mutations in the DpnI gene cassette of Streptococcus pneumoniae indicated that the two genes it contains, dpnC and dpnD, were transcribed from an adjacent promoter and that only dpnC was necessary for expression of the DpnI endonuclease. Large amounts of the DpnI endonuclease were produced from the cloned cassette in an Escherichia coli expression system, and the enzyme was purified to homogeneity. The DpnI endonuclease is composed of a single polypeptide of 30 kDa, which, as shown by NH2-terminal sequencing of the protein, is encoded by the entire dpnC open reading frame. The native protein sedimented as a monomer of 30 kDa in 0.5 M NaCl. A protein composed of a 20-kDa polypeptide, which is presumably encoded by dpnD, was also produced in large amounts. It was partially purified, but its function is unknown. Examination of the predicted amino acid sequence of DpnI revealed a potential metal-containing, DNA-binding finger structure. It is suggested that this structure provides the specificity for recognition of the methylated DNA sequence, 5'-GmATC-3', that is cleaved by the DpnI endonuclease.
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265. |
Houri H,
Tabatabaei SR,
Saee Y,
Fallah F,
Rahbar M,
Karimi A,
( 2017 ) Distribution of capsular types and drug resistance patterns of invasive pediatric Streptococcus pneumoniae isolates in Teheran, Iran. PMID : 28131730 : DOI : 10.1016/j.ijid.2017.01.020 Abstract >>
To explore the serotype distribution and drug resistance patterns of invasive pneumococcal isolates from children under 5 years of age. During a 32-month period, 585 clinical samples (including blood, cerebrospinal fluid (CSF), and synovial fluid) from children suspected of having meningitis, sepsis, pneumonia, or septic arthritis were analyzed using the BACTEC culture system. Positive cultures were examined using biochemical tests and lytA amplification for the identification of pneumococcal strains. The confirmed pneumococcal isolates were examined to determine capsular types using a modified sequential multiplex PCR and susceptibility to antimicrobial agents. Fifty-three pneumococcal isolates were detected in the 585 clinical samples: 21 (39.6%) blood samples and 32 (60.4%) CSF samples. The most frequent serotype was 23F (24.5%), followed by serotypes 19F (18.9%), 19A (7.5%), and 9V (7.5%). Twenty-one percent of pneumococcal isolates were penicillin-non-susceptible and serotype 19A was significantly associated with resistance to penicillin. This study indicated that the 13-valent pneumococcal conjugate vaccine (PCV13) could cover the majority of the invasive pneumococcal isolates. Drug-resistant and multidrug-resistant Streptococcus pneumoniae strains are circulating in Iran. Therefore, public immunization of infants using PCV13 is recommended to reduce the incidence of pneumococcal disease and pneumococcal-resistant strains in Teheran.
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266. |
Robb M,
Hobbs JK,
Woodiga SA,
Shapiro-Ward S,
Suits MD,
McGregor N,
Brumer H,
Yesilkaya H,
King SJ,
Boraston AB,
( 2017 ) Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence. PMID : 28056108 : DOI : 10.1371/journal.ppat.1006090 PMC : PMC5215778 Abstract >>
The carbohydrate-rich coating of human tissues and cells provide a first point of contact for colonizing and invading bacteria. Commensurate with N-glycosylation being an abundant form of protein glycosylation that has critical functional roles in the host, some host-adapted bacteria possess the machinery to process N-linked glycans. The human pathogen Streptococcus pneumoniae depolymerizes complex N-glycans with enzymes that sequentially trim a complex N-glycan down to the Man3GlcNAc2 core prior to the release of the glycan from the protein by endo-�]-N-acetylglucosaminidase (EndoD), which cleaves between the two GlcNAc residues. Here we examine the capacity of S. pneumoniae to process high-mannose N-glycans and transport the products. Through biochemical and structural analyses we demonstrate that S. pneumoniae also possesses an �\-(1,2)-mannosidase (SpGH92). This enzyme has the ability to trim the terminal �\-(1,2)-linked mannose residues of high-mannose N-glycans to generate Man5GlcNAc2. Through this activity SpGH92 is able to produce a substrate for EndoD, which is not active on high-mannose glycans with �\-(1,2)-linked mannose residues. Binding studies and X-ray crystallography show that NgtS, the solute binding protein of an ABC transporter (ABCNG), is able to bind Man5GlcNAc, a product of EndoD activity, with high affinity. Finally, we evaluated the contribution of EndoD and ABCNG to growth of S. pneumoniae on a model N-glycosylated glycoprotein, and the contribution of these enzymes and SpGH92 to virulence in a mouse model. We found that both EndoD and ABCNG contribute to growth of S. pneumoniae, but that only SpGH92 and EndoD contribute to virulence. Therefore, N-glycan processing, but not transport of the released glycan, is required for full virulence in S. pneumoniae. To conclude, we synthesize our findings into a model of N-glycan processing by S. pneumoniae in which both complex and high-mannose N-glycans are targeted, and in which the two arms of this degradation pathway converge at ABCNG.
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267. |
Moreau C,
Terrasse R,
Thielens NM,
Vernet T,
Gaboriaud C,
Di Guilmi AM,
( 2017 ) Deciphering Key Residues Involved in the Virulence-promoting Interactions between Streptococcus pneumoniae and Human Plasminogen. PMID : 28011643 : DOI : 10.1074/jbc.M116.764209 PMC : PMC5313095 Abstract >>
Bacterial pathogens recruit circulating proteins to their own surfaces, co-opting the host protein functions as a mechanism of virulence. Particular attention has focused on the binding of plasminogen (Plg) to bacterial surfaces, as it has been shown that this interaction contributes to bacterial adhesion to host cells, invasion of host tissues, and evasion of the immune system. Several bacterial proteins are known to serve as receptors for Plg including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytoplasmic enzyme that appears on the cell surface in this moonlighting role. Although Plg typically binds to these receptors via several lysine-binding domains, the specific interactions that occur have not been documented in all cases. However, identification of the relevant residues could help define strategies for mitigating the virulence of important human pathogens, such as Streptococcus pneumoniae (Sp). To shed light on this question, we have described a combination of peptide-spot array screening, competition and SPR assays, high-resolution crystallography, and mutational analyses to characterize the interaction between SpGAPDH and Plg. We identified three SpGAPDH lysine residues that were instrumental in defining the kinetic and thermodynamic parameters of the interaction. Altogether, the integration of the data presented in this work allows us to propose a structural model for the molecular interaction of the SpGAPDH-Plg complex.
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268. |
Dowson CG,
Hutchison A,
Brannigan JA,
George RC,
Hansman D,
Liñares J,
Tomasz A,
Smith JM,
Spratt BG,
( 1989 ) Horizontal transfer of penicillin-binding protein genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. PMID : 2813426 : DOI : 10.1073/pnas.86.22.8842 PMC : PMC298386 Abstract >>
Resistance to penicillin in clinical isolates of Streptococcus pneumoniae has occurred by the development of altered penicillin-binding proteins (PBPs) that have greatly decreased affinity for the antibiotic. We have investigated the origins of penicillin-resistant strains by comparing the sequences of the transpeptidase domain of PBP2B from 6 penicillin-sensitive and 14 penicillin-resistant strains. In addition we have sequenced part of the amylomaltase gene from 2 of the sensitive and 6 of the resistant strains. The sequences of the amylomaltase gene of all of the strains and of the PBP2B gene of the penicillin-sensitive strain show that S. pneumoniae is genetically very uniform. In contrast the PBP2B genes of the penicillin-resistant strains show approximately equal to 14% sequence divergence from those of the penicillin-sensitive strains and the development of penicillin resistance has involved the replacement, presumably by transformation, of the original PBP2B gene by a homologous gene from an unknown source. This genetic event has occurred on at least two occasions, involving different sources, to produce the two classes of altered PBP2B genes found in penicillin-resistant strains of S. pneumoniae. There is considerable variation among the PBP2B genes of the resistant strains that may have arisen by secondary transformation events accompanied by mismatch repair subsequent to their original introductions into S. pneumoniae.
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269. |
Corsini B,
Aguinagalde L,
Ruiz S,
Domenech M,
Antequera ML,
Fenoll A,
García P,
García E,
Yuste J,
( 2016 ) Immunization with LytB protein of Streptococcus pneumoniae activates complement-mediated phagocytosis and induces protection against pneumonia and sepsis. PMID : 27840016 : DOI : 10.1016/j.vaccine.2016.11.001 Abstract >>
The cell wall glucosaminidase LytB of Streptococcus pneumoniae is a surface exposed protein involved in daughter cell separation, biofilm formation and contributes to different aspects of the pathogenesis process. In this study we have characterized the antibody responses after immunization of mice with LytB in the presence of alhydrogel as an adjuvant. Enzyme-linked immunosorbent assays measuring different subclasses of immunoglobulin G, demonstrated that the antibody responses to LytB were predominantly IgG1 and IgG2b, followed by IgG3 and IgG2a subclasses. Complement-mediated immunity against two different pneumococcal serotypes was investigated using sera from immunized mice. Immunization with LytB increased the recognition of S. pneumoniae by complement components C1q and C3b demonstrating that anti-LytB antibodies trigger activation of the classical pathway. Phagocytosis assays showed that serum containing antibodies to LytB stimulates neutrophil-mediated phagocytosis against S. pneumoniae. Animal models of infection including invasive pneumonia and sepsis were performed with two different clinical isolates. Vaccination with LytB increased bacterial clearance and induced protection demonstrating that LytB might be a good candidate to be considered in a future protein-based vaccine against S. pneumoniae.
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270. |
Xu Z,
von Grafenstein S,
Walther E,
Fuchs JE,
Liedl KR,
Sauerbrei A,
Schmidtke M,
( 2016 ) Sequence diversity of NanA manifests in distinct enzyme kinetics and inhibitor susceptibility. PMID : 27125351 : DOI : 10.1038/srep25169 PMC : PMC4850393 Abstract >>
Streptococcus pneumoniae is the leading pathogen causing bacterial pneumonia and meningitis. Its surface-associated virulence factor neuraminidase A (NanA) promotes the bacterial colonization by removing the terminal sialyl residues from glycoconjugates on eukaryotic cell surface. The predominant role of NanA in the pathogenesis of pneumococci renders it an attractive target for therapeutic intervention. Despite the highly conserved activity of NanA, our alignment of the 11 NanAs revealed the evolutionary diversity of this enzyme. The amino acid substitutions we identified, particularly those in the lectin domain and in the insertion domain next to the catalytic centre triggered our special interest. We synthesised the representative NanAs and the mutagenized derivatives from E. coli for enzyme kinetics study and neuraminidase inhibitor susceptibility test. Via molecular docking we got a deeper insight into the differences between the two major variants of NanA and their influence on the ligand-target interactions. In addition, our molecular dynamics simulations revealed a prominent intrinsic flexibility of the linker between the active site and the insertion domain, which influences the inhibitor binding. Our findings for the first time associated the primary sequence diversity of NanA with the biochemical properties of the enzyme and with the inhibitory efficiency of neuraminidase inhibitors.
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271. |
Simões AS,
Tavares DA,
Rolo D,
Ardanuy C,
Goossens H,
Henriques-Normark B,
Linares J,
de Lencastre H,
Sá-Leão R,
( 2016 ) lytA-based identification methods can misidentify Streptococcus pneumoniae. PMID : 27107535 : DOI : 10.1016/j.diagmicrobio.2016.03.018 Abstract >>
During surveillance studies we detected, among over 1500 presumptive pneumococci, 11 isolates displaying conflicting or novel results when characterized by widely accepted phenotypic (optochin susceptibility and bile solubility) and genotypic (lytA-BsaAI-RFLP and MLST) identification methods. We aimed to determine the genetic basis for the unexpected results given by lytA-BsaAI-RFLP and investigate the accuracy of the WHO recommended lytA real-time PCR assay to classify these 11 isolates. Three novel lytA-BsaAI-RFLP signatures were found (one in pneumococcus and two in S. mitis). In addition, one pneumococcus displayed the atypical lytA-BsaAI-RFLP signature characteristic of non-pneumococci and two S. pseudopneumoniae displayed the typical lytA-BsaAI-RFLP pattern characteristic of pneumococci. lytA real-time PCR misidentified these three isolates. In conclusion, identification of pneumococci by lytA real-time PCR, and other lytA-based methodologies, may lead to false results. This is of particular relevance in the increasingly frequent colonization studies relying solely on culture-independent methods.
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272. |
Singh AK,
Osman AS,
Woodiga SA,
White P,
Mahan JD,
King SJ,
( 2016 ) Defining the role of pneumococcal neuraminidases and O-glycosidase in pneumococcal haemolytic uraemic syndrome. PMID : 27469261 : DOI : 10.1099/jmm.0.000322 Abstract >>
The host and bacterial factors that lead to development of pneumococcal haemolytic uraemic syndrome (pHUS) remain poorly defined; however, it is widely believed that pneumococcal exposure of the Thomsen-Friedenreich antigen (T-antigen) on host surfaces is a key step in pathogenesis. Two enzymatic activities encoded by pneumococci determine the level of T-antigen exposed. Neuraminidases cleave terminal sialic acid to expose the T-antigen which is subsequently cleaved by O-glycosidase Eng. While a handful of studies have examined the role of neuraminidases in T-antigen exposure, no studies have addressed the potential role of O-glycosidase. This study used 29 pHUS isolates from the USA and 31 serotype-matched controls. All isolates contained eng, and no significant correlation between enzymatic activity and disease state (pHUS and blood non-pHUS isolates) was observed. A prior study from Taiwan suggested that neuraminidase NanC contributes to the development of pHUS. However, we observed no difference in nanC distribution. Similar to previously published data, we found no significant correlation between neuraminidase activity and disease state. Accurate quantification of these enzymatic activities from bacteria grown in whole blood is currently impossible, but we confirmed that there were no significant correlations between disease state and neuraminidase and O-glycosidase transcript levels after incubation in blood. Genomic sequencing of six pHUS isolates did not identify any genetic elements possibly contributing to haemolytic uraemic syndrome. These findings support the hypothesis that while exposure of T-antigen may be an important step in disease pathogenesis, host factors likely play a substantial role in determining which individuals develop haemolytic uraemic syndrome after pneumococcal invasive disease.
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273. |
Liu EY,
Chang JC,
Lin JC,
Chang FY,
Fung CP,
( 2016 ) Important Mutations Contributing to High-Level Penicillin Resistance in Taiwan19F-14, Taiwan23F-15, and Spain23F-1 of Streptococcus pneumoniae Isolated from Taiwan. PMID : 27042760 : DOI : 10.1089/mdr.2015.0261 Abstract >>
Penicillin-resistant Streptococcus pneumoniae is a serious concern worldwide. In this study, we analyzed the cause of �]-lactam resistance in pandemic multidrug-resistant clones. A total of 41 penicillin-nonsusceptible clinical isolates were collected from 1996 to 2012. Sero- and molecular typing confirmed that these isolates were clonal types of Taiwan19F-14, Taiwan23F-15, and Spain23F-1. Sero-switching was found in four isolates. All isolates were multidrug resistant. Sequencing analysis of the penicillin binding proteins (PBPs) was performed on PBP1a, 2b, and 2x, and a large number of mutations were identified in comparing to clinical penicillin-susceptible isolates and the recipient strain R6 used for homologous recombination. The T451A substitution was the key amino acid in PBP2b that contributed to penicillin resistance. T338A in PBP2x played a role in resistance and reached the highest level of resistance when combined with other mutations in PBP2x. High-level penicillin resistance could not be obtained without the combination of mutations in PBP1a with PBP2b and 2x. The amino acid substitutions in PBP1a, 2b, and 2x were the crucial factors for �]-lactam resistance.
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274. |
Burton RL,
Geno KA,
Saad JS,
Nahm MH,
( 2016 ) Pneumococcus with the "6E" cps Locus Produces Serotype 6B Capsular Polysaccharide. PMID : 26818670 : DOI : 10.1128/JCM.03194-15 PMC : PMC4809907 Abstract >>
Genetic studies of serogroup 6 isolates ofStreptococcus pneumoniaeidentified putative serotype 6E. Although its capsular polysaccharide structure has not been elucidated, putative serotype 6E is described in an increasing number of studies as a potentially new serotype. We show here that SPEC6B, which is widely used as a target strain for serotype 6B opsonophagocytosis assays, has the genetic features of the putative serotype 6E but produces capsular polysaccharide identical to 6B capsular polysaccharide as determined by one-dimensional (1D) and 2D nuclear magnetic resonance (NMR). Thus, putative serotype 6E is a mere genetic variant of serotype 6B. Also, SPEC6B is appropriate as a target strain for serotype 6B opsonophagocytosis assays. This example illustrates the difficulties of assigning new bacterial serotypes based on genetic findings alone.
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275. |
Kawaguchiya M,
Urushibara N,
Kobayashi N,
( 2017 ) Multidrug Resistance in Non-PCV13 Serotypes of Streptococcus pneumoniae in Northern Japan, 2014. PMID : 27257915 : DOI : 10.1089/mdr.2016.0054 Abstract >>
Since the implementation of routine PCV13 immunization in Japan, nonvaccine serotypes (NVTs) have been increasing among clinical isolates of Streptococcus pneumoniae. In this study, susceptibility to 18 antibiotics was tested for all the 231 isolates with NVTs, which were collected from children <16 years of age in northern Japan in 2014 (July-November). High resistance rates were observed for macrolides (>90.9%), tetracycline (91.3%), and clindamycin (75.3%), while penicillin (PEN) nonsusceptibility (PNSP; MIC ?0.12 �gg/ml) was detected in 42.9% of the pneumococci [39.4%; PEN-intermediate S. pneumoniae (PISP), 3.5%; PEN-resistant S. pneumoniae (PRSP)]. All serotype 15A isolates were PRSP (MIC, ?2 �gg/ml) or PISP, and PNSP was prevalent in also serotypes 23A (96.9%), 6C (41%), and 35B (33.3%). Overall, 42.0% of the isolates showed multidrug resistance (MDR). Sequence types (STs) determined for 20 PNSP isolates with NVTs were ST63 (15A), STs 242 or 5832 (6C), STs 338 or 5242 (23A), and ST558 (35B). All the PNSP isolates possessed tet(M), and erm(B) or mefA(A/E), and 70% of them were gPRSP having three altered genes pbp1a, pbp2x, and pbp2b. Among alterations in transpeptidase-coding region of penicillin-binding proteins (PBPs), two substitutions of T371S in the STMK motif and TSQF574-577NTGY in PBP1a were common to all PRSP isolates. The present study showed the spread of PNSP in NVTs 15A, 23A, 6C, and 35B, and the emergence of the MDR international clone Sweden15A-ST63 in northern Japan.
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276. |
Prudhomme M,
Martin B,
Mejean V,
Claverys JP,
( 1989 ) Nucleotide sequence of the Streptococcus pneumoniae hexB mismatch repair gene: homology of HexB to MutL of Salmonella typhimurium and to PMS1 of Saccharomyces cerevisiae. PMID : 2676973 : DOI : 10.1128/jb.171.10.5332-5338.1989 PMC : PMC210370 Abstract >>
The Hex mismatch repair system of Streptococcus pneumoniae acts both during transformation (a recombination process that directly produces heteroduplex DNA) to correct donor strands and after DNA replication to remove misincorporated nucleotides. The hexB gene product is one of at least two proteins required for mismatch repair in this organism. The nucleotide sequence of a 2.7-kilobase segment from the S. pneumoniae chromosome that includes the 1.95-kilobase hexB gene was determined. The gene encodes a 73.5-kilodalton protein (649 residues). The spontaneous hex Rx chromosomal mutant allele with which a mutator phenotype has been associated is shown to result from a single base substitution (TAC to TAA) leading to a truncated HexB polypeptide (484 residues). The HexB protein is homologous to the MutL protein, which is required for methyl-directed mismatch repair in Salmonella typhimurium and Escherichia coli, and to the PMS1 gene product, which is likely to be involved in a mismatch correction system in Saccharomyces cerevisiae. The conservation of HexB-like proteins among procaryotic and eucaryotic organisms indicates that these proteins play an important common role in the repair process. This finding also suggests that the Hex, Mut, and PMS systems evolved from a common ancestor and that functionally similar mismatch repair systems could be widespread among procaryotic as well as eucaryotic organisms.
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277. |
Laible G,
Hakenbeck R,
Sicard MA,
Joris B,
Ghuysen JM,
( 1989 ) Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506. PMID : 2615650 : DOI : 10.1111/j.1365-2958.1989.tb00115.x Abstract >>
Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin-binding proteins (PBPs) which reduce their affinity for beta-lactam antibiotics. PBP2x is one of the high-Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime-resistant laboratory mutants. In this study, we have sequenced a 2564 base-pair chromosomal fragment from the penicillin-sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base-pair open reading frame was found which coded for a protein having an Mr of 82.35kD, a value which is in good agreement with the Mr of 80-85 kD measured by SDS-gel electrophoresis of the PBP2x protein itself. The N-terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime-resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the beta-lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from beta-lactam-resistant strains are known. The penicillin-binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N-terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin-resistant Staphylococcus aureus PBP2'. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low-Mr PBPs.
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278. |
Walther E,
Richter M,
Xu Z,
Kramer C,
von Grafenstein S,
Kirchmair J,
Grienke U,
Rollinger JM,
Liedl KR,
Slevogt H,
Sauerbrei A,
Saluz HP,
Pfister W,
Schmidtke M,
( 2015 ) Antipneumococcal activity of neuraminidase inhibiting artocarpin. PMID : 25592264 : DOI : 10.1016/j.ijmm.2014.12.004 PMC : PMC5730044 Abstract >>
Streptococcus (S.) pneumoniae is a major cause of secondary bacterial pneumonia during influenza epidemics. Neuraminidase (NA) is a virulence factor of both pneumococci and influenza viruses. Bacterial neuraminidases (NAs) are structurally related to viral NA and susceptible to oseltamivir, an inhibitor designed to target viral NA. This prompted us to evaluate the antipneumococcal potential of two NA inhibiting natural compounds, the diarylheptanoid katsumadain A and the isoprenylated flavone artocarpin. Chemiluminescence, fluorescence-, and hemagglutination-based enzyme assays were applied to determine the inhibitory efficiency (IC(50) value) of the tested compounds towards pneumococcal NAs. The mechanism of inhibition was studied via enzyme kinetics with recombinant NanA NA. Unlike oseltamivir, which competes with the natural substrate of NA, artocarpin exhibits a mixed-type inhibition with a Ki value of 9.70 �gM. Remarkably, artocarpin was the only NA inhibitor (NAI) for which an inhibitory effect on pneumococcal growth (MIC: 0.99-5.75 �gM) and biofilm formation (MBIC: 1.15-2.97 �gM) was observable. In addition, we discovered that the bactericidal effect of artocarpin can reduce the viability of pneumococci by a factor of >1000, without obvious harm to lung epithelial cells. This renders artocarpin a promising natural product for further investigations.
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279. |
Hilty M,
Wüthrich D,
Salter SJ,
Engel H,
Campbell S,
Sá-Leão R,
de Lencastre H,
Hermans P,
Sadowy E,
Turner P,
Chewapreecha C,
Diggle M,
Pluschke G,
McGee L,
Eser ?K,
Low DE,
Smith-Vaughan H,
Endimiani A,
Küffer M,
Dupasquier M,
Beaudoing E,
Weber J,
Bruggmann R,
Hanage WP,
Parkhill J,
Hathaway LJ,
Mühlemann K,
Bentley SD,
( 2014 ) Global phylogenomic analysis of nonencapsulated Streptococcus pneumoniae reveals a deep-branching classic lineage that is distinct from multiple sporadic lineages. PMID : 25480686 : DOI : 10.1093/gbe/evu263 PMC : PMC4986459 Abstract >>
The surrounding capsule of Streptococcus pneumoniae has been identified as a major virulence factor and is targeted by pneumococcal conjugate vaccines (PCV). However, nonencapsulated S. pneumoniae (non-Ec-Sp) have also been isolated globally, mainly in carriage studies. It is unknown if non-Ec-Sp evolve sporadically, if they have high antibiotic nonsusceptiblity rates and a unique, specific gene content. Here, whole-genome sequencing of 131 non-Ec-Sp isolates sourced from 17 different locations around the world was performed. Results revealed a deep-branching classic lineage that is distinct from multiple sporadic lineages. The sporadic lineages clustered with a previously sequenced, global collection of encapsulated S. pneumoniae (Ec-Sp) isolates while the classic lineage is comprised mainly of the frequently identified multilocus sequences types (STs) ST344 (n = 39) and ST448 (n = 40). All ST344 and nine ST448 isolates had high nonsusceptiblity rates to �]-lactams and other antimicrobials. Analysis of the accessory genome reveals that the classic non-Ec-Sp contained an increased number of mobile elements, than Ec-Sp and sporadic non-Ec-Sp. Performing adherence assays to human epithelial cells for selected classic and sporadic non-Ec-Sp revealed that the presence of a integrative conjugative element (ICE) results in increased adherence to human epithelial cells (P = 0.005). In contrast, sporadic non-Ec-Sp lacking the ICE had greater growth in vitro possibly resulting in improved fitness. In conclusion, non-Ec-Sp isolates from the classic lineage have evolved separately. They have spread globally, are well adapted to nasopharyngeal carriage and are able to coexist with Ec-Sp. Due to continued use of PCV, non-Ec-Sp may become more prevalent.
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280. |
Saxena S,
Khan N,
Dehinwal R,
Kumar A,
Sehgal D,
( 2015 ) Conserved surface accessible nucleoside ABC transporter component SP0845 is essential for pneumococcal virulence and confers protection in vivo. PMID : 25689507 : DOI : 10.1371/journal.pone.0118154 PMC : PMC4331430 Abstract >>
Streptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis and meningitis. Surface accessible proteins of S. pneumoniae are being explored for the development of a protein-based vaccine in order to overcome the limitations of existing polysaccharide-based pneumococcal vaccines. To identify a potential vaccine candidate, we resolved surface-associated proteins of S. pneumoniae TIGR4 strain using two-dimensional gel electrophoresis followed by immunoblotting with antisera generated against whole heat-killed TIGR4. Ten immunoreactive spots were identified by mass spectrometric analysis that included a putative lipoprotein SP0845. Analysis of the inferred amino acid sequence of sp0845 homologues from 36 pneumococcal strains indicated that SP0845 was highly conserved (>98% identity) and showed less than 11% identity with any human protein. Our bioinformatic and functional analyses demonstrated that SP0845 is the substrate-binding protein of an ATP-binding cassette (ABC) transporter that is involved in nucleoside uptake with cytidine, uridine, guanosine and inosine as the preferred substrates. Deletion of the gene encoding SP0845 renders pneumococci avirulent suggesting that it is essential for virulence. Immunoblot analysis suggested that SP0845 is expressed in in vitro grown pneumococci and during mice infection. Immunofluorescence microscopy and flow cytometry data indicated that SP0845 is surface exposed in encapsulated strains and accessible to antibodies. Subcutaneous immunization with recombinant SP0845 induced high titer antibodies in mice. Hyperimmune sera raised against SP0845 promoted killing of encapsulated pneumococcal strains in a blood bactericidal assay. Immunization with SP0845 protected mice from intraperitoneal challenge with heterologous pneumococcal serotypes. Based on its surface accessibility, role in virulence and ability to elicit protective immunity, we propose that SP0845 may be a potential candidate for a protein-based pneumococcal vaccine.
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281. |
Owen CD,
Lukacik P,
Potter JA,
Sleator O,
Taylor GL,
Walsh MA,
( 2015 ) Streptococcus pneumoniae NanC: STRUCTURAL INSIGHTS INTO THE SPECIFICITY AND MECHANISM OF A SIALIDASE THAT PRODUCES A SIALIDASE INHIBITOR. PMID : 26370075 : DOI : 10.1074/jbc.M115.673632 PMC : PMC4646021 Abstract >>
Streptococcus pneumoniae is an important human pathogen that causes a range of disease states. Sialidases are important bacterial virulence factors. There are three pneumococcal sialidases: NanA, NanB, and NanC. NanC is an unusual sialidase in that its primary reaction product is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, also known as DANA), a nonspecific hydrolytic sialidase inhibitor. The production of Neu5Ac2en from �\2-3-linked sialosides by the catalytic domain is confirmed within a crystal structure. A covalent complex with 3-fluoro-�]-N-acetylneuraminic acid is also presented, suggesting a common mechanism with other sialidases up to the final step of product formation. A conformation change in an active site hydrophobic loop on ligand binding constricts the entrance to the active site. In addition, the distance between the catalytic acid/base (Asp-315) and the ligand anomeric carbon is unusually short. These features facilitate a novel sialidase reaction in which the final step of product formation is direct abstraction of the C3 proton by the active site aspartic acid, forming Neu5Ac2en. NanC also possesses a carbohydrate-binding module, which is shown to bind �\2-3- and �\2-6-linked sialosides, as well as N-acetylneuraminic acid, which is captured in the crystal structure following hydration of Neu5Ac2en by NanC. Overall, the pneumococcal sialidases show remarkable mechanistic diversity while maintaining a common structural scaffold.
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282. |
Na IY,
Baek JY,
Park IH,
Kim DH,
Song JH,
Ko KS,
( 2015 ) pspK gene prevalence and characterization of non-typable Streptococcus pneumonia isolates from Asian countries. PMID : 25750083 : DOI : 10.1099/mic.0.000073 Abstract >>
Recently, it has been reported that some non-typable (NT) Streptococcus pneumoniae isolates from Korea and other countries contained a novel gene pspK in the capsular polysaccharide synthesis (cps) locus. In this study, we investigated the presence of pspK in 120 NT S. pneumoniae isolates from 12 Asian countries; isolate characteristics were also examined. The presence of pspK was assayed by PCR. Clonality of NT S. pneumoniae isolates containing pspK was investigated by MLST and PFGE. Antimicrobial susceptibility testing was performed and the structure of pspK was also determined. Nineteen NT isolates (15.8 %) were identified as containing pspK: two isolates from Korea, four from Vietnam, two from Hong Kong, eight from Thailand, and one each from Taiwan, the Philippines and Saudi Arabia. Seven isolates from Korea, Vietnam and Thailand were identified as ST1106, whereas just one or two belonged to ST310, ST393, ST10137, ST2754 or ST4136. All but one of the ST1106 NT isolates showed non-susceptibility to penicillin, and all isolates were resistant to cefuroxime, erythromycin, clindamycin and trimethoprim/sulfamethoxazole. The structure of pspK was similar amongst 20 isolates, which had a R1-R2-like region and a variable number of repeats in the repetitive region. However, one isolate (P05-11) from the Philippines lacked the R1-R2 region. NT S. pneumoniae isolates containing pspK were distributed across several Asian countries. Although MLST analysis suggested that most pspK-containing NT S. pneumoniae isolates may have emerged independently, ST1106 isolates with the selective advantage of antimicrobial resistance may have disseminated clonally throughout the countries.
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283. |
Poyart-Salmeron C,
Trieu-Cuot P,
Carlier C,
Courvalin P,
( 1989 ) Molecular characterization of two proteins involved in the excision of the conjugative transposon Tn1545: homologies with other site-specific recombinases. PMID : 2551683 : PMC : PMC401188 Abstract >>
Excision is probably the initial and rate-limiting step of the movements of conjugative transposons of Gram-positive bacteria such as Tn916 and Tn1545. We have shown, by molecular cloning and DNA sequencing, that a 2058 bp Sau3A right-junction fragment of transposon Tn1545 specifies two gene products that are involved in the excision of the element. The DNA sequence of these genes, designated orf1 and orf2, has been determined and the corresponding proteins, ORF1 and ORF2, have been identified in a bacterial cell-free coupled transcription-translation system. These proteins are freely diffusible since they are able to trans-complement in vivo a deletion derivative of Tn1545 defective for excision. Using an in vivo complementation assay, we have demonstrated that ORF2 alone is able to catalyse excision and that ORF1 strongly stimulates the activity of ORF2. We also found that ORF1 and ORF2 display local homology with, respectively, proteins Xis and Int from lamboid phages, which suggests that these excision systems have a common origin. Based on the functional properties of the integrase of bacteriophage lambda, on the analysis of the nucleotide sequence of the junction fragments and of the target before insertion and after excision, a model is proposed for ORF2-catalysed excision of Tn1545 and related conjugative transposons.
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284. |
Hole?ková N,
Doubravová L,
Massidda O,
Molle V,
Buriánková K,
Benada O,
Kofro?ová O,
Ulrych A,
Branny P,
( 2014 ) LocZ is a new cell division protein involved in proper septum placement in Streptococcus pneumoniae. PMID : 25550321 : DOI : 10.1128/mBio.01700-14 PMC : PMC4281919 Abstract >>
How bacteria control proper septum placement at midcell, to guarantee the generation of identical daughter cells, is still largely unknown. Although different systems involved in the selection of the division site have been described in selected species, these do not appear to be widely conserved. Here, we report that LocZ (Spr0334), a newly identified cell division protein, is involved in proper septum placement in Streptococcus pneumoniae. We show that locZ is not essential but that its deletion results in cell division defects and shape deformation, causing cells to divide asymmetrically and generate unequally sized, occasionally anucleated, daughter cells. LocZ has a unique localization profile. It arrives early at midcell, before FtsZ and FtsA, and leaves the septum early, apparently moving along with the equatorial rings that mark the future division sites. Consistently, cells lacking LocZ also show misplacement of the Z-ring, suggesting that it could act as a positive regulator to determine septum placement. LocZ was identified as a substrate of the Ser/Thr protein kinase StkP, which regulates cell division in S. pneumoniae. Interestingly, homologues of LocZ are found only in streptococci, lactococci, and enterococci, indicating that this close phylogenetically related group of bacteria evolved a specific solution to spatially regulate cell division. Bacterial cell division is a highly ordered process regulated in time and space. Recently, we reported that the Ser/Thr protein kinase StkP regulates cell division in Streptococcus pneumoniae, through phosphorylation of several key proteins. Here, we characterized one of the StkP substrates, Spr0334, which we named LocZ. We show that LocZ is a new cell division protein important for proper septum placement and likely functions as a marker of the cell division site. Consistently, LocZ supports proper Z-ring positioning at midcell. LocZ is conserved only among streptococci, lactococci, and enterococci, which lack homologues of the Min and nucleoid occlusion effectors, indicating that these bacteria adapted a unique mechanism to find their middle, reflecting their specific shape and symmetry.
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285. |
Yun KW,
Lee H,
Choi EH,
Lee HJ,
( 2015 ) Diversity of Pneumolysin and Pneumococcal Histidine Triad Protein D of Streptococcus pneumoniae Isolated from Invasive Diseases in Korean Children. PMID : 26252211 : DOI : 10.1371/journal.pone.0134055 PMC : PMC4529296 Abstract >>
Pneumolysin (Ply) and pneumococcal histidine triad protein D (PhtD) are candidate proteins for a next-generation pneumococcal vaccine. We aimed to analyze the genetic diversity and antigenic heterogeneity of Ply and PhtD for 173 pneumococci isolated from invasive diseases in Korean children. Allele was designated based on the variation of amino acid sequence. Antigenicity was predicted by the amino acid hydrophobicity of the region. There were seven and 39 allele types for the ply and phtD genes, respectively. The nucleotide sequence identity was 97.2%-99.9% for ply and 91.4%-98.0% for phtD gene. Only minor variations in hydrophobicity were noted among the antigenicity plots of Ply and PhtD. Overall, the allele types of the ply and phtD genes were remarkably homogeneous, and the antigenic diversity of the corresponding proteins was very limited. The Ply and PhtD could be useful antigens for universal pneumococcal vaccines.
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286. |
Shaik MM,
Lombardi C,
Maragno Trindade D,
Fenel D,
Schoehn G,
Di Guilmi AM,
Dessen A,
( 2015 ) A structural snapshot of type II pilus formation in Streptococcus pneumoniae. PMID : 26198632 : DOI : 10.1074/jbc.M115.647834 PMC : PMC4566232 Abstract >>
Pili are fibrous appendages expressed on the surface of a vast number of bacterial species, and their role in surface adhesion is important for processes such as infection, colonization, andbiofilm formation. The human pathogen Streptococcus pneumoniae expresses two different types of pili, PI-1 and PI-2, both of which require the concerted action of structural proteins and sortases for their polymerization. The type PI-1 streptococcal pilus is a complex, well studied structure, but the PI-2 type, present in a number of invasive pneumococcal serotypes, has to date remained less well understood. The PI-2 pilus consists of repeated units of a single protein, PitB, whose covalent association is catalyzed by cognate sortase SrtG-1 and partner protein SipA. Here we report the high resolution crystal structures of PitB and SrtG1 and use molecular modeling to visualize a "trapped" 1:1 complex between the two molecules. X-ray crystallography and electron microscopy reveal that the pneumococcal PI-2 backbone fiber is formed by PitB monomers associated in head-to-tail fashion and that short, flexible fibers can be formed even in the absence of coadjuvant proteins. These observations, obtained with a simple pilus biosynthetic system, are likely to be applicable to other fiber formation processes in a variety of Gram-positive organisms.
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287. |
Park IH,
Geno KA,
Yu J,
Oliver MB,
Kim KH,
Nahm MH,
( 2015 ) Genetic, biochemical, and serological characterization of a new pneumococcal serotype, 6H, and generation of a pneumococcal strain producing three different capsular repeat units. PMID : 25589550 : DOI : 10.1128/CVI.00647-14 PMC : PMC4340893 Abstract >>
Streptococcus pneumoniae clinical isolates were recently described that produced capsular polysaccharide with properties of both serotypes 6A and 6B. Their hybrid serological property correlated with mutations affecting the glycosyltransferase WciP, which links rhamnose to ribitol by an �\(1-3) linkage for serotypes 6A and 6C and an �\(1-4) linkage for serotypes 6B and 6D. The isolates had mutations in the triad residues of WciP that have been correlated with enzyme specificity. The canonical triad residues of WciP are Ala192-Ser195-Arg254 for serotypes 6A and 6C and Ser192-Asn195-Gly254 for serotypes 6B and 6D. To prove that the mutations in the triad residues are responsible for the hybrid serotype, we introduced the previously described Ala192-Cys195-Arg254 triad into a 6A strain and found that the change made WciP bispecific, resulting in 6A and 6B repeat unit expression, although 6B repeat unit production was favored over production of 6A repeat units. Likewise, this triad permitted a 6C strain to express 6C and 6D repeat units. With reported bispecificity in WciN, which adds either glucose or galactose as the second sugar in the serogroup 6 repeat unit, the possibility exists for a strain to simultaneously produce all four serogroup 6 repeat units; however, when genes encoding both bispecific enzymes were introduced into a 6A strain, only 6A, 6B, and 6D repeat units were detected serologically. Nonetheless, this may be the first example of a bacterial polysaccharide with three different repeat units. This strategy of expressing multiple repeat units in a single polymer is a novel approach to broadening vaccine coverage by eliminating the need for multiple polysaccharide sources to cover multiple serogroup members.
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288. |
Manso AS,
Chai MH,
Atack JM,
Furi L,
De Ste Croix M,
Haigh R,
Trappetti C,
Ogunniyi AD,
Shewell LK,
Boitano M,
Clark TA,
Korlach J,
Blades M,
Mirkes E,
Gorban AN,
Paton JC,
Jennings MP,
Oggioni MR,
( 2014 ) A random six-phase switch regulates pneumococcal virulence via global epigenetic changes. PMID : 25268848 : DOI : 10.1038/ncomms6055 PMC : PMC4190663 Abstract >>
Streptococcus pneumoniae (the pneumococcus) is the world's foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or 'phases') that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.
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289. |
Yun KW,
Cho EY,
Choi EH,
Lee HJ,
( 2014 ) Capsular polysaccharide gene diversity of pneumococcal serotypes 6A, 6B, 6C, and 6D. PMID : 25220816 : DOI : 10.1016/j.ijmm.2014.08.004 Abstract >>
This study was performed to better understand the genetic diversity and evolutionary relatedness of pneumococcal serotypes 6A, 6B, 6C, and 6D. Multi-locus sequence typing (MLST) was performed for 160 serogroup 6 isolates from clinical specimens collected from children between 1991 and 2010. We identified 38 sequence types (STs) comprising five clonal complexes with 12 singletons. Although most STs were confined to a single serotype, some STs were shared by two serotypes, and one ST was shared by three serotypes. Many STs of serotype 6A showed genetic relatedness with those of serotype 6C or 6D in eBURST analysis. Five capsular polysaccharide (cps) genes - wchA, wciO, wciP, wzy, and wzx - were analysed in 74 isolates from our clinical samples and in 36 isolates from GenBank. There were several profiles and clades in each serotype on the analysis of the concatenated sequences of the five cps genes. Small genetic distances between serotypes 6A and 6B and between serotypes 6C and 6D were observed while serotype 6B with an indel sequence formed a distinct clade. When comparing the individual cps genes between the serotypes, there was also a high level of similarity in the wchA and wciO gene sequences between serotype 6C and serotype 6D. On the other hand, serotypes 6A and 6D had the most highly similar wzy and wzx gene sequences. The wzy sequences of serotype 6C were nearly identical (99.6%) to those of serotype 6A clade II strains. In conclusion, we revealed the diversity of the genetic background and cps sequences in each pneumococcal serotype of serogroup 6. Pneumococcal serotype diversity might be attributable to complex serial mutation and recombination events.
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290. |
Gerlini A,
Colomba L,
Furi L,
Braccini T,
Manso AS,
Pammolli A,
Wang B,
Vivi A,
Tassini M,
van Rooijen N,
Pozzi G,
Ricci S,
Andrew PW,
Koedel U,
Moxon ER,
Oggioni MR,
( 2014 ) The role of host and microbial factors in the pathogenesis of pneumococcal bacteraemia arising from a single bacterial cell bottleneck. PMID : 24651834 : DOI : 10.1371/journal.ppat.1004026 PMC : PMC3961388 Abstract >>
The pathogenesis of bacteraemia after challenge with one million pneumococci of three isogenic variants was investigated. Sequential analyses of blood samples indicated that most episodes of bacteraemia were monoclonal events providing compelling evidence for a single bacterial cell bottleneck at the origin of invasive disease. With respect to host determinants, results identified novel properties of splenic macrophages and a role for neutrophils in early clearance of pneumococci. Concerning microbial factors, whole genome sequencing provided genetic evidence for the clonal origin of the bacteraemia and identified SNPs in distinct sub-units of F0/F1 ATPase in the majority of the ex vivo isolates. When compared to parental organisms of the inoculum, ex-vivo pneumococci with mutant alleles of the F0/F1 ATPase had acquired the capacity to grow at low pH at the cost of the capacity to grow at high pH. Although founded by a single cell, the genotypes of pneumococci in septicaemic mice indicate strong selective pressure for fitness, emphasising the within-host complexity of the pathogenesis of invasive disease.
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291. |
Kohler S,
Hallström T,
Singh B,
Riesbeck K,
Spart? G,
Zipfel PF,
Hammerschmidt S,
( 2015 ) Binding of vitronectin and Factor H to Hic contributes to immune evasion of Streptococcus pneumoniae serotype 3. PMID : 25181963 : DOI : 10.1160/TH14-06-0561 Abstract >>
Streptococcus pneumoniae serotype 3 strains are highly resistant to opsonophagocytosis due to recruitment of the complement inhibitor Factor H via Hic, a member of the pneumococcal surface protein C (PspC) family. In this study, we demonstrated that Hic also interacts with vitronectin, a fluid-phase regulator involved in haemostasis, angiogenesis, and the terminal complement cascade as well as a component of the extracellular matrix. Blocking of Hic by specific antiserum or genetic deletion significantly reduced pneumococcal binding to soluble and immobilised vitronectin and to Factor H, respectively. In parallel, ectopic expression of Hic on the surface of Lactococcus lactis conferred binding to soluble and immobilised vitronectin as well as Factor H. Molecular analyses with truncated Hic fragments narrowed down the vitronectin-binding site to the central core of Hic (aa 151-201). This vitronectin-binding region is separate from that of Factor H, which binds to the N-terminus of Hic (aa 38-92). Binding of pneumococcal Hic was localised to the C-terminal heparin-binding domain (HBD3) of vitronectin. However, an N-terminal region to HBD3 was further involved in Hic-binding to immobilised vitronectin. Finally, vitronectin bound to Hic was functionally active and inhibited formation of the terminal complement complex. In conclusion, Hic interacts with vitronectin and simultaneously with Factor H, and both human proteins may contribute to colonisation and invasive disease caused by serotype 3 pneumococci.
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292. |
Connaris H,
Govorkova EA,
Ligertwood Y,
Dutia BM,
Yang L,
Tauber S,
Taylor MA,
Alias N,
Hagan R,
Nash AA,
Webster RG,
Taylor GL,
( 2014 ) Prevention of influenza by targeting host receptors using engineered proteins. PMID : 24733924 : DOI : 10.1073/pnas.1404205111 PMC : PMC4035977 Abstract >>
There is a need for new approaches for the control of influenza given the burden caused by annual seasonal outbreaks, the emergence of viruses with pandemic potential, and the development of resistance to current antiviral drugs. We show that multivalent biologics, engineered using carbohydrate-binding modules specific for sialic acid, mask the cell-surface receptor recognized by the influenza virus and protect mice from a lethal challenge with 2009 pandemic H1N1 influenza virus. The most promising biologic protects mice when given as a single 1-�gg intranasal dose 7 d in advance of viral challenge. There also is sufficient virus replication to establish an immune response, potentially protecting the animal from future exposure to the virus. Furthermore, the biologics appear to stimulate inflammatory mediators, and this stimulation may contribute to their protective ability. Our results suggest that this host-targeted approach could provide a front-line prophylactic that has the potential to protect against any current and future influenza virus and possibly against other respiratory pathogens that use sialic acid as a receptor.
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293. |
Mingoia M,
Morici E,
Morroni G,
Giovanetti E,
Del Grosso M,
Pantosti A,
Varaldo PE,
( 2014 ) Tn5253 family integrative and conjugative elements carrying mef(I) and catQ determinants in Streptococcus pneumoniae and Streptococcus pyogenes. PMID : 25070090 : DOI : 10.1128/AAC.03638-14 PMC : PMC4187955 Abstract >>
The linkage between the macrolide efflux gene mef(I) and the chloramphenicol inactivation gene catQ was first described in Streptococcus pneumoniae (strain Spn529), where the two genes are located in a module designated IQ element. Subsequently, two different defective IQ elements were detected in Streptococcus pyogenes (strains Spy029 and Spy005). The genetic elements carrying the three IQ elements were characterized, and all were found to be Tn5253 family integrative and conjugative elements (ICEs). The ICE from S. pneumoniae (ICESpn529IQ) was sequenced, whereas the ICEs from S. pyogenes (ICESpy029IQ and ICESpy005IQ, the first Tn5253-like ICEs reported in this species) were characterized by PCR mapping, partial sequencing, and restriction analysis. ICESpn529IQ and ICESpy029IQ were found to share the intSp 23FST81 integrase gene and an identical Tn916 fragment, whereas ICESpy005IQ has int5252 and lacks Tn916. All three ICEs were found to lack the linearized pC194 plasmid that is usually associated with Tn5253-like ICEs, and all displayed a single copy of a toxin-antitoxin operon that is typically contained in the direct repeats flanking the excisable pC194 region when this region is present. Two different insertion sites of the IQ elements were detected, one in ICESpn529IQ and ICESpy029IQ, and another in ICESpy005IQ. The chromosomal integration of the three ICEs was site specific, depending on the integrase (intSp 23FST81 or int5252). Only ICESpy005IQ was excised in circular form and transferred by conjugation. By transformation, mef(I) and catQ were cotransferred at a high frequency from S. pyogenes Spy005 and at very low frequencies from S. pneumoniae Spn529 and S. pyogenes Spy029.
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294. |
Croucher NJ,
Hanage WP,
Harris SR,
McGee L,
van der Linden M,
de Lencastre H,
Sá-Leão R,
Song JH,
Ko KS,
Beall B,
Klugman KP,
Parkhill J,
Tomasz A,
Kristinsson KG,
Bentley SD,
( 2014 ) Variable recombination dynamics during the emergence, transmission and 'disarming' of a multidrug-resistant pneumococcal clone. PMID : 24957517 : DOI : 10.1186/1741-7007-12-49 PMC : PMC4094930 Abstract >>
Pneumococcal �]-lactam resistance was first detected in Iceland in the late 1980s, and subsequently peaked at almost 25% of clinical isolates in the mid-1990s largely due to the spread of the internationally-disseminated multidrug-resistant PMEN2 (or Spain6B-2) clone of Streptococcus pneumoniae. Whole genome sequencing of an international collection of 189 isolates estimated that PMEN2 emerged around the late 1960s, developing resistance through multiple homologous recombinations and the acquisition of a Tn5253-type integrative and conjugative element (ICE). Two distinct clades entered Iceland in the 1980s, one of which had acquired a macrolide resistance cassette and was estimated to have risen sharply in its prevalence by coalescent analysis. Transmission within the island appeared to mainly emanate from Reykjav?k and the Southern Peninsular, with evolution of the bacteria effectively clonal, mainly due to a prophage disrupting a gene necessary for genetic transformation in many isolates. A subsequent decline in PMEN2's prevalence in Iceland coincided with a nationwide campaign that reduced dispensing of antibiotics to children in an attempt to limit its spread. Specific mutations causing inactivation or loss of ICE-borne resistance genes were identified from the genome sequences of isolates that reverted to drug susceptible phenotypes around this time. Phylogenetic analysis revealed some of these occurred on multiple occasions in parallel, suggesting they may have been at least temporarily advantageous. However, alteration of 'core' sequences associated with resistance was precluded by the absence of any substantial homologous recombination events. PMEN2's clonal evolution was successful over the short-term in a limited geographical region, but its inability to alter major antigens or 'core' gene sequences associated with resistance may have prevented persistence over longer timespans.
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295. |
Lacks SA,
Lopez P,
Greenberg B,
Espinosa M,
( 1986 ) Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. PMID : 2438417 : DOI : 10.1016/0022-2836(86)90026-4 Abstract >>
The streptococcal plasmid pMV158 and its derivative pLS1 are able to replicate and confer tetracycline resistance in both Gram-positive and Gram-negative bacteria. Copy numbers of pLS1 were 24, 4 and 4 molecules per genome in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli, respectively. Replication of the streptococcal plasmids in E. coli required functional polA and recA genes. A copy-number mutation corresponding to a 332 base-pair deletion of pLS1 doubled the plasmid copy number in all three species. Determination of the complete DNA sequence of pLS1 revealed transcriptional and translational signals and four open reading frames. A putative inhibitory RNA was encoded in the region deleted by the copy-control mutation. Two putative mRNA transcripts encoded proteins for replication functions and tetracycline resistance, respectively. The repB gene encoded a trans-acting, 23,000 Mr protein necessary for replication, and the tet gene encoded a very hydrophobic, 50,000 Mr protein required for tetracycline resistance. The polypeptides corresponding to these proteins were identified by specific labeling of plasmid-encoded products. The tet gene of pLS1 was highly homologous to tet genes in two other plasmids of Gram-positive origin but different in both sequence and mode of regulation from tet genes of Gram-negative origin.
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296. |
Park IH,
Geno KA,
Sherwood LK,
Nahm MH,
Beall B,
( 2014 ) Population-based analysis of invasive nontypeable pneumococci reveals that most have defective capsule synthesis genes. PMID : 24831650 : DOI : 10.1371/journal.pone.0097825 PMC : PMC4022640 Abstract >>
Since nasopharyngeal carriage of pneumococcus precedes invasive pneumococcal disease, characteristics of carriage isolates could be incorrectly assumed to reflect those of invasive isolates. While most pneumococci express a capsular polysaccharide, nontypeable pneumococci are sometimes isolated. Carriage nontypeables tend to encode novel surface proteins in place of a capsular polysaccharide synthetic locus, the cps locus. In contrast, capsular polysaccharide is believed to be indispensable for invasive pneumococcal disease, and nontypeables from population-based invasive pneumococcal disease surveillance have not been extensively characterized. We received 14,328 invasive pneumococcal isolates through the Active Bacterial Core surveillance program during 2006-2009. Isolates that were nontypeable by Quellung serotyping were characterized by PCR serotyping, sequence analyses of the cps locus, and multilocus sequence typing. Eighty-eight isolates were Quellung-nontypeable (0.61%). Of these, 79 (89.8%) contained cps loci. Twenty-two nontypeables exhibited serotype 8 cps loci with defects, primarily within wchA. Six of the remaining nine isolates contained previously-described aliB homologs in place of cps loci. Multilocus sequence typing revealed that most nontypeables that lacked capsular biosynthetic genes were related to established non-encapsulated lineages. Thus, invasive pneumococcal disease caused by nontypeable pneumococcus remains rare in the United States, and while carriage nontypeables lacking cps loci are frequently isolated, such nontypeable are extremely rare in invasive pneumococcal disease. Most invasive nontypeable pneumococci possess defective cps locus genes, with an over-representation of defective serotype 8 cps variants.
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297. |
Calix JJ,
Brady AM,
Du VY,
Saad JS,
Nahm MH,
( 2014 ) Spectrum of pneumococcal serotype 11A variants results from incomplete loss of capsule O-acetylation. PMID : 24352997 : DOI : 10.1128/JCM.02695-13 PMC : PMC3957789 Abstract >>
Streptococcus pneumoniae is a significant bacterial pathogen that expresses >90 capsule serotypes. Conventional serotyping methods assume that each serotype is a genetically and antigenically distinct entity; however, recent investigations have revealed pneumococcal isolates that cannot be unambiguously serotyped because they share the properties of more than one serotype. Here, we employed a novel serotyping method and NMR spectroscopy to examine clinical isolates sharing properties of serotypes 11A and 11E. These ambiguous clinical isolates were provisionally named 11A variant (11Av) isolates. Serotype 11A pneumococci characteristically express capsule �]-galactose-6-O-acetylation (�]Gal6OAc) mediated by the capsule synthesis gene wcjE, while 11E strains contain loss-of-function mutations in wcjE and completely lack the expression of �]Gal6OAc. Although 11Av isolates also contained mutated wcjE alleles, 11Av clinical isolates were composed of antigenically homogeneous bacteria expressing reduced amounts of 11A-specific capsule antigen. NMR data confirmed reduced but detectable amounts of �]Gal6OAc on 11Av capsule polysaccharide. Furthermore, the transformation of strains with wcjE alleles from 11Av strains was sufficient to restore partial �]Gal6OAc in an 11E background. We conclude that, instead of being distinct entities, serotypes 11A and 11E represent two extremes of an antigenic spectrum resulting from variable capsule O-acetylation secondary to heterologous wcjE mutations. These findings challenge whether all clinically relevant pneumococci can be definitively categorized into distinct serotypes.
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298. |
Boudes M,
Sanchez D,
Graille M,
van Tilbeurgh H,
Durand D,
Quevillon-Cheruel S,
( 2014 ) Structural insights into the dimerization of the response regulator ComE from Streptococcus pneumoniae. PMID : 24500202 : DOI : 10.1093/nar/gku110 PMC : PMC4005675 Abstract >>
Natural transformation contributes to the maintenance and to the evolution of the bacterial genomes. In Streptococcus pneumoniae, this function is reached by achieving the competence state, which is under the control of the ComD-ComE two-component system. We present the crystal and solution structures of ComE. We mimicked the active and non-active states by using the phosphorylated mimetic ComE(D58E) and the unphosphorylatable ComE(D58A) mutants. In the crystal, full-length ComE(D58A) dimerizes through its canonical REC receiver domain but with an atypical mode, which is also adopted by the isolated REC(D58A) and REC(D58E). The LytTR domain adopts a tandem arrangement consistent with the two direct repeats of its promoters. However ComE(D58A) is monomeric in solution, as seen by SAXS, by contrast to ComE(D58E) that dimerizes. For both, a relative mobility between the two domains is assumed. Based on these results we propose two possible ways for activation of ComE by phosphorylation.
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299. |
Iannelli F,
Santoro F,
Oggioni MR,
Pozzi G,
( 2014 ) Nucleotide sequence analysis of integrative conjugative element Tn5253 of Streptococcus pneumoniae. PMID : 24295984 : DOI : 10.1128/AAC.01764-13 PMC : PMC3910829 Abstract >>
Conjugative transposon Tn5253, an integrative conjugative element (ICE) of Streptococcus pneumoniae carrying the cat and tet(M) genes, was shown to be 64,528 bp in size and to contain 79 open reading frames, of which only 38 could be annotated. Two distinct genetic elements were found integrated into Tn5253: Tn5251 (18,033 bp), of the Tn916-Tn1545 family of ICEs, and �[cat(pC194) (7,627 bp), which could not conjugate but was capable of intracellular mobility by excision, circularization, and integration by homologous recombination. The highest conjugation frequency of Tn5253 was observed when Streptococcus pyogenes was the donor (6.7 �� 10(-3) transconjugants/donor).
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300. |
Pinto TC,
Souza AR,
de Pina SE,
Costa NS,
Borges Neto AA,
Neves FP,
Merquior VL,
Dias CA,
Peralta JM,
Teixeira LM,
( 2013 ) Phenotypic and molecular characterization of optochin-resistant Streptococcus pneumoniae isolates from Brazil, with description of five novel mutations in the ATPC gene. PMID : 23884994 : DOI : 10.1128/JCM.01168-13 PMC : PMC3811620 Abstract >>
Optochin (Opt) susceptibility is used largely for the identification of Streptococcus pneumoniae in diagnostic laboratories. Opt-resistant (Opt(r)) S. pneumoniae isolates have been reported, however, indicating the potential for misidentification of this important pathogen. Point mutations in the atpC gene have been associated with the emergence of Opt(r) S. pneumoniae, but data on the characterization of such atypical variants of S. pneumoniae are still limited. The present report describes the results of a polyphasic approach to identifying and characterizing 26 Opt(r) S. pneumoniae isolates recovered from patients or carriers living in Brazil. Sixteen isolates consisted of heterogeneous populations, and 10 isolates were homogeneously Opt(r). The isolates had different serotypes and antimicrobial susceptibility profiles. They also presented diverse genetic characteristics, as indicated by pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem-repeat analysis (MLVA), and pspA gene typing. Except for Opt MICs (4- to 64-fold higher among Opt(r) variants), Opt(r) and Opt-susceptible (Opt(s)) subpopulations originating from the same culture had identical characteristics. Sequencing of the atpC gene of the Opt(r) variants revealed 13 different nucleotide changes distributed among eight different codons. Changes in codon 49 were the most frequent, suggesting that this might be a hot spot for optochin resistance-conferring mutations. On the other hand, five novel types of mutations in the atpC gene (Met13Ile, Gly18Ser, Gly20Ala, Ala31Val, and Ala49Gly) were identified. In silico prediction modeling indicated that the atpC gene mutations corresponded to alterations in the transmembrane region of the ATPase, leading to a higher hydrophobicity profile in �\-helix 1 and to a lower hydrophobicity profile in �\-helix 2.
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301. |
Carvalho Mda G,
Pimenta FC,
Moura I,
Roundtree A,
Gertz RE,
Li Z,
Jagero G,
Bigogo G,
Junghae M,
Conklin L,
Feikin DR,
Breiman RF,
Whitney CG,
Beall BW,
( 2013 ) Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract. PMID : 23825797 : DOI : 10.7717/peerj.97 PMC : PMC3698467 Abstract >>
We performed culture-based and PCR-based tests for pneumococcal identification and serotyping from carriage specimens collected in rural and urban Kenya. Nasopharyngeal specimens from 237 healthy children <5 years old (C-NPs) and combined nasopharyngeal/oropharyngeal specimens from 158 adults (A-NP/OPs, 118 HIV-positive) were assessed using pneumococcal isolation (following broth culture enrichment) with Quellung-based serotyping, real-time lytA-PCR, and conventional multiplexed PCR-serotyping (cmPCR). Culture-based testing from C-NPs, HIV-positive A-NP/OPs, and HIV-negative A-NP/OPs revealed 85.2%, 40.7%, and 12.5% pneumococcal carriage, respectively. In contrast, cmPCR serotypes were found in 93.2%, 98.3%, and 95.0% of these sets, respectively. Two of 16 lytA-negative C-NPs and 26 of 28 lytA-negative A-NP/OPs were cmPCR-positive for 1-10 serotypes (sts) or serogroups (sgs). A-NP/OPs averaged 5.5 cmPCR serotypes/serogroups (5.2 in HIV-positive, 7.1 in HIV-negative) and C-NPs averaged 1.5 cmPCR serotypes/serogroups. cmPCR serotypes/serogroups from lytA-negative A-NP/OPs included st2, st4, sg7F/7A, sg9N/9L, st10A, sg10F/10C/33C, st13, st17F, sg18C/18A/18B/18F, sg22F/22A, and st39. Nine strains of three non-pneumococcal species (S. oralis, S. mitis, and S. parasanguinis) (7 from A-OP, 1 from both A-NP and A-OP, and 1 from C-NP) were each cmPCR-positive for one of 7 serotypes/serogroups (st5, st13, sg15A/15F, sg10F/10C/33C, sg33F/33A/37, sg18C/18A/18B/18F, sg12F/12A/12B/ 44/46) with amplicons revealing 83.6-99.7% sequence identity to pneumococcal references. In total, 150 cmPCR amplicons from carriage specimens were sequenced, including 25 from lytA-negative specimens. Amplicon sequences derived from specimens yielding a pneumococcal isolate with the corresponding serotype were identical or highly conserved (>98.7%) with the reference cmPCR amplicon for the st, while cmPCR amplicons from lytA-negative specimens were generally more divergent. Separate testing of 56 A-OPs and 56 A-NPs revealed that ?94% of the positive cmPCR results from A-NP/OPs were from OP microbiota. In contrast, A-NPs yielded >2-fold more pneumococcal isolates than A-OPs. Verified and suspected non-pneumococcal cmPCR serotypes/serogroups appeared to be relatively rare in C-NPs and A-NPs compared to A-OPs. Our findings indicate that non-pneumococcal species can confound serotype-specific PCR and other sequence-based assays due to evolutionarily conserved genes most likely involved in biosynthesis of surface polysaccharide structures.
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302. |
Oliver MB,
Jones C,
Larson TR,
Calix JJ,
Zartler ER,
Yother J,
Nahm MH,
( 2013 ) Streptococcus pneumoniae serotype 11D has a bispecific glycosyltransferase and expresses two different capsular polysaccharide repeating units. PMID : 23737526 : DOI : 10.1074/jbc.M113.488528 PMC : PMC3724649 Abstract >>
Streptococcus pneumoniae (pneumococcus) expresses a capsular polysaccharide (CPS) that protects against host immunity and is synthesized by enzymes in the capsular polysaccharide synthesis (cps) locus. Serogroup 11 has six members (11A to -E) and the CPS structure of all members has been solved, except for serotype 11D. The cps loci of 11A and 11D differ by one codon (N112S) in wcrL, which putatively encodes a glycosyltransferase that adds the fourth sugar of the CPS repeating unit (RU). Gas chromatography and nuclear magnetic resonance analysis revealed that 11A and 11D PSs contain identical CPS RUs that contain �\Glc as the fourth sugar. However, ?25% of 11D CPS RUs contain instead �\GlcNAc as the fourth sugar, suggesting that 11D wcrL encodes a bispecific glycosyltransferase. To test the hypothesis that codon 112 of WcrL determines enzyme specificity, and therefore the fourth sugar in the RU, we generated three isogenic pneumococcal strains with 11A cps loci containing wcrL encoding Ser-112 (MBO128) or Ala-112 (MBO130). MBO128 was serologically and biochemically identical to serotype 11D. MBO130 has a unique serologic profile; has as much �\GlcNAc as 11F, 11B, and 11C CPS do; and may represent a new serotype. These findings demonstrate how pneumococci alter their CPS structure and their immunologic properties with a minimal genetic change.
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303. |
Smith A,
Johnston C,
Inverarity D,
Slack M,
Paterson GK,
Diggle M,
Mitchell T,
( 2013 ) Investigating the role of pneumococcal neuraminidase A activity in isolates from pneumococcal haemolytic uraemic syndrome. PMID : 23924664 : DOI : 10.1099/jmm.0.063479-0 Abstract >>
Streptococcus pneumoniae diseases are a rare but increasingly recognized trigger of atypical haemolytic uraemic syndrome (HUS) in young children and associated with a higher mortality rate than diarrhoea-associated HUS. This study aimed to determine the importance of neuraminidase A (NanA) and genomic diversity in the pathogenesis of pneumococcal HUS (pHUS). We investigated the nanA gene sequence, gene expression, neuraminidase activity and comparative genomic hybridization of invasive pneumococcal disease (IPD) isolates from patients with pHUS and control strains matched by serotype and sequence type (ST), isolated from patients with IPD but not pHUS. The nanA sequence of 33 isolates was determined and mutations at 142 aa positions were identified. High levels of diversity were observed within the NanA protein, with mosaic blocks, insertions and repeat regions present. When comparing nanA allelic diversity with ST and disease profile in the isolates tested, nanA alleles clustered mostly by ST. No particular nanA allele was associated with pHUS. There was no significant difference in overall neuraminidase activity between pHUS isolates and controls when induced/uninduced with N-acetylneuraminic acid. Comparative genomic hybridization showed little difference in genetic content between the pHUS isolates and the controls. Results of gene expression studies identified 12 genes differentially regulated in all pHUS isolates compared with the control. Although neuraminidase enzyme activity may be important in pHUS progression and contribute to pathogenesis, the lack of a distinction between pHUS isolates and controls suggests that host factors, such as acquired abnormalities of the alternative complement cascade in young children, may play a more significant role in the outcome of pHUS.
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304. |
Ko KS,
Baek JY,
Song JH,
( 2013 ) Capsular gene sequences and genotypes of "serotype 6E" Streptococcus pneumoniae isolates. PMID : 23824778 : DOI : 10.1128/JCM.01645-13 PMC : PMC3811659 Abstract >>
To characterize Streptococcus pneumoniae "serotype 6E," complete cps loci were sequenced. The capsular genes of "serotype 6E" isolates differed much from those of serotypes 6A and 6B. We identified 10 additional "serotype 6E" isolates, which are not confined to a restricted geographic locality. Most of these "serotype 6E" isolates belonged to sequence type 90 and its single-locus variants. The homogeneity of their genetic background and cps loci suggests a recent origin of the "serotype 6E" isolates.
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305. |
Otsuka T,
Chang B,
Wada A,
Okazaki M,
( 2013 ) Molecular epidemiology and serogroup 6 capsular gene evolution of pneumococcal carriage in a Japanese birth cohort study. PMID : 24025348 : DOI : 10.1099/jmm.0.066316-0 Abstract >>
Antibiotic resistance in Streptococcus pneumoniae is a major concern worldwide. However, it is unclear whether resistance is associated with only a few highly prevalent clones or numerous and diverse clones. We monitored 349 healthy children and obtained nasopharyngeal cultures at five time points coinciding with health check-ups (4, 7, 10, 18 and 36 months) between 2008 and 2012. A total of 497 S. pneumoniae isolates from 257 healthy children were characterized using capsular serotyping, multilocus sequence typing and antibiotic resistance genotyping (ermB, mefA/E and pbp mutations). Among these isolates, 25 serotypes and 66 sequence types (STs) were found, including 24 new STs with 11 new alleles. Although resistance was present in a variety of ST clones, most of the clones (57/66, 86.4 %) had one specific resistant or susceptible genotype. Of 233 phenotypically penicillin-non-susceptible isolates, 196 (84.1 %) belonged to only six clones, comprising ST90(6B), ST236(19F), ST242(23F), ST3787(6A), ST1437(23F) and ST338(23A) and their variants. We concluded that drug-resistant S. pneumoniae is associated with a limited number of highly prevalent clones that are capable of adapting to the community setting. Furthermore, we analysed the capsular gene evolution in serogroup 6. The strain ST2924(6D) was probably the result of recombination of a 3563 bp fragment of the capsule locus acquired by an ST2924(6C) strain from an ST90(6B) or ST2924(6B) strain. Compared with previous studies, our results showed a different recombination site (wciN and wzx) and a different cps profile (8-7-11), indicating that serogroup 6 strains have multiple sites for cps recombination as a mechanism of vaccine escape.
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306. |
Almeida ST,
de Lencastre H,
Sá-Leão R,
( 2013 ) Epidemiology and population structure of serotypes 1, 5 and 7f carried by children in Portugal from 1996-2010 before introduction of the 10-valent and 13-valent pneumococcal conjugate vaccines. PMID : 24058686 : DOI : 10.1371/journal.pone.0075442 PMC : PMC3776787 Abstract >>
Among the over 90 serotypes of Streptococcus pneumoniae described, serotypes 1, 5, and 7F account for a significant proportion of invasive disease worldwide and are now covered by the most recent 10- and 13-valent pneumococcal conjugate vaccines (PCVs). The epidemiology of these serotypes in carriage remains poorly studied because they are rarely detected. We aimed to gain insights into the epidemiology and population structure of serotypes 1, 5 and 7F carried by children in Portugal before PCV10 and PCV13 became widely used. Isolates obtained in cross-sectional studies carried out over a 15-year period (1996-2010) were retrospectively pooled and characterized. Of 5,123 pneumococci obtained, 70 were associated with serotypes 1 (n = 21), 5 (n = 7), and 7F (n = 42). The highest prevalence detected was 3.3% for serotype 1 in 2006, 1% for serotype 5 in 2009, and 3.3% for serotype 7F in 2006; Serotype 1 was associated with PMEN international clones Sweden(1)-28(ST306) and Sweden(1)-40(ST304); serotype 5 was associated with Colombia(5)-19(ST289); and serotype 7F was associated with Netherlands(7F)-39(ST191). All these isolates were fully susceptible. Most carriers of serotypes 1 (86%), 5 (86%), and 7F (91%) were older than two years but a significant association with older age was only observed for serotype 7F (p = 0.006). Evidence for cross-transmission was obtained. In conclusion, we were able to detect and characterize the rarely carried serotypes 1, 5, and 7F among healthy children in Portugal. These data will constitute an important baseline for upcoming surveillance studies aimed to establish the impact of novel PCVs targeting these serotypes in carriage.
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307. |
Park IH,
Kim KH,
Andrade AL,
Briles DE,
McDaniel LS,
Nahm MH,
( 2012 ) Nontypeable pneumococci can be divided into multiple cps types, including one type expressing the novel gene pspK. PMID : 22532557 : DOI : 10.1128/mBio.00035-12 PMC : PMC3340917 Abstract >>
Although virulence of Streptococcus pneumoniae is associated with its capsule, some pathogenic S. pneumoniae isolates lack capsules and are serologically nontypeable (NT). We obtained 64 isolates that were identified as NT "pneumococci" (i.e., bacteria satisfying the conventional definition but without the multilocus sequence typing [MLST]-based definition of S. pneumoniae) by the traditional criteria. All 64 were optochin sensitive and had lytA, and 63 had ply. Twelve isolates had cpsA, suggesting the presence of a conventional but defective capsular polysaccharide synthesis (cps) locus. The 52 cpsA-negative isolates could be divided into three null capsule clades (NCC) based on aliC (aliB-like ORF1), aliD (aliB-like ORF2), and our newly discovered gene, pspK, in their cps loci. pspK encodes a protein with a long alpha-helical region containing an LPxTG motif and a YPT motif known to bind human pIgR. There were nine isolates in NCC1 (pspK(+) but negative for aliC and aliD), 32 isolates in NCC2 (aliC(+) aliD(+) but negative for pspK), and 11 in NCC3 (aliD(+) but negative for aliC and pspK). Among 52 cpsA-negative isolates, 41 were identified as S. pneumoniae by MLST analysis. All NCC1 and most NCC2 isolates were S. pneumoniae, whereas all nine NCC3 and two NCC2 isolates were not S. pneumoniae. Several NCC1 and NCC2 isolates from multiple individuals had identical MLST and cps regions, showing that unencapsulated S. pneumoniae can be infectious among humans. Furthermore, NCC1 and NCC2 S. pneumoniae isolates could colonize mice as well as encapsulated S. pneumoniae, although S. pneumoniae with an artificially disrupted cps locus did not. Moreover, an NCC1 isolate with pspK deletion did not colonize mice, suggesting that pspK is critical for colonization. Thus, PspK may provide pneumococci a means of surviving in the nasopharynx without capsule. IMPORTANCE The presence of a capsule is critical for many pathogenic bacteria, including pneumococci. Reflecting the pathogenic importance of the pneumococcal capsule, pneumococcal vaccines are designed to elicit anticapsule antibodies. Additional evidence for the pathogenic importance of the pneumococcal capsule is the fact that in pneumococci all the genes necessary for capsule production are together in one genetic locus, which is called the cps locus. However, there are occasional pathogenic pneumococci without capsules, and how they survive in the host without the capsule is unknown. Here, we show that in these acapsular pneumococci, the cps loci have been replaced with various novel genes and they can colonize mouse nasopharynges as well as capsulated pneumococci. Since the genes that replace the cps loci are likely to be important in host survival, they may show new and/or alternative capsule-independent survival mechanisms used by pneumococci.
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308. |
Oliver MB,
van der Linden MP,
Küntzel SA,
Saad JS,
Nahm MH,
( 2013 ) Discovery of Streptococcus pneumoniae serotype 6 variants with glycosyltransferases synthesizing two differing repeating units. PMID : 23897812 : DOI : 10.1074/jbc.M113.480152 PMC : PMC3764802 Abstract >>
Streptococcus pneumoniae is a persistent, opportunistic commensal of the human nasopharynx and is the leading cause of community-acquired pneumonia. It expresses an anti-phagocytic capsular polysaccharide (PS). Genetic variation of the capsular PS synthesis (cps) locus is the molecular basis for structural and antigenic heterogeneity of capsule types (serotypes). Serogroup 6 has four known members (6A-6D) with distinct serologic properties, homologous cps loci, and structurally similar PSs. cps of serotypes 6A/6B have wciN�\, encoding �\-1,3-galactosyltransferase, whereas serotypes 6C/6D have wciN�] encoding �\-1,3-glucosyltransferase. Two atypical serogroup 6 isolates (named 6X11 and 6X12) have been discovered recently in Germany. Flow cytometric studies using monoclonal antibodies show that 6X11 has serologic properties of 6B/6D, whereas 6X12 has 6A/6C. NMR studies of their capsular PSs revealed that 6X11 and 6X12 have two different repeating units with a distribution of ~40:60 6B:6D and 75:25 6A:6C PS, respectively. Sequencing of the wciN�\ gene in 6X12 and 6X11 revealed single and double nucleotide substitutions, respectively, resulting in the amino acid changes A150T and D38N. Substitution of alanine with threonine at position 150 in a 6A strain was associated with hybrid serologic and chemical profiles like 6X12. The hybrid serotypes represented by 6X12 and 6X11 strains are now named serotypes 6F and 6G. Single amino acid changes in cps genes encoding glycosyltransferases can alter substrate specificities, permit biosynthesis of heterogeneous capsule repeating units, and result in new hybrid capsule types that may differ in their interaction with the immune system of the host.
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309. |
Leung MH,
Bryson K,
Freystatter K,
Pichon B,
Edwards G,
Charalambous BM,
Gillespie SH,
( 2012 ) Sequetyping: serotyping Streptococcus pneumoniae by a single PCR sequencing strategy. PMID : 22553238 : DOI : 10.1128/JCM.06384-11 PMC : PMC3405617 Abstract >>
The introduction of pneumococcal conjugate vaccines necessitates continued monitoring of circulating strains to assess vaccine efficacy and replacement serotypes. Conventional serological methods are costly, labor-intensive, and prone to misidentification, while current DNA-based methods have limited serotype coverage requiring multiple PCR primers. In this study, a computer algorithm was developed to interrogate the capsulation locus (cps) of vaccine serotypes to locate primer pairs in conserved regions that border variable regions and could differentiate between serotypes. In silico analysis of cps from 92 serotypes indicated that a primer pair spanning the regulatory gene cpsB could putatively amplify 84 serotypes and differentiate 46. This primer set was specific to Streptococcus pneumoniae, with no amplification observed for other species, including S. mitis, S. oralis, and S. pseudopneumoniae. One hundred thirty-eight pneumococcal strains covering 48 serotypes were tested. Of 23 vaccine serotypes included in the study, most (19/22, 86%) were identified correctly at least to the serogroup level, including all of the 13-valent conjugate vaccine and other replacement serotypes. Reproducibility was demonstrated by the correct sequetyping of different strains of a serotype. This novel sequence-based method employing a single PCR primer pair is cost-effective and simple. Furthermore, it has the potential to identify new serotypes that may evolve in the future.
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310. |
Croucher NJ,
Mitchell AM,
Gould KA,
Inverarity D,
Barquist L,
Feltwell T,
Fookes MC,
Harris SR,
Dordel J,
Salter SJ,
Browall S,
Zemlickova H,
Parkhill J,
Normark S,
Henriques-Normark B,
Hinds J,
Mitchell TJ,
Bentley SD,
( 2013 ) Dominant role of nucleotide substitution in the diversification of serotype 3 pneumococci over decades and during a single infection. PMID : 24130509 : DOI : 10.1371/journal.pgen.1003868 PMC : PMC3794909 Abstract >>
Streptococcus pneumoniae of serotype 3 possess a mucoid capsule and cause disease associated with high mortality rates relative to other pneumococci. Phylogenetic analysis of a complete reference genome and 81 draft sequences from clonal complex 180, the predominant serotype 3 clone in much of the world, found most sampled isolates belonged to a clade affected by few diversifying recombinations. However, other isolates indicate significant genetic variation has accumulated over the clonal complex's entire history. Two closely related genomes, one from the blood and another from the cerebrospinal fluid, were obtained from a patient with meningitis. The pair differed in their behaviour in a mouse model of disease and in their susceptibility to antimicrobials, with at least some of these changes attributable to a mutation that up-regulated the patAB efflux pump. This indicates clinically important phenotypic variation can accumulate rapidly through small alterations to the genotype.
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311. |
Wyres KL,
van Tonder A,
Lambertsen LM,
Hakenbeck R,
Parkhill J,
Bentley SD,
Brueggemann AB,
( 2013 ) Evidence of antimicrobial resistance-conferring genetic elements among pneumococci isolated prior to 1974. PMID : 23879707 : DOI : 10.1186/1471-2164-14-500 PMC : PMC3726389 Abstract >>
Antimicrobial resistance among pneumococci has greatly increased over the past two to three decades. Resistance to tetracycline (tet(M)), chloramphenicol (cat) and macrolides (erm(B) and/or mef(A/E)) is generally conferred by acquisition of specific genes that are associated with mobile genetic elements, including those of the Tn916 and Tn5252 families. The first tetracycline-, chloramphenicol- and macrolide-resistant pneumococci were detected between 1962 and 1970; however, until now the oldest pneumococcus shown to harbour Tn916 and/or Tn5252 was isolated in 1974. In this study the genomes of 38 pneumococci isolated prior to 1974 were probed for the presence of tet(M), cat, erm(B), mef(A/E) and int (integrase) to indicate the presence of Tn916/Tn5252-like elements. Two Tn916-like, tet(M)-containing, elements were identified among pneumococci dated 1967 and 1968. The former element was highly similar to that of the PMEN1 multidrug-resistant, globally-distributed pneumococcal reference strain, which was isolated in 1984. The latter element was associated with a streptococcal phage. A third, novel genetic element, designated ICESpPN1, was identified in the genome of an isolate dated 1972. ICESpPN1 contained a region of similarity to Tn5252, a region of similarity to a pneumococcal pathogenicity island and novel lantibiotic synthesis/export-associated genes. These data confirm the existence of pneumococcal Tn916 elements in the first decade within which pneumococcal tetracycline resistance was described. Furthermore, the discovery of ICESpPN1 demonstrates the dynamic variability of pneumococcal genetic elements and is contrasted with the evidence for Tn916 stability.
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312. |
Boers SA,
van der Reijden WA,
Jansen R,
( 2012 ) High-throughput multilocus sequence typing: bringing molecular typing to the next level. PMID : 22815712 : DOI : 10.1371/journal.pone.0039630 PMC : PMC3399827 Abstract >>
Multilocus sequence typing (MLST) is a widely used system for typing microorganisms by sequence analysis of housekeeping genes. The main advantage of MLST in comparison to other typing techniques is the unambiguity and transferability of sequence data. However, a main disadvantage is the high cost of DNA sequencing. Here we introduce a high-throughput MLST (HiMLST) method that employs next-generation sequencing (NGS) technology (Roche 454), to generate large quantities of high-quality MLST data at low costs. The HiMLST protocol consists of two steps. In the first step MLST target genes are amplified by PCR in multi-well plates. During this PCR the amplicons of each bacterial isolate are provided with a unique DNA barcode, the multiplex identifier (MID). In the second step all amplicons are pooled and sequenced in a single NGS-run. The MLST profile of each individual isolate can be retrieved easily using its unique MID. With HiMLST we have profiled 575 isolates of Legionella pneumophila, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pneumoniae in mixed species HiMLST experiments. In conclusion, the introduction of HiMLST paves the way for a broad employment of the MLST as a high-quality and cost-effective method for typing microbial species.
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313. |
Beilharz K,
Nováková L,
Fadda D,
Branny P,
Massidda O,
Veening JW,
( 2012 ) Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP. PMID : 22431591 : DOI : 10.1073/pnas.1119172109 PMC : PMC3326482 Abstract >>
How the human pathogen Streptococcus pneumoniae coordinates cell-wall synthesis during growth and division to achieve its characteristic oval shape is poorly understood. The conserved eukaryotic-type Ser/Thr kinase of S. pneumoniae, StkP, previously was reported to phosphorylate the cell-division protein DivIVA. Consistent with a role in cell division, GFP-StkP and its cognate phosphatase, GFP-PhpP, both localize to the division site. StkP localization depends on its penicillin-binding protein and Ser/Thr-associated domains that likely sense uncross-linked peptidoglycan, because StkP and PhpP delocalize in the presence of antibiotics that target the latest stages of cell-wall biosynthesis and in cells that have stopped dividing. Time-lapse microscopy shows that StkP displays an intermediate timing of recruitment to midcell: StkP arrives shortly after FtsA but before DivIVA. Furthermore, StkP remains at midcell longer than FtsA, until division is complete. Cells mutated for stkP are perturbed in cell-wall synthesis and display elongated morphologies with multiple, often unconstricted, FtsA and DivIVA rings. The data show that StkP plays an important role in regulating cell-wall synthesis and controls correct septum progression and closure. Overall, our results indicate that StkP signals information about the cell-wall status to key cell-division proteins and in this way acts as a regulator of cell division.
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314. |
Nahm MH,
Yother J,
Abeygunwardana C,
Larson TR,
( 2012 ) Biochemical, genetic, and serological characterization of two capsule subtypes among Streptococcus pneumoniae Serotype 20 strains: discovery of a new pneumococcal serotype. PMID : 22736767 : DOI : 10.1074/jbc.M112.380451 PMC : PMC3431705 Abstract >>
The bacterial pathogen Streptococcus pneumoniae expresses one of over 90 structurally distinct polysaccharide (PS) capsule serotypes. Prior PS structural analyses of the vaccine-associated serotype 20 do not agree with reports describing the genes that mediate capsule synthesis. Furthermore, using immunized human sera-based assays, serological differences were recently noted among strains typed as serotype 20. We examined the capsule structures of two serologically dissimilar serotype 20 strains, 20�\ and 20�], by extensive biochemical analysis. 20�\ PS was composed of the previously described serotype 20 hexasaccharide repeat unit, whereas the 20�] PS was composed of a novel heptasaccharide repeat unit containing an extra branching �\-glucose residue. Genetic analysis of the subtypes revealed that 20�\ may have arisen from a 20�] progenitor following loss of function mutation to the glycosyltransferase gene whaF. Conventional serotyping methods using rabbit polyclonal or mouse monoclonal antibodies were unable to distinguish the subtypes. However, genetic analysis of multiple "serotype 20" clinical isolates revealed that all strains contain the 20�] genotype. We propose naming bacteria that express the previously described 20�\ capsule structure 20A and bacteria that express the novel 20�] capsule structure 20B, a new pneumococcal serotype.
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315. |
Moreno AT,
Oliveira ML,
Ho PL,
Vadesilho CF,
Palma GM,
Ferreira JM,
Ferreira DM,
Santos SR,
Martinez MB,
Miyaji EN,
( 2012 ) Cross-reactivity of antipneumococcal surface protein C (PspC) antibodies with different strains and evaluation of inhibition of human complement factor H and secretory IgA binding via PspC. PMID : 22336290 : DOI : 10.1128/CVI.05706-11 PMC : PMC3318277 Abstract >>
Pneumococcal surface protein C (PspC) is an important candidate for a cost-effective vaccine with broad coverage against pneumococcal diseases. Previous studies have shown that Streptococcus pneumoniae is able to bind to both human factor H (FH), an inhibitor of complement alternative pathway, and human secretory IgA (sIgA) via PspC. PspC was classified into 11 groups based on variations of the gene. In this work, we used three PspC fragments from different groups (PspC3, PspC5, and PspC8) to immunize mice for the production of antibodies. Immunization with PspC3 induced antibodies that recognized the majority of the clinical isolates as analyzed by Western blotting of whole-cell extracts and flow cytometry of intact bacteria, while anti-PspC5 antibodies showed cross-reactivity with the paralogue pneumococcal surface protein A (PspA), and anti-PspC8 antibodies reacted only with the PspC8-expressing strain. Most of the isolates tested showed strong binding to FH and weaker interaction with sIgA. Preincubation with anti-PspC3 and anti-PspC5 IgG led to some inhibition of binding of FH, and preincubation with anti-PspC3 partially inhibited sIgA binding in Western blotting. The analysis of intact bacteria through flow cytometry showed only a small decrease in FH binding after incubation of strain D39 with anti-PspC3 IgG, and one clinical isolate showed inhibition of sIgA binding by anti-PspC3 IgG. We conclude that although anti-PspC3 antibodies were able to recognize PspC variants from the majority of the strains tested, partial inhibition of FH and sIgA binding through anti-PspC3 antibodies in vitro could be observed for only a restricted number of isolates.
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316. |
Virolainen A,
Toropainen M,
Nahm MH,
Lambertsen L,
( 2012 ) From Quellung to multiplex PCR, and back when needed, in pneumococcal serotyping. PMID : 22692742 : DOI : 10.1128/JCM.00689-12 PMC : PMC3421528 Abstract >>
All currently available vaccines against Streptococcus pneumoniae are based on selections of the over 90 different serotypes, which underlines the importance of serotyping for surveillance and vaccine efficacy monitoring. In this study, we modified and validated a PCR-based scheme for deducing the serotypes of the invasive pneumococci isolated in Finland. For validation, 170 isolates were serotyped using the new protocol with six sequential multiplex PCRs for the deduction of serotypes, supplemented with Quellung testing when needed. The results were compared with those obtained by traditional serotyping methods. We found that 98.8% (168/170) of the isolates were correctly serotyped by the new protocol. Subsequently, the scheme was taken into regular use for serotyping the invasive pneumococci isolated in Finland for serotype-specific surveillance purposes and has been applied in the serotyping of more than 1,500 invasive isolates so far. The sequential multiplex PCRs (mPCRs) have given a result for over 99% of the isolates and allowed us to both handle samples in bulk and noticeably reduce the cost of reagents. While serotyping primarily by PCR is precise and effective, Quellung testing remains the most reliable way to discover possible discrepancies between the DNA deduced and the phenotypic serotype of an isolate. Since implementing the protocol for regular use, two serotype 19F PCR-positive isolates were found to be serotype 19A by the Quellung reaction. While a rare occurrence, this is an important observation, which prompted a revision of our serotyping protocol to prevent possible underreporting of serotype 19A, a potential replacement serotype following large-scale vaccination.
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317. |
Calix JJ,
Dagan R,
Pelton SI,
Porat N,
Nahm MH,
( 2012 ) Differential occurrence of Streptococcus pneumoniae serotype 11E between asymptomatic carriage and invasive pneumococcal disease isolates reflects a unique model of pathogen microevolution. PMID : 22267713 : DOI : 10.1093/cid/cir953 PMC : PMC3284216 Abstract >>
Streptococcus pneumoniae is a commensal colonizer of the human nasopharynx (NP) that causes disease after evasion of host defenses and dissemination. Pneumococcal strains expressing the newly identified serotype 11E arise from antigenically similar 11A progenitors by genetic inactivation of the O-acetyltransferase gene wcjE. Each 11E strain contains a distinct mutation to wcjE, suggesting that 11E strains are not transmitted among hosts despite their recovery from multiple patients with pneumococcal disease. We investigated whether the presumed lack of transmission of serotype 11E is consistent with its inability to survive in the NP. More than 400 pneumococcal carriage, middle ear, conjunctiva, and blood isolates, serotyped as 11A by Quellung reaction, were reexamined for reactivity to 11A- and 11E-specific antibodies. We confirmed serotyping of isolates with sequencing of wcjE alleles. Serotype 11E strains were statistically more likely to occur among blood (4 of 15), conjunctiva (1 of 14), or middle ear (2 of 21) isolates than among carriage isolates (2 of 355). All 11E isolates contained unique mutations that putatively decrease wcjE expression. The lack of a circulating 11E clone and the increased occurrence of 11E strains among disease isolates supports the idea that serotype 11E independently arises during infection after initial colonization with a serotype 11A progenitor. Factors encountered in the NP likely contribute to relative rarity of 11E among carriage isolates, whereas selective pressures in deeper tissues possibly promote 11E emergence. These findings illustrate a novel model of microevolution that transpires during the span of a single encounter with serotype 11A, highlighting the adaptability of bacterial pathogens within hosts.
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318. |
Denapaite D,
Hakenbeck R,
( 2011 ) A new variant of the capsule 3 cluster occurs in Streptococcus pneumoniae from deceased wild chimpanzees. PMID : 21969869 : DOI : 10.1371/journal.pone.0025119 PMC : PMC3182177 Abstract >>
The presence of new Streptococcus pneumoniae clones in dead wild chimpanzees from the Ta? National Park, C?te d'Ivoire, with previous respiratory problems has been demonstrated recently by DNA sequence analysis from samples obtained from the deceased apes. In order to broadenour understanding on the relatedness of these pneumococcal clones to those from humans, the gene locus responsible for biosynthesis of the capsule polysaccharide (CPS) has now been characterized. DNA sequence analysis of PCR fragments identified a cluster named cps3(Ta?) containing the four genes typical for serotype 3 CPS, but lacking a 5'-region of ?2 kb which is degenerated in other cps3 loci and not required for type 3 biosynthesis. CPS3 is composed of a simple disaccharide repeat unit comprising glucose and glucuronic acid (GlcUA). The two genes ugd responsible for GlcUA synthesis and wchE encoding the type 3 synthase are essential for CPS3 biosynthesis, whereas both, galU and the 3'-truncated gene pgm are not required due to the presence of homologues elsewhere in the genome. The DNA sequence of cps3(Ta?) diverged considerably from those of other cps3 loci. Also, the gene pgm(Ta?) represents a full length version with a nonsense mutation at codon 179. The two genes ugd(Ta?) and wchE(Ta?) including the promoter region were transformed into a nonencapsulated laboratory strain S. pneumoniae R6. Transformants which expressed type 3 capsule polysaccharide were readily obtained, documenting that the gene products are functional. In summary, the data indicate that cps3(Ta?) evolved independent from other cps3 loci, suggesting the presence of specialized serotype 3 S. pneumoniae clones endemic to the Ta? National Park area.
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319. |
Schuster C,
Dobrinski B,
Hakenbeck R,
( 1990 ) Unusual septum formation in Streptococcus pneumoniae mutants with an alteration in the D,D-carboxypeptidase penicillin-binding protein 3. PMID : 2228972 : DOI : 10.1128/jb.172.11.6499-6505.1990 PMC : PMC526838 Abstract >>
An internal 630-bp DNA fragment of the gene encoding penicillin-binding protein 3 (PBP 3) (dacA) of Streptococcus pneumoniae was identified in a lambda gt11 gene bank screened with anti-PBP 3 antiserum. The deduced 210-amino-acid sequence showed a high degree of homology to the low-molecular-weight PBPs 5 and 6 of Escherichia coli and Bacillus subtilis PBP 5. Viable mutants lacking a C-terminal part of PBP 3 were obtained after a plasmid containing the dacA fragment was integrated into the PBP 3 gene by homologous recombination. The truncated PBP 3* was still active in terms of beta-lactam binding. Most PBP 3 was found in the growth medium, indicating that membrane anchoring of PBP 3 is provided by the C terminus, as has been shown for other D,D-carboxypeptidases. The mutant cells grew with a slower generation time than the wild type in the shape of irregular enlarged spheres. In addition, as revealed by electron microscopy, cell separation was severely affected, septa were found unevenly distributed at multiple sites within the cells, and the murein layer appeared variable in thickness.
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320. |
Trieu-Cuot P,
Poyart-Salmeron C,
Carlier C,
Courvalin P,
( 1990 ) Nucleotide sequence of the erythromycin resistance gene of the conjugative transposon Tn1545. PMID : 2163525 : DOI : 10.1093/nar/18.12.3660 PMC : PMC331044 Abstract >>
N/A
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321. |
Sistek V,
Boissinot M,
Boudreau DK,
Huletsky A,
Picard FJ,
Bergeron MG,
( 2012 ) Development of a real-time PCR assay for the specific detection and identification of Streptococcus pseudopneumoniae using the recA gene. PMID : 22022828 : DOI : 10.1111/j.1469-0691.2011.03684.x Abstract >>
We sequenced the evolutionarily conserved genes 16S rRNA, atpD, tuf, and recA from Streptococcus pseudopneumoniae, Streptococcus pneumoniae, Streptococcus mitis, and Streptococcus oralis. Phylogenetic analysis revealed that recA provided good resolution between these species, including discrimination of the novel species S. pseudopneumoniae. By contrast, the more conserved 16S rRNA, tuf and atpD are not sufficiently discriminatory. Therefore, recA sequences were used to develop a real-time PCR assay with a locked nucleic acid-mediated TaqMan probe for the specific detection and identification of S. pseudopneumoniae. The PCR assay showed excellent specificity and a detection limit of <10 genome copies for the detection and identification of S. pseudopneumoniae strains, which makes it a promising tool for molecular identification and epidemiological studies. In conclusion, this article describes for the first time a PCR assay for the specific identification of S. pseudopneumoniae.
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322. |
Kawai Y,
Marles-Wright J,
Cleverley RM,
Emmins R,
Ishikawa S,
Kuwano M,
Heinz N,
Bui NK,
Hoyland CN,
Ogasawara N,
Lewis RJ,
Vollmer W,
Daniel RA,
Errington J,
( 2011 ) A widespread family of bacterial cell wall assembly proteins. PMID : 21964069 : DOI : 10.1038/emboj.2011.358 PMC : PMC3243631 Abstract >>
Teichoic acids and acidic capsular polysaccharides are major anionic cell wall polymers (APs) in many bacteria, with various critical cell functions, including maintenance of cell shape and structural integrity, charge and cation homeostasis, and multiple aspects of pathogenesis. We have identified the widespread LytR-Cps2A-Psr (LCP) protein family, of previously unknown function, as novel enzymes required for AP synthesis. Structural and biochemical analysis of several LCP proteins suggest that they carry out the final step of transferring APs from their lipid-linked precursor to cell wall peptidoglycan (PG). In Bacillus subtilis, LCP proteins are found in association with the MreB cytoskeleton, suggesting that MreB proteins coordinate the insertion of the major polymers, PG and AP, into the cell wall.
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323. |
Ko KS,
Baek JY,
Song JH,
( 2012 ) Multidrug-resistant Streptococcus pneumoniae serotype 6D clones in South Korea. PMID : 22170935 : DOI : 10.1128/JCM.05895-11 PMC : PMC3295089 Abstract >>
To investigate the characteristics of main Streptococcus pneumoniae clones of serotype 6D (ST282 and ST3171) in South Korea, antimicrobial susceptibility testing was performed, and 11 genes around the cps locus were sequenced on ST282(6D), ST3171(6D), and ST81(6A) isolates. The antimicrobial susceptibility patterns were very similar between clones belonging to the same clonal complex, ST81(6A) and ST282(6D); nonsusceptibilities to penicillin and cefuroxime, high MICs of ceftriaxone, and high resistance rates to trimethoprim-sulfamethoxazole. However, ST3171(6D) isolates showed resistance to only macrolides and clindamycin. The sequences of 11 genes around the cps locus indicated the same genetic backgrounds between the ST81(6A) and ST282(6D) isolates. On the other hand, ST3171(6D) isolates showed nucleotide and amino acid differences from ST81(6A) and ST282(6D) isolates in most genes, indicating a different genetic background. The mosaic structure of dexB gene in ST282(6D) isolates indicated that recombination might occur in the dexB gene. Our results suggest that the multidrug-resistant ST282(6D) pneumococcal clone has emerged by serial genetic recombination, including capsular switch.
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324. |
( 1996 ) Penicillin-binding proteins as resistance determinants in clinical isolates of Streptococcus pneumoniae. PMID : 9158756 : DOI : 10.1089/mdr.1996.2.177 Abstract >>
Altered penicillin-binding proteins (PBPs) with reduced affinity for penicillin are encoded by mosaic genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Generally, members of one bacterial clone contain the same mosaic gene. We report here on a serotype 19A clone of penicillin- and multiple-resistant S. pneumoniae prevalent in Hungary, members of which are exceptionally diverse in terms of PBP properties. The pbp2x gene of four 19A isolates was sequenced, and a distinct mosaic structure detected in each case. The pbp2x genes also differed from a homologous gene of a high-level penicillin-resistant S. mitis from Hungary. The contribution of PBPs to resistance development was studied on transformation experiments using the laboratory strain R6 as recipient, and PBP genes from the type 19A isolate Hu11. pbp2x and pbp2b function as primary resistance determinants for different beta-lactams. Secondary transformation with pbp1a increased the resistance level considerably for penicillins and cefotaxime. Chromosomal DNA of a high-level penicillin- and cefotaxime-resistant S. mitis from Hungary also transformed the R6 strain to increased resistance levels, and PBP2x and PBP2b functioned as primary resistance determinants as above. In contrast, high-level cefotaxime resistance appeared to be due to a low affinity PBP2a, indicating that this PBP can also function as a resistance determinant.
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325. |
( 1997 ) Scavengase p20: a novel family of bacterial antioxidant enzymes. PMID : 9141476 : DOI : 10.1016/s0014-5793(97)00302-5 Abstract >>
A novel antioxidant enzyme designated scavengase p20 was identified in various pathogenic bacteria through database searching for sequences strikingly homologous to a recently discovered Escherichia coli thiol peroxidase p20. The direct biochemical evidence for the existence of scavengase p20 in Haemophilus influenzae, Streptococcus pneumoniae and Helicobacter pylori was provided by protein microsequencing and by in vitro assays for antioxidant activities. Overlapping genes encoding scavengase p20 and superoxide dismutase were recognized in H. pylori and their functional implications are discussed.
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326. |
( 1997 ) mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae. PMID : 9333056 : PMC : PMC164101 Abstract >>
Recently, it was shown that a significant number of erythromycin-resistant Streptococcus pneumoniae and Streptococcus pyogenes strains contain a determinant that mediates resistance via a putative efflux pump. The gene encoding the erythromycin-resistant determinant was cloned and sequenced from three strains of S. pneumoniae bearing the M phenotype (macrolide resistant but clindamycin and streptogramin B susceptible). The DNA sequences of mefE were nearly identical, with only 2-nucleotide differences between genes from any two strains. When the mefE sequences were compared to the mefA sequence from S. pyogenes, the two genes were found to be closely related (90% identity). Strains of S. pneumoniae were constructed to confirm that mefE is necessary to confer erythromycin resistance and to explore the substrate specificity of the pump; no substrates other than 14- and 15-membered macrolides were identified.
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327. |
( 1997 ) Characterization of the locus encoding the Streptococcus pneumoniae type 19F capsular polysaccharide biosynthetic pathway. PMID : 9157246 : DOI : 10.1046/j.1365-2958.1997.2551624.x Abstract >>
We have previously reported the nucleotide sequence of the first six genes of the Streptococcus pneumoniae type 19F capsular polysaccharide biosynthesis locus (cps19f). In this study we used plasmid insertion/rescue and inverse polymerase chain reaction (PCR) to clone an additional 10 kb downstream region containing the remainder of the cps19f locus, which was then subjected to sequence analysis. The cps19f locus is located in the S. pneumoniae chromosome between dexB and aliA, and consists of 15 open reading frames (ORFs), designated cps19fA to cps19fO, that appear to be arranged as a single transcriptional unit. Insertion-duplication mutants in seven out of the nine new ORFs have been constructed in a smooth type 19F strain, all of which resulted in a rough (nonencapsulated) phenotype, confirming that the operon is essential for capsule production. Comparison with sequence databases has allowed us to propose functions for 12 of the cps19f gene products, and a biosynthetic pathway for type 19F capsular polysaccharide. T7 expression studies confirmed that cps19fH, cps19fK, cps19fL, cps19fM and cps19fN directed the production of polypeptides of the expected size in Escherichia coli. The function of the cps19fK product was confirmed by its ability to complement a mutation in nfrC (rffE) in E. coli, as judged by restoration of sensitivity to bacteriophage N4. Interestingly, the last four genes of the locus (cps19fL-O) exhibit very strong homology (up to 70% amino acid identity) to a portion of the Shigella flexneri rfb gene cluster encoding biosynthesis of dTDP-rhamnose. When expressed in E. coli, cps19fL-O were capable of complementing a mutation deleting the respective Shigella flexneri homologues. Southern hybridization analysis indicated that cps19fA and cps19fB were the only cps genes found in all 16 S. pneumoniae serotypes/groups tested. The region from cps19fG to cps19fK was found only in members of serogroup 19, and, within this, cps19fl was unique to type 19F.
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328. |
( 1997 ) Site-specific nicking in vitro at ori T by the DNA relaxase of Tn5252. PMID : 9073581 : DOI : 10.1006/plas.1996.1268 Abstract >>
Tn5252 is a promiscuous streptococcal element capable of madiating horizontal spread of multiple antibiotic resistance. To begin understanding the functional role of a transfer-related region in Tn5252, its nucleotide sequence was determined. Sequence of this 3. 3-kb DNA segment revealed the presence of six open reading frames. The predicted amino acid sequence of one of the open reading frames, ORF9, showed similarity to a predicted protein product of the lactococcal conjugative plasmid, pC1528. The deduced primary protein sequence of another, ORF4, showed strong structural similarity to conserved regions of various prokaryotic DNA relaxases that initiate conjugal transfer by strand- and site-specific cleavage at the transfer origin. A hybrid protein containing the ORF4 protein fused to the carboxyl terminal end of maltose binding protein was purified from Escherichia coli and found to specifically nick plasmids carrying a 2-kb DNA segment derived from the transposon. The nicking reaction is protein concentration-dependent. These results imply that the conjugative transposition of Tn5252 may involve rolling circle replication and transfer of a unique DNA strand.
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329. |
( 1997 ) Molecular and genetic characterization of the capsule biosynthesis locus of Streptococcus pneumoniae type 19B. PMID : 9244289 : DOI : 10.1128/jb.179.15.4953-4958.1997 PMC : PMC179348 Abstract >>
We have previously reported the nucleotide sequence of the Streptococcus pneumoniae type 19F capsular polysaccharide synthesis locus (cps19f), which consists of 15 open reading frames (ORFs) designated cps19fA to -O. Hybridization analysis indicated that close homologs for cps19fA to -H and cps19fK to -O were found in type 19B, but there were no homologs for cps19fI and -J. In this study we used long-range PCR to amplify and clone a 10.5-kb section of the S. pneumoniae type 19B capsule locus (cps19b) between cps19bH and cps19bK. This region of the cps19b locus is 4 kb larger than that in the cps19f locus and replaces cps19fI and cps19fJ with five new ORFs, designated cps19bP, -I, -Q, -R, and -J. We have proposed functions for four of the protein products, including functional homologs of Cps19fI and Cps19fJ. Transformation of a S. pneumoniae mutant containing an interrupted type 19F capsule locus with the 10.5-kb cps19b PCR product converted the recipient strain to type 19B. Southern hybridization analysis indicated that cps19bP, -I, -Q, -R, and -J are unique to type 19B and the closely related type 19C.
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330. |
( 1997 ) Mechanism of sulfonamide resistance in clinical isolates of Streptococcus pneumoniae. PMID : 9333035 : PMC : PMC164080 Abstract >>
The genetic basis of sulfonamide resistance in six clinical isolates of Streptococcus pneumoniae was demonstrated to be 3- or 6-bp duplications within sulA, the chromosomal gene encoding dihydropteroate synthase. The duplications all result in repetition of one or two amino acids in the region from Arg58 to Tyr63, close to but distinct from the sul-d mutation, a duplication previously reported in a resistant laboratory strain (P. Lopez, M. Espinosa, B. Greenberg, and S. A. Lacks, J. Bacteriol. 169:4320-4326, 1987). Six sulfonamide-susceptible clinical isolates lacked such duplications. The role of the duplications in conferring sulfonamide resistance was confirmed by transforming 319- or 322-bp PCR fragments into the chromosome of a susceptible recipient. Two members of a clone of serotype 9V, one susceptible and one resistant to sulfonamide, which are highly related by other criteria, were shown to have sulA sequences that differ in 7.2% of nucleotides in addition to the duplication responsible for resistance. It is postulated that horizontal gene exchange has been involved in the acquisition (or loss) of resistance within this clone. However, five of the six resistant isolates have distinct duplications and other sequence polymorphisms, suggesting that resistance has arisen independently on many occasions.
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331. |
( 1997 ) TN5252: a model for complex streptococcal conjugative transposons. PMID : 9331826 : DOI : 10.1007/978-1-4899-1825-3_242 Abstract >>
N/A
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332. |
( 1997 ) A mutation in the D,D-carboxypeptidase penicillin-binding protein 3 of Streptococcus pneumoniae contributes to cefotaxime resistance of the laboratory mutant C604. PMID : 9145848 : PMC : PMC163829 Abstract >>
Cefotaxime resistance in laboratory mutant C604 of Streptococcus pneumoniae, for which the MIC is 1.5 microg/ml, is independent of alterations in high-molecular-mass penicillin-binding protein (PBP) 1a. Instead, a point mutation in PBP 3, the D,D-carboxypeptidase of this organism, caused a reduced affinity for penicillin and contributed to the decreased susceptibility. The mutation Thr-242 to Ile was located directly adjacent to the triad Lys-239-Thr-Gly, a position known to be important for beta-lactam interaction with high-molecular-mass PBPs and beta-lactamases. This mutation was absent in the PBP 3's of four genetically distinct clinical isolates resistant to high levels of penicillin. None of the pbp3 genes had a mosaic structure, but in three cases there was evidence for a site-specific recombination event within a BOX element immediately downstream of pbp3.
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( 1997 ) Functional analysis of glycosyltransferases encoded by the capsular polysaccharide biosynthesis locus of Streptococcus pneumoniae serotype 14. PMID : 9235953 : DOI : 10.1074/jbc.272.31.19502 Abstract >>
Bacteria belonging to the species Streptococcus pneumoniae vary in their capsule. Presently, 90 capsular serotypes are known, all possessing their own specific polysaccharide structure. Little is known about the biosynthesis of these capsular polysaccharides. The cps locus of S. pneumoniae serotype 14 was cloned. So far, 7 open reading frames have been sequenced, cps14B to cps14H. The gene products are similar to proteins involved in bacterial polysaccharide biosynthesis, both of Gram-negative and -positive micro-organisms. Gene-specific mutants were created for cps14D to cps14H by insertional mutagenesis. All mutants no longer agglutinated with a monoclonal antibody against type 14 capsule polysaccharides. The biosynthetic function of cps14E and cps14G was determined by analysis of the intermediates in the synthesis of the oligosaccharide subunit, formed in membrane preparations of the wild-type and mutant strains and in membrane preparations of Escherichia coli expressing the pneumococcal glycosyltransferases. The enzyme encoded by cps14E is a glucosyl-1-phosphate transferase that links glucose to a lipid carrier, the first step in the biosynthesis of the type 14 repeating unit. The gene product of cps14G encodes a beta-1,4-galactosyltransferase, the enzyme responsible for the second step in the subunit synthesis, the transfer of galactose to lipid-linked glucose.
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( 1997 ) Capsular polysaccharide synthesis in Streptococcus pneumoniae serotype 14: molecular analysis of the complete cps locus and identification of genes encoding glycosyltransferases required for the biosynthesis of the tetrasaccharide subunit. PMID : 9383201 : DOI : 10.1046/j.1365-2958.1997.5791940.x Abstract >>
We have reported previously on seven genes (cps14B-H) of Streptococcus pneumoniae serotype 14, which are part of the type 14 capsular polysaccharide synthesis (cps14) locus. This study describes the cloning and sequencing of the remaining part of the cps14 locus. The entire cps14 gene cluster consists of 12 open reading genes (cps14A to cps14L), which appear to be arranged as a single transcriptional unit. The flanking regions of the cps14 locus contain vestiges of insertion elements. Moreover, a 115-bp-long repeated DNA element, which is also present in several other intergenic regions on the pneumococcal chromosome, has been identified upstream of cps14A. All 12 open reading frames (ORFs) were inactivated by the insertion of a tetracycline resistance cassette. The cps14A to cps14J and cps14L mutants were unencapsulated, whereas only a limited amount of capsular polysaccharide was expressed by a cps14K insertion mutant. Comparison with DNA and protein sequences available in databases allowed us to predict functions for four out of the five new cps14 gene products. The biosynthetic function of Cps14I was determined experimentally by analysis of intermediates in the synthesis of the type 14 tetrasaccharide subunit, catalysed by membrane preparations of Escherichia coli expressing pneumococcal glycosyltransferases. The cps14I gene encodes the beta-1,3-N-acetylglucosaminyltransferase activity necessary for the addition of the third sugar in the synthesis of the type 14 repeating unit. The activity encoded by cps14J was established using a synthetic glycosyltransferase acceptor: cps14J encodes a beta-1,4-galactosyltransferase, which requires beta-linked GlcNAc as an acceptor. Thus, Cps14J is responsible for the addition of the last (fourth) sugar in the synthesis of the type 14 subunit.
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( 1997 ) A novel resistance mechanism against beta-lactams in Streptococcus pneumoniae involves CpoA, a putative glycosyltransferase. PMID : 9150233 : DOI : 10.1128/jb.179.10.3342-3349.1997 PMC : PMC179116 Abstract >>
Piperacillin resistance in Streptococcus pneumoniae was mediated by mutations in a novel gene, cpoA, that also confer transformation deficiency and a decrease in penicillin-binding protein la. cpoA is part of an operon located downstream of the primary sigma factor of S. pneumoniae. The deduced protein, CpoA, and the peptide encoded by the adjacent 3' open reading frame contained domains homologous to glycosyltransferases of procaryotes and eucaryotes that act on membrane-associated substrates, such as enzymes functioning in lipopolysaccharide core biosynthesis of gram-negative bacteria, RodD of Bacillus subtilis, which is involved in teichoic acid biosynthesis, and the human PIG-A protein, which is required for early steps of glycosylphosphatidylinositol anchor biosynthesis. This suggests that the cpo operon has a similar function related to cell surface components.
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( 1997 ) The com locus controls genetic transformation in Streptococcus pneumoniae. PMID : 9157240 : DOI : 10.1046/j.1365-2958.1997.2481617.x Abstract >>
Genetic exchange by natural transformation in Streptococcus pneumoniae occurs in a cell-density dependent process and is initiated by a small extracellular signalling molecule, the competence-stimulating peptide (CSP). comC, the gene for this peptide, has previously been identified and encodes a 44 amino acid pre-peptide that is apparently processed to an active molecule that consists of the C-terminal 17 amino acids. We have sequenced the region adjacent to comC and shown that it is the first gene of an operon, com, consisting of two downstream elements, comD and comE, which encode members of the two-component family of sensor regulators. Null mutants with defects in either comC or comD were transformation deficient and failed to respond to exogenous CSP. A comC mutant did not exhibit any detectable CSP activity, while a comD mutant that contained an intact comC produced minimal CSP activity. In mixed-culture experiments consisting of isogenic pairs of pneumococci (Csp+ and Csp-), we showed that induction of competence by quorum sensing was independent of CSP. Northern analysis showed that com was transcribed as a single polycistronic message, while analysis of strains with transcriptional fusions showed that com was constitutively expressed under conditions that both promoted or repressed the development of competence. Finally, we showed genetically and biochemically a CSP-dependent transcription of rec, a competence-induced locus, and that ComD and ComE are required for this CSP-dependent expression.
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( 1997 ) Contribution of novel choline-binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae. PMID : 9364908 : DOI : 10.1111/j.1365-2958.1997.mmi494.x Abstract >>
The surface of Streptococcus pneumoniae is decorated with a family of choline-binding proteins (CBPs) that are non-covalently bound to the phosphorylcholine of the teichoic acid. Two examples (PspA, a protective antigen, and LytA, the major autolysin) have been well characterized. We identified additional CPBs and characterized a new CBP, CbpA, as an adhesin and a determinant of virulence. Using choline immobilized on a solid matrix, a mixture of proteins from a pspA-deficient strain of pneumococcus was eluted in a choline-dependent fashion. Antisera to these proteins passively protected mice challenged in the peritoneum with a lethal dose of pneumococci. The predominant component of this mixture, CbpA, is a 75-kDa surface-exposed protein that reacts with human convalescent antisera. The deduced sequence from the corresponding gene showed a chimeric architecture with a unique N-terminal region and a C-terminal domain consisting of 10 repeated choline-binding domains nearly identical to PspA. A cbpA-deficient mutant showed a >50% reduction in adherence to cytokine-activated human cells and failed to bind to immobilized sialic acid or lacto-N-neotetraose, known pneumococcal ligands on eukaryotic cells. Carriage of this mutant in an animal model of nasopharyngeal colonization was reduced 100-fold. There was no difference between the parent strain and this mutant in an intraperitoneal model of sepsis. These data for CbpA extend the important functions of the CBP family to bacterial adherence and identify a pneumococcal vaccine candidate.
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( 1997 ) SpsA, a novel pneumococcal surface protein with specific binding to secretory immunoglobulin A and secretory component. PMID : 9350867 : DOI : 10.1046/j.1365-2958.1997.5391899.x Abstract >>
The interaction of pathogenic bacteria with host serum and matrix proteins is a common strategy to enhance their virulence. Streptococcus pneumoniae colonizes the human upper respiratory tract in healthy individuals and is also able to cause invasive diseases. Here, we describe a novel pneumococcal surface protein, SpsA, capable of binding specifically to human secretory immunoglobulin A (SIgA). The dissociation constant of SIgA binding to SpsA was 9.3 x 10(-9) M. Free secretory component (SC) also binds to S. pneumoniae, whereas serum IgA does not, suggesting that pneumococcal binding to SIgA is mediated by the SC. To our knowledge, this is the first defined interaction of SC with a prokaryotic protein. The spsA gene encodes a polypeptide of 523 amino acids with a predicted molecular mass of 59 151 Da. The SIgA- or SC-binding domain is located in the N-terminal part of SpsA and exhibits no significant homology to any other proteins. The purified SIgA-binding domain of SpsA could completely inhibit the binding of SIgA to pneumococci. SpsA was expressed by 73% of the tested S. pneumoniae isolates and was substantially conserved between different serotypes. The interaction between S. pneumoniae and SC via SpsA represents a novel biological interaction that might increase virulence by the impairment of bacterial clearance.
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( 1997 ) Solution structure of an rRNA methyltransferase (ErmAM) that confers macrolide-lincosamide-streptogramin antibiotic resistance. PMID : 9187657 : Abstract >>
The Erm family of methyltransferases is responsible for the development of resistance to the macrolide-lincosamide-streptogramin type B (MLS) antibiotics. These enzymes methylate an adenine of 23S ribosomal RNA that prevents the MLS antibiotics from binding to the ribosome and exhibiting their antibacterial activity. Here we describe the three-dimensional structure of an Erm family member, ErmAM, as determined by NMR spectroscopy. The catalytic domain of ErmAM is structurally similar to that found in other methyltransferases and consists of a seven-stranded beta-sheet flanked by alpha-helices and a small two-stranded beta-sheet. In contrast to the catalytic domain, the substrate binding domain is different from other methyltransferases and adopts a novel fold that consists of four alpha-helices.
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( 1997 ) Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics. PMID : 9244281 : DOI : 10.1128/jb.179.15.4901-4908.1997 PMC : PMC179340 Abstract >>
To understand the biochemical basis of resistance of bacteria to beta-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k(cat)/Km values for the two purified enzymes (3,400 M(-1) s(-1) for S-PBP2x and 11.2 M(-1) s(-1) for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of beta-lactam hydrolytic activity. Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.
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( 1996 ) Identification of the tetracycline resistance gene, tet(O), in Streptococcus pneumoniae. PMID : 9124862 : PMC : PMC163643 Abstract >>
Five isolates of Streptococcus pneumoniae resistant to tetracycline but lacking tet(M) were studied. The tetracycline resistance gene, tet(O), was detected for the first time in the pneumococcus. The gene was amplified and sequenced and found to share 99% nucleotide sequence identity and 99, 99, and 98% deduced amino acid sequence identity with the tet(O) resistance genes of Streptococcus mutans, Campylobacter coli, and Campylobacter jejuni, respectively.
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( 1993 ) Evolution of penicillin resistance in Streptococcus pneumoniae; the role of Streptococcus mitis in the formation of a low affinity PBP2B in S. pneumoniae. PMID : 8412708 : DOI : 10.1111/j.1365-2958.1993.tb01723.x Abstract >>
Penicillin-resistant strains of Streptococcus pneumoniae possess forms of penicillin-binding proteins (PBPs) that have a low affinity for penicillin compared to those from penicillin-sensitive strains. PBP genes from penicillin-resistant isolates are very variable and have a mosaic structure composed of blocks of nucleotides that are similar to those found in PBP genes from penicillin-sensitive isolates and blocks that differ by up to 21%. These chromosomally encoded mosaic genes have presumably arisen following transformation and homologous recombination with PBP genes from a number of closely related species. This study shows that PBP2B genes from many penicillin-resistant isolates of S. pneumoniae contain blocks of nucleotides originating from Streptococcus mitis. In several instances it would appear that this material alone is sufficient to produce a low affinity PBP2B. In other examples PBP2B genes possess blocks of nucleotides from S. mitis and at least one additional unidentified species. Mosaic structure was also found in the PBP2B genes of penicillin-sensitive isolates of S. mitis or S. pneumoniae. These mosaics did not confer penicillin resistance but nevertheless reveal something of the extent to which localized recombination occurs in these naturally transformable streptococci.
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( 1993 ) Molecular epidemiology of penicillin-resistant pneumococci isolated in Nairobi, Kenya. PMID : 8406829 : PMC : PMC281170 Abstract >>
A total of 26% of the pneumococci isolated from an outpatient clinic in Nairobi, Kenya, during 1991 to 1992 had intermediate levels of penicillin resistance. Gene fingerprinting and DNA sequencing were used to distinguish the penicillin-binding protein (PBP) 1A, 2B, and 2X genes in 23 resistant isolates. Isolates were grouped into those that had identical forms of each of the three PBP genes (fingerprint groups) and those that had identical rRNA gene restriction patterns (ribotypes). Both methods divided the isolates into 11 groups. In a few cases, horizontal gene transfer appeared to have distributed an identical altered PBP gene into different pneumococcal lineages. Eight isolates were indistinguishable by ribotyping or multilocus enzyme electrophoresis and contained identical PBP 1A genes. Although these isolates were therefore members of the same clone, they were divided into two fingerprint groups which contained different PBP 2X and 2B genes. Presumably, members of this clone have acquired different altered PBP 2X and 2B genes on two separate occasions. One of these fingerprint groups contained isolates of serotype 14, whereas the other contained isolates of both serotypes 14 and 7. The identification of isolates in the latter group that are identical by all criteria, except serotype, implies the occurrence of a change in serotype. The predominant serotypes of the penicillin-resistant pneumococci from Nairobi were serotypes 14 and 19. In both cases, isolates of the same serotype which required the same MIC of penicillin were not members of a single clone, indicating that identity of serotype and MIC are not sufficient criteria for defining clones of resistant pneumococci even when the bacteria are isolated from a single clinic.
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( 1996 ) Characterization of the Streptococcus pneumoniae immunoglobulin A1 protease gene (iga) and its translation product. PMID : 8926055 : PMC : PMC174323 Abstract >>
Bacterial immunoglobulin A1 (IgA1) proteases constitute a very heterogenous group of extracellular endopeptidases which specifically cleave human IgA1 in the hinge region. Here we report that the IgA1 protease gene, iga, of Streptococcus pneumoniae is homologous to that of Streptococcus sanguis. By using the S. sanguis iga gene as hybridization probe, the corresponding gene from a clinical isolate of S. pneumoniae was isolated in an Escherichia coli lambda phage library. A lysate of E. coli infected with hybridization-positive recombinant phages possessed IgA1-cleaving activity. The complete sequence of the S. pneumoniae iga gene was determined. An open reading frame with a strongly biased codon usage and having the potential of encoding a protein of 1,927 amino acids with a molecular mass of 215,023 Da was preceded by a potential -10 promoter sequence and a putative Shine-Dalgarno sequence. A putative signal peptide was found in the N-terminal end of the protein. The amino acid sequence similarity to the S. sanguis IgA1 protease indicated that the pneumococcal IgA1 protease is a Zn-metalloproteinase. The primary structures of the two streptococcal IgA1 proteases were quite different in the N-terminal parts, and both proteins contained repeat structures in this region. Using a novel assay for IgA1 protease activity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we demonstrated that the secreted IgA1 protease was present in several different molecular forms ranging in size from approximately 135 to 220 kDa. In addition, interstrain differences in the sizes of the pneumococcal IgA1 proteases were detected. Southern blot analyses suggested that the S. pneumoniae iga gene is highly heterogenous within the species.
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( 1997 ) Decoration of lipopolysaccharide with phosphorylcholine: a phase-variable characteristic of Haemophilus influenzae. PMID : 9038301 : PMC : PMC175073 Abstract >>
Choline, although not a nutritional requirement for Haemophilus influenzae, is taken up from the growth medium and incorporated into its lipopolysaccharide (LPS). Incorporated choline is in the form of phosphorylcholine (ChoP) based on the reactivity with the monoclonal antibody with specificity for this structure, TEPC-15. Incorporation of [3H]choline from the growth medium and expression of the TEPC-15 epitope undergo high-frequency phase variation, characteristic of other LPS structures in this species. The expression and phase variation of ChoP require a previously identified locus involved in LPS biosynthesis, lic1. The first gene in lic1, licA, contains a translational switch based on variation in the number of intragenic tandem repeats of the sequence 5'-CAAT-3'. The full-length LicA polypeptide resembles choline kinases of eucaryotes, suggesting that the pathway for choline incorporation into the H. influenzae glycolipid has similarities to the pathway for choline incorporation in eucaryotic lipid synthesis. The display of ChoP, a host-like structure, renders the organism more rather than less susceptible to the bactericidal activity of human serum. The increased serum sensitivity of variants with ChoP correlates with higher serum immunoglobulin G titers to LPS containing this structure. ChoP appears to be a cell surface feature common to a number of pathogens of the human respiratory tract, including Streptococcus pneumoniae and mycoplasmas. In the case of H. influenzae, its primary contribution to pathogenesis does not appear to be antigenic variation to evade host humoral clearance mechanisms.
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( 1996 ) Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae. PMID : 8945574 : PMC : PMC174516 Abstract >>
psaA encodes a 37-kDa putative pneumococcal surface adhesin. Although its complete nucleotide sequence has been determined, its contribution to the pathogenicity of Streptococcus pneumoniae has not previously been assessed. In this study, we used a PCR-amplified internal fragment of the psaA gene from S. pneumoniae type 2 strain D39 cloned in pVA891, to direct the construction of D39 derivatives in which the psaA gene had been specifically interrupted, by insertion-duplication mutagenesis. Two independent D39 psaA mutants (PsaA-(1) and PsaA-(2)) were significantly less virulent (as judged by intranasal or intraperitoneal challenge of mice) than either the wild-type D39 strain or a derivative of PsaA-(1) in which the psaA gene had been reconstituted by back-transformation with an intact copy of the cloned gene. pVA891-directed mutagenesis of an open reading frame (designated ORF3) immediately 3' to psaA or insertion of pVA891 between psaA and ORF3 had no impact on intranasal virulence. However, a small but significant difference in virulence was observed between these two derivatives and the parental D39 strain in a low-dose intraperitoneal challenge model, suggesting that the ORF3 product may also contribute to pathogenesis. Adherence of PsaA-(1) to A549 cells (type II pneumocytes) was only 9% of that for D39, while the ORF3-negative strain exhibited intermediate adherence (23%). This is the first functional evidence that PsaA is an adhesin. Sequence analysis of the psaA gene from D39 indicated significant deviation from that previously published for the homolog from S. pneumoniae R36A. The deduced amino acid sequences of mature PsaA from the two strains had only 81% homology, with the bulk of the variation occurring in the amino-terminal portion.
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( 1996 ) Cloning and characterization of nanB, a second Streptococcus pneumoniae neuraminidase gene, and purification of the NanB enzyme from recombinant Escherichia coli. PMID : 8759848 : DOI : 10.1128/jb.178.16.4854-4860.1996 PMC : PMC178267 Abstract >>
Streptococcus pneumoniae is believed to produce more than one form of neuraminidase, but there has been uncertainty as to whether this is due to posttranslational modification of a single gene product or the existence of more than one neuraminidase-encoding gene. Only one stable pneumococcal neuraminidase gene (designated nanA) has been described. In the present study, we isolated and characterized a second neuraminidase gene (designated nanB), which is located close to nanA on the pneumococcal chromosome (approximately 4.5kb downstream). nanB was located on an operon separate from that of nanA, which includes at least five other open reading frames. NanB has a predicted size of 74.5 kDa after cleavage of a 29-amino-acid signal peptide. There was negligible amino acid homology between NanA and NanB, but NanB did exhibit limited homology with the sialidase of Clostridium septicum. NanB was purified from recombinant Escherichia coli and found to have a pH optimum of 4.5, compared with 6.5 to 7.0 for NanA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggested that NanB has a molecular size of approximately 65 kDa. The discrepancy between this estimate and the size predicted from the nucleotide sequence is most likely a consequence of C-terminal processing or anomalous electrophoretic behavior.
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( 1996 ) Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance. PMID : 8763932 : DOI : 10.1128/jb.178.14.4060-4069.1996 PMC : PMC178161 Abstract >>
DNA topoisomerase IV mediates chromosome segregation and is a potential target for antibacterial agents including new antipneumococcal fluoroquinolones. We have used hybridization to a Staphylococcus aureus gyrB probe in concert with chromosome walking to isolate the Streptococcus pneumoniae parE-parC locus, lying downstream of a putative new insertion sequence and encoding 647-residue ParE and 823-residue ParC subunits of DNA topoisomerase IV. These proteins exhibited greatest homology respectively to the GrlB (ParE) and GrlA (ParC) subunits of S. aureus DNA topoisomerase IV. When combined, whole-cell extracts of Escherichia coli strains expressing S. pneumoniae ParC or ParE proteins reconstituted a salt-insensitive ATP-dependent decatenase activity characteristic of DNA topoisomerase IV. A second gyrB homolog isolated from S. pneumoniae encoded a 648-residue protein which we identified as GyrB through its close homology both to counterparts in S. aureus and Bacillus subtilis and to the product of the S. pneumoniae nov-1 gene that confers novobiocin resistance. gyrB was not closely linked to gyrA. To examine the role of DNA topoisomerase IV in fluoroquinolone action and resistance in S. pneumoniae, we isolated mutant strains stepwise selected for resistance to increasing concentrations of ciprofloxacin. We analysed four low-level resistant mutants and showed that Ser-79 of ParC, equivalent to resistance hotspots Ser-80 of GrlA and Ser-84 of GyrA in S. aureus, was in each case substituted with Tyr. These results suggest that DNA topoisomerase IV is an important target for fluoroquinolones in S. pneumoniae and establish this organism as a useful gram-positive system for resistance studies.
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( 1996 ) ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype. PMID : 8891124 : PMC : PMC163513 Abstract >>
The genes encoding the ParC and ParE subunits of topoisomerase IV of Streptococcus pneumoniae, together with the region encoding amino acids 46 to 172 (residue numbers are as in Escherichia coli) of the pneumococcal GyrA subunit, were partially characterized. The gyrA gene maps to a physical location distant from the gyrB and parC loci on the chromosome, whereas parC is closely linked to parE. Ciprofloxacin-resistant (Cpr) clinical isolates of S. pneumoniae had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level Cpr) or in both quinolone resistance-determining regions of ParC and GyrA (high-level Cpr). Mutations were found in residue positions equivalent to the serine at position 83 and the aspartic acid at position 87 of the E. coli GyrA subunit. Transformation experiments suggest that ParC is the primary target of ciprofloxacin. Mutation in parC appears to be a prerequisite before mutations in gyrA can influence resistance levels.
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( 1996 ) Regulation of competence for genetic transformation in Streptococcus pneumoniae by an auto-induced peptide pheromone and a two-component regulatory system. PMID : 8878046 : DOI : 10.1046/j.1365-2958.1996.501417.x Abstract >>
The regulation of competence for genetic transformation in Streptococcus pneumoniae depends on a quorum-sensing system, but the only molecular elements of the system whose specific role have been identified are an extracellular peptide signal and an ABC-transporter required for its export. Here we show that transcription of comC, the gene encoding a predicted 41-residue precursor peptide that is thought to be processed and secreted as the 17-residue mature competence activator, increased approximately 40-fold above its basal level of expression in response to exogenous synthetic activator, consistent with earlier experiments indicating that the activator acts autocatalytically. We also describe two new genes, comD and comE, that encode members of histidine protein kinase and response-regulator families and are linked to comC. Disruption of comE abolished both response to synthetic activator peptide and endogenous competence induction.
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( 1996 ) Monoclonal antibodies that recognize a common pneumococcal protein with similarities to streptococcal group A surface glyceraldehyde-3-phosphate dehydrogenase. PMID : 8751897 : PMC : PMC174261 Abstract >>
Monoclonal antibodies (MAbs) against clinical isolates of Streptococcus pneumoniae were produced in a search for common pneumococcal proteins. One of the fusions generated two MAbs, 174,B-8 (immunoglobulin G2a) and 177,D-8 (immunoglobulin G1), which by Western blotting (immunoblotting) stained with a main band of 40 kDa found in all isolates of S. pneumoniae examined. Cross-reactivity studies with streptococci other than pneumococci revealed very weak or moderate reactions with the MAbs. The 40-kDa protein was isolated by immunoaffinity chromatography and subsequent preparative Western blotting. N-terminal amino acid sequencing showed 90% amino acid sequence homology with a surface-located glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes. This protein has also been reported to exhibit binding to mammalian proteins such as fibronectin, which may serve as host receptors. The epitopes for MAbs 174,B-8 and 177,D-8 reacting with the pneumococcal analog were not accessible to antibody binding in live bacteria but were exposed after heat killing. The MAbs showed negligible cross-reactions with S. pyogenes.
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( 1996 ) The capsule polysaccharide synthesis locus of streptococcus pneumoniae serotype 14: Identification of the glycosyl transferase gene cps14E. PMID : 8682774 : DOI : 10.1128/jb.178.13.3736-3741.1996 PMC : PMC232630 Abstract >>
To identify a chromosomal region of Streptococcus pneumoniae serotype 14 involved in capsule polysaccharide synthesis, two strategies were used: (i) Tn916 mutagenesis, followed by the characterization of four unencapsulated mutants, and (ii) cross-hybridization with a capsule polysaccharide synthesis gene (cps) probe from S. agalactiae, which has a structurally similar capsule. The two approaches detected the same chromosomal region consisting of two adjacent EcoRI fragments. One of these EcoRI fragments was cloned and hybridized with a cosmid library. This resulted in clone cMKO2. A similar cosmid clone was obtained from an unencapsulated Tn916 mutant, Spnl4.H. Sequence analysis of the two cosmid clones revealed that in the Tn916 mutant, a gene, cps14E, which is homologous to other bacterial genes encoding glycosyl transferases, had been inactivated. An open reading frame immediately downstream of cps14E, designated cps14F, shows no significant homology with any known genes or proteins. A functional assay showed that cps14E encodes a glycosyl transferase and that a gene-specific knockout mutant lacks this enzyme activity, whereas inactivation of cps14F does not have this effect.
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( 1996 ) Directly repeated insertion of 9-nucleotide sequence detected in penicillin-binding protein 2B gene of penicillin-resistant Streptococcus pneumoniae. PMID : 8723477 : PMC : PMC163302 Abstract >>
We investigated the molecular mechanism of 50 penicillin-resistant Streptococcus pneumoniae strains (penicillin: MIC, > or = 0.125 microgram/ml) having neither class A nor class B mutations in the penicillin-binding protein 2B gene (pbp2b). An analysis of the nucleotide sequences of the pbp2b genes from seven strains revealed an unique direct repeat of 9 nucleotides (TGGTATACT) between active-site serine (residue 385) and Ser-X-Asn (residues 442 to 444) motifs. The same insertion was detected in 13 strains.
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( 1995 ) Streptococcus pneumoniae type 3 encodes a protein highly similar to the human glutamate decarboxylase (GAD65). PMID : 8566695 : DOI : 10.1016/0378-1097(95)00346-7 Abstract >>
A 2.5-kb ScaI fragment of the type 3 pneumococcal strain 406 DNA containing a 1425-nucleotide open reading frame (gadA) and encoding a 475-amino acid protein (M(r) 54,427) was characterised. The gene gadA was expressed in Salmonella typhimurium. Pulsed-field gel electrophoresis and Southern blotting analysis of DNAs prepared from several pneumococcal serotypes showed that only those clinical isolates belonging to serotype 3 harbour the gadA gene. Sequence comparison of GadA with proteins included in the data banks revealed the highest similarity with human glutamate decarboxylase (GAD65) (59% similarity, 28% identity). Auto-antibodies to GAD65 have been associated with the onset of insulin-dependent diabetes mellitus. Interestingly, several epitopes of GAD65 that have been identified as immunodominant are particularly well conserved in the pneumococcal GadA.
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( 1996 ) Characterization of conjugative transposon Tn5251 of Streptococcus pneumoniae. PMID : 8595862 : DOI : 10.1111/j.1574-6968.1996.tb07994.x Abstract >>
Tn5251 belongs to the Tn916-Tn1545 family of conjugative transposons (CT) and was found integrated into CT Tn5252, to form the composite element Tn5253 of Streptococcus pneumoniae. We show that Tn5251 is identical in structure and size to Tn916. DNA sequence analysis of a 4,419-bp segment containing the tet(M) gene showed that only 73 nucleotides out of 4,419 were different in the two CT. Essentially all differences (66/73) were clustered in a 688-bp segment of tet(M), which was 90% identical to Tn916 and 100% identical to the tet(M) genes of Tn1545 from S. pneumoniae and pOZ101 from Neisseria gonorrhoeae. DNA sequence analysis of the Tn5251/Tn5252 junction fragments allowed us (i) to determine Tn5251 termini, (ii) to define the 6-bp coupling sequences flanking the CT, and (iii) to infer the structure of the integration site (attB) of Tn5251 into Tn5252. Conjugal transfer of Tn5251 independent from Tn5253 could not be detected, even if we could show excision and formation of Tn5251 circular intermediates at a level of 5.4 copies per 10(6) chromosomes.
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( 1993 ) Horizontal spread of an altered penicillin-binding protein 2B gene between Streptococcus pneumoniae and Streptococcus oralis. PMID : 8354467 : DOI : 10.1111/j.1574-6968.1993.tb06345.x Abstract >>
The region encoding the transpeptidase domain of the penicillin-binding protein 2B (PBP 2B) gene of two penicillin-resistant clinical isolates of Streptococcus oralis was > 99.6% identical in nucleotide sequence to that of a penicillin-resistant serotype 6 isolate of Streptococcus pneumoniae. The downstream 849 base pairs of these genes were identical. Analysis of the data indicates that the PBP gene has probably been transferred from S. pneumoniae into S. oralis, rather than vice versa, and shows that one region of this resistance gene has been distributed horizontally both within S. pneumoniae and into two different viridans group streptococci.
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357. |
( 1994 ) Mosaic pbpX genes of major clones of penicillin-resistant Streptococcus pneumoniae have evolved from pbpX genes of a penicillin-sensitive Streptococcus oralis. PMID : 7934893 : DOI : 10.1111/j.1365-2958.1994.tb01089.x Abstract >>
Penicillin-resistant clinical isolates of Streptococcus pneumoniae contain mosaic penicillin-binding protein (PBP) genes that encode PBPs with decreased affinity for beta-lactam antibiotics. The mosaic blocks are believed to be the result of gene transfer of homologous PBP genes from related penicillin-resistant species. We have now identified a gene homologous to the pneumococcal PBP2x gene (pbpX) in a penicillin-sensitive Streptococcus oralis isolate M3 from South Africa that diverged by almost 20% from pbpX of penicillin-sensitive pneumococci, and a central sequence block of a mosaic pbpX gene of Streptococcus mitis strain NCTC 10712. In contrast, it differed by only 2-4% of the 1 to 1.5 kb mosaic block in pbpX genes of three genetically unrelated penicillin-resistant S. pneumoniae isolates, two of them representing clones of serotype 6B and 23F, which are prevalent in Spain and are also already found in other countries. With low concentrations of cefotaxime, transformants of the sensitive S. pneumoniae R6 strain could be selected containing pbpX genes from either S. mitis NCTC 10712 or S. oralis M3, demonstrating that genetic exchange can already occur between beta-lactam-sensitive species. These data are in agreement with the assumption that PBPs as penicillin-resistance determinants have evolved by the accumulation of point mutations in genes of sensitive commensal species.
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358. |
( 1994 ) A neuraminidase from Streptococcus pneumoniae has the features of a surface protein. PMID : 8063384 : PMC : PMC303019 Abstract >>
A gene from Streptococcus pneumoniae (nanA), with features entirely consistent with a neuraminidase gene, has been sequenced. High levels of neuraminidase activity were obtained after cloning of this gene, without flanking sequences, into a high-expression vector. RNA hybridization studies have shown that the gene is transcribed by a virulent pneumococcus strain. The predicted molecular weight of the protein and certain amino acid sequences are typical of other neuraminidases. NanA contains the four copies of the sequence SXDXGXTW that is present in all the bacterial neuraminidases previously described. Kyte and Doolittle analysis showed that NanA is a hydrophilic protein with hydrophobic domains at the N terminus and the C terminus. A putative signal peptide was found in the N terminus of this protein, indicating that the protein is exported from the pneumococcus. The C terminus has the features of the anchor motif found in other surface proteins from gram-positive bacteria. Electron microscopy studies showed the presence of neuraminidase associated with the cell surface of the pneumococcus.
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359. |
( 1994 ) Isolation, characterization, and nucleotide sequence of IS1202, an insertion sequence of Streptococcus pneumoniae. PMID : 8021229 : DOI : 10.1128/jb.176.14.4437-4443.1994 PMC : PMC205658 Abstract >>
A comparative hybridization protocol was used to isolate a small segment of DNA present in the Streptococcus pneumoniae type 19F strain SSZ but absent from strain Rx1, a nonencapsulated derivative of the type 2 strain D39. This segment of DNA is a 1,747-bp insertion sequence, designated IS1202, flanked by 23-bp imperfect inverted repeats and containing a single open reading frame sufficient to encode a 54.4-kDa polypeptide. A 27-bp target sequence is duplicated at either end of the element. IS1202 is not related to any of the currently known insertion elements and is the first reported for S. pneumoniae. Although found predominantly in type 19F strains in up to five copies, it has also been shown to be present in the chromosomes of pneumococci belonging to other serotypes. One of the four IS1202 copies in the encapsulated strain SSZ is located 1,009 bp downstream of the dexB gene, and transformation studies reveal that it is also closely linked to the type 19F capsular polysaccharide synthesis (cps) locus.
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360. |
( 1993 ) Genetic identification of exported proteins in Streptococcus pneumoniae. PMID : 7934910 : DOI : 10.1111/j.1365-2958.1993.tb01233.x Abstract >>
A strategy was developed to mutate and genetically identify exported proteins in Streptococcus pneumoniae. Vectors were created and used to screen pneumococcal DNA in Escherichia coli and S. pneumoniae for translational gene fusions to alkaline phosphatase (PhoA). Twenty five PhoA+ pneumococcal mutants were isolated and the loci from eight of these mutants showed similarity to known exported or membrane-associated proteins. Homologues were found to: (i) protein-dependent peptide permeases, (ii) penicillin-binding proteins, (iii) Clp proteases, (iv) two-component sensor regulators, (v) the phosphoenolpyruvate: carbohydrate phosphotransferases permeases, (vi) membrane-associated dehydrogenases, (vii) P-type (E1E2-type) cation transport ATPases, (viii) ABC transporters responsible for the translocation of the RTX class of bacterial toxins. Unexpectedly one PhoA+ mutant contained a fusion to a member of the DEAD protein family of ATP-dependent RNA helicases suggesting export of these proteins.
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361. |
( 1994 ) Identification and nucleotide sequence analysis of a transfer-related region in the streptococcal conjugative transposon Tn5252. PMID : 8051031 : DOI : 10.1128/jb.176.16.5145-5150.1994 PMC : PMC196358 Abstract >>
To obtain a functional map of Tn5252, a 47.5-kb streptococcal conjugative transposon, a series of defined deletion and insertion mutations were introduced within the transposon. Interruptions at several regions were found to affect the conjugal transposition functions of the element in filter-mating experiments. The nucleotide sequence of the left terminus of Tn5252 showed two open reading frames, ORF1 and ORF2, adjoining the att site. The organization of this region and the structure of the predicted integrase encoded by ORF1 were found to be similar to those of other site-specific recombination systems.
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362. |
( 1993 ) Cloning and sequencing of a gene involved in the synthesis of the capsular polysaccharide of Streptococcus pneumoniae type 3. PMID : 8510646 : DOI : 10.1007/bf00281617 Abstract >>
A 4.5 kb ScaI chromosomal DNA fragment of a clinical isolate of Streptococcus pneumoniae serotype 3 was cloned in Escherichia coli. Combined genetic and molecular analyses have allowed the localization, in a 781 bp EcoRV subfragment, of a gene (cap3-1) directly responsible for the transformation of an unencapsulated, serotype 3 mutant to the capsulated phenotype. Comparison of the deduced amino acid sequence of CAP3-1 with the protein sequences compiled in the data banks revealed that the CAP3-1 polypeptide was highly similar to the amino-terminus of the GDP-mannose dehydrogenase of Pseudomonas aeruginosa, an enzyme that participates in the synthesis of the mucoid polysaccharide of this species. In addition, the 32 N-terminal amino acids of CAP3-1 perfectly matched structures common to NAD(+)-binding domains of many dehydrogenases. Our results indicate that the 4.5 kb ScaI fragment might also contain genes common to 13 different pneumococcal serogroups or serotypes tested. To the best of our knowledge, this is the first time that a gene of the capsular complex of S. pneumoniae has been cloned and sequenced. The findings reported here provide new insights for the study of the molecular biology of the main virulence factor responsible for the pathogenesis of pneumococcal infections and might represent a basic step in the identification of cross-reactive antigens that should allow the preparation of new and improved vaccines.
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363. |
( 1995 ) Sequence and transcriptional analysis of a DNA region involved in the production of capsular polysaccharide in Streptococcus pneumoniae type 3. PMID : 8566758 : DOI : 10.1016/0378-1119(95)00657-5 Abstract >>
The nucleotide (nt) sequence of a 9704-bp EcoRI fragment of Streptococcus pneumoniae (Sp) type-3 DNA has been determined and found to contain one partial and five complete open reading frames (ORFs). One of these ORFs corresponds to the cap3 A gene coding for the UDP-glucose (UDPGlc) dehydrogenase which is directly responsible for the transformation of some unencapsulated serotype-3 Sp mutants to the encapsulated phenotype [Arrecubieta et al., J. Bacteriol. 176 (1994) 6375-6383]. The two ORFs downstream from this gene (cap3B and cap3C) encode proteins with molecular masses of 49 and 34 kDa. Analysis of the deduced amino acid (aa) sequences of Cap3B and Cap3C shows homology to polysaccharide synthases and UDPG1c pyrophosphorylases, respectively. Furthermore, genetic complementation analysis showed that cap3C restored the galU defect of an Escherichia coli mutant. Northern blots have shown that cap3A, cap3B and cap3C constitute a single transcriptional unit, and primer extension analysis has revealed that the transcription start point is preceded by a nt sequence identical to the sigma 70 consensus promoter sequence of E. coli. The sequence upstream from this cluster also has a high degree of similarity with genes postulated to be essential for capsular production in several Gram+ bacteria. However, Northern blot analysis and insertion-duplication mutagenesis indicated that genes located in this region are not necessary for type-3 capsule production in the Sp strain 406.
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364. |
( 1994 ) A two-component signal-transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae. PMID : 8065267 : DOI : 10.1111/j.1365-2958.1994.tb01038.x Abstract >>
Penicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin-binding protein (PBP) variants with decreased affinities for beta-lactam antibiotics. Cefotaxime-resistant laboratory mutants, selected after several steps on increasing concentrations of this beta-lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, ciaR and ciaH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal-transducing proteins). Two hydrophobic peptide regions divided the histidine kinase CiaH into two putative domains: an N-terminal extracellular sensor part, and an intracellular C-terminal domain with the conserved His-226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime-resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime-resistant mutants were each located in the C-terminal half of CiaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated ciaH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal-transducing system cia is involved in early steps of competence regulation.
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365. |
Lacks SA,
Greenberg B,
Lopez P,
( 1995 ) A cluster of four genes encoding enzymes for five steps in the folate biosynthetic pathway of Streptococcus pneumoniae. PMID : 7798151 : DOI : 10.1128/jb.177.1.66-74.1995 PMC : PMC176557 Abstract >>
Two genes, sulB and sulC, in a folate biosynthetic gene cluster of Streptococcus pneumoniae were identified after determination of the DNA sequence between two previously reported genes, sulA and sulD, in a cloned segment of chromosomal DNA containing a mutation to sulfonamide resistance. The gene products, SulB and SulC, correspond to polypeptides of 49 and 21 kDa, respectively. SulC has GTP cyclohydrolase activity and catalyzes the first step in the folate biosynthetic pathway. SulB apparently has dihydrofolate synthetase activity in that it complements a folC mutant of Escherichia coli and thus catalyzes the last step in the pathway. Prior work showed that SulA, a dihydropteroate synthase, and SulD, a bifunctional enzyme, catalyze three intervening steps. Mapping of the mRNA transcribed from the operon was consistent with its beginning at a promoter with a -35 site (gTGtCc) and an extended -10 site (T-TG-TAaAAT) and its termination at the end of a hairpin structure, which would give a transcript 3,745 nucleotides in length. SulC showed a considerable conservation of sequence by comparison with proven or putative GTP cyclohydrolases from four unrelated species, with 38 to 53% of the residues being identical. A similar comparison of SulB with dihydrofolate synthetases showed an identity of only 26 to 37%. Overall, comparisons of the five folate biosynthetic enzymes in different species suggest that S. pneumoniae is related more closely to other gram-positive bacteria, less closely to eucaryotes, and least closely to the gram-negative E. coli. The varied arrangements of folate biosynthetic genes in different species imply an early evolutionary period of fluidity in genomic rearrangement.
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366. |
Smith AM,
Klugman KP,
( 1995 ) Alterations in penicillin-binding protein 2B from penicillin-resistant wild-type strains of Streptococcus pneumoniae. PMID : 7785985 : DOI : 10.1128/aac.39.4.859 PMC : PMC162643 Abstract >>
The 1.5-kb transpeptidase-encoding region (TER) of penicillin-binding protein (PBP) 2B was amplified and sequenced from 18 penicillin-resistant isolates of Streptococcus pneumoniae, with each isolate representing a different DNA fingerprint profile of the TER. PBP 2B TERs from penicillin-resistant isolates revealed extensive sequence divergence from the penicillin-susceptible R6 strain, differing by up to 170 nucleotide substitutions and resulting in up to 38 alterations in the amino acid sequence of the protein. All penicillin-resistant isolates showed sequence divergence within a +/- 300-bp area at the center of the PBP 2B TER. Although a number of amino acid substitutions were found within this central area of PBP 2B, only two substitutions were common to all resistant isolates, namely, Thr-252 replacement by Ala and Glu-282 replacement by Gly. These two substitutions appear to be essentially associated with a decreased affinity of PBP 2B for penicillin. A second block of divergent nucleotide sequence was prominent amongst isolates with high levels of resistance. This was a +/- 100-bp area of the TER around nucleotide 1300 and included the substitution of Gly for Asp-431, which was the only amino acid substitution within this area that was common to all isolates. These data may assist in the definition of the structural changes in the penicillin-binding site of PBP 2B associated with penicillin resistance in S. pneumoniae.
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367. |
( 1994 ) Molecular characterization of cap3A, a gene from the operon required for the synthesis of the capsule of Streptococcus pneumoniae type 3: sequencing of mutations responsible for the unencapsulated phenotype and localization of the capsular cluster on the pneumococcal chromosome. PMID : 7929009 : DOI : 10.1128/jb.176.20.6375-6383.1994 PMC : PMC196979 Abstract >>
The complete nucleotide sequence of the cap3A gene of Streptococcus pneumoniae, which is directly responsible for the transformation of some unencapsulated, serotype 3 mutants to the encapsulated phenotype, has been determined. This gene encodes a protein of 394 amino acids with a predicted M(r) of 44,646. Twelve independent cap3A mutations have been mapped by genetic transformation, and three of them have been sequenced. Sequence comparisons revealed that cap3A was very similar (74.4%) to the hasB gene of Streptococcus pyogenes, which encodes a UDP-glucose dehydrogenase (UDP-GlcDH) that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, the donor substances in the pneumococcal type 3 capsular polysaccharide. Furthermore, a PCR-generated cap3A+ gene restored encapsulation in our cap3A mutants as well as in a mutant previously characterized as deficient in UDP-GlcDH (R. Austrian, H. P. Bernheimer, E.E.B. Smith, and G.T. Mills, J. Exp. Med. 110:585-602, 1959). These results support the conclusion that cap3A codes for UDP-GlcDH. We have also identified a region upstream of cap3A that should contain common genes necessary for the production of capsule of any type. Pulsed-field gel electrophoresis and Southern blotting showed that the capsular genes specific for serotype 3 are located near the genes encoding PBP 2X and PBP 1A in the S. pneumoniae chromosome, whereas copies of the common genes (or part of them) appear to be present in different locations in the genome.
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368. |
( 1994 ) Molecular basis of the optochin-sensitive phenotype of pneumococcus: characterization of the genes encoding the F0 complex of the Streptococcus pneumoniae and Streptococcus oralis H(+)-ATPases. PMID : 7934882 : DOI : 10.1111/j.1365-2958.1994.tb01045.x Abstract >>
The gene responsible for the optochin-sensitive (OptS) phenotype of Streptococcus pneumoniae has been characterized. Sequence comparisons indicated that the genes involved encoded the subunits of the F0 complex of an H(+)-ATPase. Sequence analysis and transformation experiments showed that the atpC gene is responsible for the optochin-sensitive resistant (OptS/OptR) phenotype. Our results also show that natural as well as laboratory OptR isolates have arisen by point mutations that produce different amino acid changes at positions 48, 49 or 50 of the ATPase c subunit. The nucleotide sequence of the F0 complex of the Streptococcus oralis ATPase has also been determined. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (OptS) and M222 (an OptR strain produced by interspecies recombination between pneumococcus and S. oralis), and S. oralis revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin specifically inhibited the membrane-bound ATPase activity of the S. pneumoniae wild-type (OptS) strains, and found a 100-fold difference between OptS and OptR strains, both in growth inhibition and in membrane ATPase resistance.
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369. |
( 1994 ) Nucleotide sequence analysis of genes essential for capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 19F. PMID : 7960118 : PMC : PMC303279 Abstract >>
Previous studies have shown that the capsular polysaccharide synthesis (cps) locus of the type 19F Streptococcus pneumoniae strain SSZ was closely linked to a copy of the insertion sequence IS1202 (J.K. Morona, A. Guidolin, R. Morona, D. Hansman, and J.C. Paton, J. Bacteriol. 176:4437-4443, 1994). In the present study, we used plasmid insertion and rescue and inverse PCR to clone 6,322 bp of flanking DNA upstream of IS1202. Sequence analysis indicated that this region contains six complete open reading frames (ORFs) and one partial ORF that are arranged as a single transcriptional unit. Chromosomal disruption of any of these ORFs in a smooth-type 19F strain leads to a rough (unencapsulated) phenotype, indicating that this operon is essential for capsule production. The ORFs have therefore been designated cps19fA to cps19fG, where cps19fA is the first gene of the type 19F cps locus. Furthermore, many of the gene products from this incomplete operon exhibit strong similarities to proteins known to be involved in the production of capsular polysaccharide, exopolysaccharide, teichoic acid, enterobacterial common antigen, and lipopolysaccharide from numerous other bacterial species. This has allowed us to propose functions for many of the type 19F cps gene products. Southern hybridization studies reveal that cps19fA and cps19fB are conserved among all 12 pneumococcal serotypes tested, whereas genes downstream of cps19fB are conserved among some, but not all, of the serotypes tested.
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370. |
Muñoz R,
Bustamante M,
de la Campa AG,
( 1995 ) Ser-127-to-Leu substitution in the DNA gyrase B subunit of Streptococcus pneumoniae is implicated in novobiocin resistance. PMID : 7608096 : DOI : 10.1128/jb.177.14.4166-4170.1995 PMC : PMC177155 Abstract >>
We report the cloning of the gyrB gene from Streptococcus pneumoniae 533 that carries the nov-1 allele. The gyrB gene codes for a protein homologous to the gyrase B subunit of archaebacteria and eubacteria. The same amino acid substitution (Ser-127 to Leu) confers novobiocin resistance on four isolates of S. pneumoniae. This amino acid position is equivalent to Val-120 of Escherichia coli GyrB, a residue that lies inside the ATP-binding domain as revealed by the crystal structure of the protein.
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371. |
Dillard JP,
Vandersea MW,
Yother J,
( 1995 ) Characterization of the cassette containing genes for type 3 capsular polysaccharide biosynthesis in Streptococcus pneumoniae. PMID : 7869055 : DOI : 10.1084/jem.181.3.973 PMC : PMC2191931 Abstract >>
The capsular polysaccharide is the major virulence factor of Streptococcus pneumoniae. Previously, we identified and cloned a region from the S. pneumoniae chromosome specific for the production of type 3 capsular polysaccharide. Now, by sequencing the region and characterizing mutations genetically and in an in vitro capsule synthesis assay, we have assigned putative functions to the products of the type-specific genes. Using DNA from the right end of the region in mapping studies, we have obtained further evidence indicating that the capsule genes of each serotype are contained in a gene cassette located adjacent to this region. We have cloned the region flanking the left end of the cassette from the type 3 chromosome and have found that it is repeated in the S. pneumoniae chromosome. The DNA sequence and hybridization data suggest a model for recombination of the capsule gene cassettes that not only describes the replacement of capsule genes, but also suggests an explanation for binary capsule type formation, and the creation of novel capsule types.
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372. |
Coffey TJ,
Daniels M,
McDougal LK,
Dowson CG,
Tenover FC,
Spratt BG,
( 1995 ) Genetic analysis of clinical isolates of Streptococcus pneumoniae with high-level resistance to expanded-spectrum cephalosporins. PMID : 7574521 : DOI : 10.1128/aac.39.6.1306 PMC : PMC162732 Abstract >>
Streptococcus pneumoniae CS109 and CS111 were isolated in the United States in 1991 and have high levels of resistance to expanded-spectrum cephalosporins (MICs of 8 and 32 micrograms of cefotaxime per ml, respectively). CS109, but not CS111, also showed high-level resistance to penicillin. As both strains expressed the serotype 23F capsule, were very closely related in overall genotype, and possessed identical or closely related mosaic pbp1a, pbp2x, and pbp2b genes, it is likely that they have arisen from a recent common ancestor. High-level resistance to expanded-spectrum cephalosporins was entirely due to alterations of penicillin-binding proteins (PBPs) 1a and 2x, since a mixture of the cloned pbp1a and pbp2x genes from the resistant strains could transform the susceptible strain R6 to the full level of cephalosporin resistance of the clinical isolates. Both PBP1a and PBP2x of these strains were more resistant to inhibition by cephalosporins than those of typical highly penicillin-resistant isolates. The pbp1a genes of CS109 and CS111 were identical in sequence, and the fourfold difference in their levels of resistance to cephalosporins was due to a Thr-550-->Ala substitution at the residue following the conserved Lys-Ser-Gly motif of PBP2x. This substitution was also the major cause of the 16-fold-lower resistance of CS111 to penicillin. The pbp2x gene of CS111, in an appropriate genetic background, could provide resistance to 16 micrograms of cefotaxime per ml but only to 0.12 microgram of benzylpenicillin per ml. Removal of the codon 550 mutation resulted in a pbp2x gene that provided resistance to 4 microgram of cefotaxime per ml and 4 microgram of benzylpenicillin per ml. The Thr-550-->Ala substitution in CS111 therefore appears to provide increased resistance to expanded-spectrum cephalosporins but a loss of resistance to penicillin.
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373. |
Sampson JS,
O'Connor SP,
Stinson AR,
Tharpe JA,
Russell H,
( 1994 ) Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins. PMID : 7505262 : PMC : PMC186105 Abstract >>
Gene psaA, which encodes the Streptococcus pneumoniae 37-kDa protein, was cloned in Escherichia coli, and its complete nucleotide sequence was determined. Analysis of the sequence of the 2.4-kb cloned fragment revealed three open reading frames (ORFs). ORF2, which is 933 bp long, was identified as psaA. The two other ORFs identified flank psaA. ORF1, located upstream of psaA, is 836 nucleotides long and encodes a protein with a calculated molecular mass of 29,843 Da. The sequence for ORF3, located downstream of psaA, was only partially determined. Northern (RNA) blot analysis of pneumococcal RNA suggests that psaA is transcribed as part of a polycistronic message. Analysis of the primary structure of the protein encoded by this gene indicated significant similarity to two previously reported streptococcal proteins, SsaB (80% similarity) and FimA (92.3% similarity), from S. sanguis and S. parasanguis, respectively. These two homologous proteins have been shown to be associated with bacterial adhesion, and the possibility of a similar role for PsaA is hypothesized.
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374. |
Håvarstein LS,
Coomaraswamy G,
Morrison DA,
( 1995 ) An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. PMID : 7479953 : DOI : 10.1073/pnas.92.24.11140 PMC : PMC40587 Abstract >>
Competence for genetic transformation in Streptococcus pneumoniae has been known for three decades to arise in growing cultures at a critical cell density, in response to a secreted protease-sensitive signal. We show that strain CP1200 produces a 17-residue peptide that induces cells of the species to develop competence. The sequence of the peptide was found to be H-Glu-Met-Arg-Leu-Ser-Lys-Phe-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg- Lys-Lys-OH. A synthetic peptide of the same sequence was shown to be biologically active in small quantities and to extend the range of conditions suitable for development of competence. Cognate codons in the pneumococcal chromosome indicate that the peptide is made ribosomally. As the gene encodes a prepeptide containing the Gly-Gly consensus processing site found in peptide bacteriocins, the peptide is likely to be exported by a specialized ATP-binding cassette transport protein as is characteristic of these bacteriocins. The hypothesis is presented that this transport protein is encoded by comA, previously shown to be required for elaboration of the pneumococcal competence activator.
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375. |
Saluja SK,
Weiser JN,
( 1995 ) The genetic basis of colony opacity in Streptococcus pneumoniae: evidence for the effect of box elements on the frequency of phenotypic variation. PMID : 7565084 : DOI : 10.1111/j.1365-2958.1995.tb02294.x Abstract >>
Streptococcus pneumoniae undergoes spontaneous phase variation in colony morphology. Differences in colony opacity have previously been shown to correlate with differences in the ability of organisms to colonize the mucosal surface of the nasopharynx in an animal model. The genetic basis of opacity variation was identified in transformation experiments. A DNA library, from a strain that varies at high frequency, was screened to identify a single clone capable of transforming a transparent recipient strain which varies at low frequency to an opaque phenotype. Analysis of this opacity locus revealed two genes, glpD and glpF, with similarity to genes required for glycerol metabolism in other bacteria. Following the pneumococcal glpF, repetitive intergenic elements, boxes A and C, were identified. These stem-loop-forming elements were not present in the same locus of the recipient strain. Although not required for phase variation in colony opacity, the box element was necessary for expression of phase variation at high frequency. Introduction of the box elements during transformation affected colony morphology, possibly by altering expression of a putative regulatory gene downstream from the box element. Mutagenesis within this region confirmed the contribution of the putative regulatory gene to the expression of colony opacity. Growth characteristics of strains generated in this study provide additional evidence for an association of differences in cell wall autolysis and variation in colony opacity.
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376. |
Zhou L,
Hui FM,
Morrison DA,
( 1995 ) Characterization of IS1167, a new insertion sequence in Streptococcus pneumoniae. PMID : 7597107 : DOI : 10.1006/plas.1995.1014 Abstract >>
A new insertion sequence in Streptococcus pneumoniae was identified as a 1435-bp segment of the genome containing 24-bp terminal inverted repeats and flanked by 8-bp direct repeats. A copy of the element, named IS1167, adjacent to the comAB genes was sequenced; seven additional copies were found in the genome of strain CP1200 and relatives descended from strain R36A. Among 22 independent pneumococcal isolates, 11 contained copies of elements hybridizing to IS1167 in nine distinct restriction fragment patterns, with 3-12 copies each. The bulk of the element was occupied by two overlapping open reading frames, encoding basic proteins which together exhibited strong similarity to the full length of the putative transposase of the staphylococcal transposable element, IS1181, as well as significant similarity to those of seven additional known or putative insertion sequences related to the mycobacterial element, IS1096.
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377. |
Sabelnikov AG,
Greenberg B,
Lacks SA,
( 1995 ) An extended -10 promoter alone directs transcription of the DpnII operon of Streptococcus pneumoniae. PMID : 7541838 : DOI : 10.1006/jmbi.1995.0366 Abstract >>
The genetic cassette encoding the DpnII restriction-modification system of Streptococcus pneumoniae gave transcription products of approximately 2.7 and 1.8 kilobases. The larger, mRNA1, covered both of the methylase genes, dpnM and dpnA, and the endonuclease gene dpnB; the smaller, mRNA2, covered only the dpnA and dpnB genes. Transcription of mRNA1 was shown to begin at the translation start site for dpnM, thereby producing an mRNA without any apparent ribosome-binding site for translation of the DpnM methylase. The promoter for mRNA1 was shown by base substitution and deletion analysis to consist of an extended -10 site, TaTGgTATAAT, with no required -35 site. A possible promoter further upstream with close matches to a -35 site and a nonextended -10 site was not used. A survey of 36 proven and putative promoters used by S. pneumoniae revealed that 61% of them contained the full -10 extension, although, other than the dpnM promoter, they matched at a -35 site, as well. It appears that, unlike those found in Escherichia coli, S. pneumoniae promoters frequently require an extended -10 site, and such a site can function naturally without a -35 site.
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378. |
Martin B,
García P,
Castanié MP,
Claverys JP,
( 1995 ) The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction. PMID : 7538190 : DOI : 10.1111/j.1365-2958.1995.tb02250.x Abstract >>
The recently identified recA gene of the naturally transformable bacterium Streptococcus pneumoniae has been further characterized by constructing a recA null mutation and by investigating its regulation. The recA mutation has been shown to confer both DNA repair (as judged from sensitivity to u.v. and methyl methane sulphonate) and recombination deficiencies. Plasmid transformation into the recA mutant was also drastically reduced. Western blotting established that recA gene expression is increased several fold at the onset of competence for genetic transformation. Increased expression was associated with the appearance of a recA-specific transcript, approximately 5.7 kb long. This transcript indicated that recA is part of a competence-inducible (cin) operon. The major (about 4.3 kb) transcript detected from non-competent cells did not include cinA, the first gene in the operon, suggesting that this gene could be specifically required at some stage in the transformation process. Detection of small amounts of the 5.7 kb polycistronic mRNA in cells treated with mitomycin C suggested that the operon could also be damage inducible. In addition, mitomycin C treatment of a recA- lysogenic strain did not lead to prophage induction and cell lysis. This is unlike the situation of a recA+ lysogen. Together these results demonstrate that RecA controls lysogenic induction and suggest the existence of a SOS repair system in S. pneumoniae.
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379. |
Watson DA,
Kapur V,
Musher DM,
Jacobson JW,
Musser JM,
( 1995 ) Identification, cloning, and sequencing of DNA essential for encapsulation of Streptococcus pneumoniae. PMID : 7549771 : Abstract >>
This paper reports the cloning and sequencing of a region of DNA from Streptococcus pneumoniae serotype 3 surrounding transposon Tn916, insertion of which was previously shown to result in lack of expression of the extracellular capsule. Sequence analysis revealed that the transposon inserted into a consensus insertion site 71 bp from the 5' end of the cloned fragment. Within the clone, 3' downstream regions from two different pneumococcal lytA genes were identified, as well as a putative 194 AA open reading frame (ORF1). Moreover, two copies of the repeat element BOX, oriented in opposite directions, were located immediately 3' of orf1. Within the region bounded by the first pair of internal sequencing primers, analysis revealed that the fragment amplified by PCR was always of the same size. Moreover, Southern blotting showed that for all serotypes examined to date, homology exists with the cloned fragment. These results indicate that this region of the chromosome is highly conserved and, taken together with other independently derived data, suggest that interruptions or deletions within this DNA lead to unencapsulation.
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380. |
Manna S,
Dunne EM,
Ortika BD,
Pell CL,
Kama M,
Russell FM,
Mungun T,
Mulholland EK,
Hinds J,
Satzke C,
( 2018 ) Discovery of a Streptococcus pneumoniae serotype 33F capsular polysaccharide locus that lacks wcjE and contains a wcyO pseudogene. PMID : 30395578 : DOI : 10.1371/journal.pone.0206622 PMC : PMC6218050 Abstract >>
As part of large on-going vaccine impact studies in Fiji and Mongolia, we identified 25/2750 (0.9%) of nasopharyngeal swabs by microarray that were positive for Streptococcus pneumoniae contained pneumococci with a divergent 33F capsular polysaccharide locus (designated '33F-1'). We investigated the 33F-1 capsular polysaccharide locus to better understand the genetic variation and its potential impact on serotyping results. Whole genome sequencing was conducted on ten 33F-1 pneumococcal isolates. Initially, sequence reads were used for molecular serotyping by PneumoCaT. Phenotypic typing of 33F-1 isolates was then performed using the Quellung reaction and latex agglutination. Genome assemblies were used in phylogenetic analyses of each gene in the capsular locus to investigate genetic divergence. All ten pneumococcal isolates with the 33F-1 cps locus typed as 33F by Quellung and latex agglutination. Unlike the reference 33F capsule locus sequence, DNA microarray and PneumoCaT analyses found that 33F-1 pneumococci lack the wcjE gene, and instead contain wcyO with a frameshift mutation. Phylogenetic analyses found the wzg, wzh, wzd, wze, wchA, wciG and glf genes in the 33F-1 cps locus had higher DNA sequence similarity to homologues from other serotypes than to the 33F reference sequence. We have discovered a novel genetic variant of serotype 33F, which lacks wcjE and contains a wcyO pseudogene. This finding adds to the understanding of molecular epidemiology of pneumococcal serotype diversity, which is poorly understood in low and middle-income countries.
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381. |
Priebe SD,
Hadi SM,
Greenberg B,
Lacks SA,
( 1988 ) Nucleotide sequence of the hexA gene for DNA mismatch repair in Streptococcus pneumoniae and homology of hexA to mutS of Escherichia coli and Salmonella typhimurium. PMID : 3275608 : DOI : 10.1128/jb.170.1.190-196.1988 PMC : PMC210625 Abstract >>
The Hex system of heteroduplex DNA base mismatch repair operates in Streptococcus pneumoniae after transformation and replication to correct donor and nascent DNA strands, respectively. A functionally similar system, called Mut, operates in Escherichia coli and Salmonella typhimurium. The nucleotide sequence of a 3.8-kilobase segment from the S. pneumoniae chromosome that includes the 2.7-kilobase hexA gene was determined. An open reading frame that could encode a 17-kilodalton polypeptide (OrfC) was located just upstream of the gene encoding a polypeptide of 95 kilodaltons corresponding to HexA. Shine-Dalgarno sequences and putative promoters were identified upstream of each protein start site. Insertion mutations showed that only HexA functioned in mismatch repair and that the promoter for hexA transcription was located within the OrfC-coding region. The HexA polypeptide contains a consensus sequence for ATP- or GTP-binding sites in proteins. Comparison of the entire HexA protein sequence to that of MutS of S. typhimurium, which was determined by Haber et al. in the accompanying paper (L. T. Haber, P. P. Pang, D. I. Sobell, J. A. Mankovitch, and G. C. Walker, J. Bacteriol. 170:197-202, 1988), showed the proteins to be homologous, inasmuch as 36% of their amino acid residues were identical. This homology indicates that the Hex and Mut systems of mismatch repair evolved from an ancestor common to the gram-positive streptococci and the gram-negative enterobacteria. It is the first direct evidence linking the two systems.
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382. |
Lopez P,
Espinosa M,
Greenberg B,
Lacks SA,
( 1987 ) Sulfonamide resistance in Streptococcus pneumoniae: DNA sequence of the gene encoding dihydropteroate synthase and characterization of the enzyme. PMID : 3114239 : DOI : 10.1128/jb.169.9.4320-4326.1987 PMC : PMC213747 Abstract >>
A chromosomal gene of Streptococcus pneumoniae carrying a spontaneous mutation to sulfonamide resistance was identified. Comparison of its DNA sequence with the wild-type sequence showed that the mutation, sul-d, consisted of an insert of 6 base pairs, a repeat of an adjacent 6-base-pair segment. The gene encoded a 34-kilodalton polypeptide, SulA, which as a dimer or trimer constituted the enzyme dihydropteroate synthase. This was shown by enzyme activity measurements, expression in minicells of Bacillus subtilis, and the amino-terminal sequence of the polypeptide product. Subcloning of the gene in an Escherichia coli expression vector allowed purification of the enzyme to 80% homogeneity in a single step and at high yield. Although a deleted plasmid, pLS83, produced the mutant dihydropteroate synthase, it did not confer sulfonamide resistance in vivo. It is suggested that the SulA polypeptide is also a component of an enzyme that acts in another step of folate biosynthesis and that this step is inhibited in vivo by either free or conjugated sulfonamides.
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383. |
Caillaud F,
Trieu-Cuot P,
Carlier C,
Courvalin P,
( 1987 ) Nucleotide sequence of the kanamycin resistance determinant of the pneumococcal transposon Tn1545: evolutionary relationships and transcriptional analysis of aphA-3 genes. PMID : 3039302 : DOI : 10.1007/bf00331623 Abstract >>
The nucleotide sequence of the kanamycin resistance determinant aphA-3 encoded by transposon Tn1545 from Streptococcus pneumoniae was determined and compared to those of plasmids pJH1 and pIP1433 from Streptococcus faecalis and Campylobacter coli, respectively. The three sequences were found to be identical and differed by two substitutions and the deletion of a codon from that of plasmid pSH2 from Staphylococcus aureus. Comparison of the 5' noncoding sequences indicated that the regions containing the aphA-3 gene in pJH1 and in Tn1545 evolved independently by deletion from a sequence similar to that found in pIP1433. In the latter plasmid, aphA-3 is transcribed from a promoter, P1, which is flanked by two 12-base pair direct repeats. The rearrangement observed in pJH1 removed one of these recombinogenic sites and altered the -10 and 3' flanking sequences of P1. The promoter thus generated. P1', allows expression of similar level of kanamycin resistance as P1. However, fusion experiments carried out with a promotorless chloramphenicol acetyltransferase gene indicated that the canonical promoter P1 is significantly less efficient than P1'. From analysis of the thermodynamic properties of these promoters, we conclude that this difference in strength reflects the melting properties of the -10 sequences. The transition from pIP1433 to pJH1 may correspond to the progression of a molecule structurally unstable to a more stable one combined with the need to maintain an efficient promoter upstream of the aphA-3 gene. The deletion event in Tn1545, which occurred between the two 12-base pair directly repeated sequences, removed P1 in its entirety.(ABSTRACT TRUNCATED AT 250 WORDS)
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384. |
Sikanyika M,
Aragão D,
McDevitt CA,
Maher MJ,
( 2019 ) The structure and activity of the glutathione reductase from Streptococcus pneumoniae. PMID : 30605126 : DOI : 10.1107/S2053230X18016527 Abstract >>
The glutathione reductase (GR) from Streptococcus pneumoniae is a flavoenzyme that catalyzes the reduction of oxidized glutathione (GSSG) to its reduced form (GSH) in the cytoplasm of this bacterium. The maintenance of an intracellular pool of GSH is critical for the detoxification of reactive oxygen and nitrogen species and for intracellular metal tolerance to ions such as zinc. Here, S. pneumoniae GR (SpGR) was overexpressed and purified and its crystal structure determined at 2.56 ? resolution. SpGR shows overall structural similarity to other characterized GRs, with a dimeric structure that includes an antiparallel �]-sheet at the dimer interface. This observation, in conjunction with comparisons with the interface structures of other GR enzymes, allows the classification of these enzymes into three classes. Analyses of the kinetic properties of SpGR revealed a significantly higher value for Km(GSSG) (231.2 �� 24.7 ?M) in comparison to other characterized GR enzymes.
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385. |
Bernardo-García N,
Mahasenan KV,
Batuecas MT,
Lee M,
Hesek D,
Petrá?ková D,
Doubravová L,
Branny P,
Mobashery S,
Hermoso JA,
( 2018 ) Allostery, Recognition of Nascent Peptidoglycan, and Cross-linking of the Cell Wall by the Essential Penicillin-Binding Protein 2x of Streptococcus pneumoniae. PMID : 29357220 : DOI : 10.1021/acschembio.7b00817 Abstract >>
Transpeptidases, members of the penicillin-binding protein (PBP) families, catalyze cross-linking of the bacterial cell wall. This transformation is critical for the survival of bacteria, and it is the target of inhibition by �]-lactam antibiotics. We report herein our structural insights into catalysis by the essential PBP2x of Streptococcus pneumoniae by disclosing a total of four X-ray structures, two computational models based on the crystal structures, and molecular-dynamics simulations. The X-ray structures are for the apo PBP2x, the enzyme modified covalently in the active site by oxacillin (a penicillin antibiotic), the enzyme modified by oxacillin in the presence of a synthetic tetrasaccharide surrogate for the cell-wall peptidoglycan, and a noncovalent complex of cefepime (a cephalosporin antibiotic) bound to the active site. A prerequisite for catalysis by transpeptidases, including PBP2x, is the molecular recognition of nascent peptidoglycan strands, which harbor pentapeptide stems. We disclose that the recognition of nascent peptidoglycan by PBP2x takes place by complexation of one pentapeptide stem at an allosteric site located in the PASTA domains of this enzyme. This binding predisposes the third pentapeptide stem in the same nascent peptidoglycan strand to penetration into the active site for the turnover events. The complexation of the two pentapeptide stems in the same peptidoglycan strand is a recognition motif for the nascent peptidoglycan, critical for the cell-wall cross-linking reaction.
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386. |
Rezaei Javan R,
van Tonder AJ,
King JP,
Harrold CL,
Brueggemann AB,
( 2018 ) Genome Sequencing Reveals a Large and Diverse Repertoire of Antimicrobial Peptides. PMID : 30210481 : DOI : 10.3389/fmicb.2018.02012 PMC : PMC6120550 Abstract >>
Competition among bacterial members of the same ecological niche is mediated by bacteriocins: antimicrobial peptides produced by bacterial species to kill other bacteria. Bacteriocins are also promising candidates for novel antimicrobials. Streptococcus pneumoniae (the "pneumococcus") is a leading cause of morbidity and mortality worldwide and a frequent colonizer of the human nasopharynx. Here, 14 newly discovered bacteriocin gene clusters were identified among >6,200 pneumococcal genomes. The molecular epidemiology of the bacteriocin clusters was investigated using a large global and historical pneumococcal dataset dating from 1916. These analyses revealed extraordinary bacteriocin diversity among pneumococci and the majority of bacteriocin clusters were also found in other streptococcal species. Genomic hotspots for the integration of different bacteriocin gene clusters were discovered. Experimentally, bacteriocin genes were transcriptionally active when the pneumococcus was under stress and when two strains were co-cultured in broth. These findings reveal much more diversity among bacterial defense mechanisms than previously appreciated, which fundamentally broaden our understanding of bacteriocins relative to intraspecies and interspecies nasopharyngeal competition and bacterial population structure.
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387. |
Mannarelli BM,
Balganesh TS,
Greenberg B,
Springhorn SS,
Lacks SA,
( 1985 ) Nucleotide sequence of the Dpn II DNA methylase gene of Streptococcus pneumoniae and its relationship to the dam gene of Escherichia coli. PMID : 2989823 : DOI : 10.1073/pnas.82.13.4468 PMC : PMC391122 Abstract >>
The structural gene (dpnM) for the Dpn II DNA methylase of Streptococcus pneumoniae, which is part of the Dpn II restriction system and methylates adenine in the sequence 5'-G-A-T-C-3', was identified by subcloning fragments of a chromosomal segment from a Dpn II-producing strain in an S. pneumoniae host/vector cloning system and demonstrating function of the gene also in Bacillus subtilis. Determination of the nucleotide sequence of the gene and adjacent DNA indicates that it encodes a polypeptide of 32,903 daltons. A putative promoter for transcription of the gene lies within a hundred nucleotides of the polypeptide start codon. Comparison of the coding sequence to that of the dam gene of Escherichia coli, which encodes a similar methylase, revealed 30% of the amino acid residues in the two enzymes to be identical. This homology presumably reflects a common origin of the two genes prior to the divergence of Gram-positive and Gram-negative bacteria. It is suggested that the restriction function of the gene is primitive, and that the homologous restriction system in E. coli has evolved to play an accessory role in heteroduplex DNA base mismatch repair.
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388. |
Yang HB,
Hou WT,
Cheng MT,
Jiang YL,
Chen Y,
Zhou CZ,
( 2018 ) Structure of a MacAB-like efflux pump from Streptococcus pneumoniae. PMID : 29335499 : DOI : 10.1038/s41467-017-02741-4 PMC : PMC5768738 Abstract >>
The spr0693-spr0694-spr0695 operon of Streptococcus pneumoniae encodes a putative ATP-binding cassette (ABC)-type efflux pump involved in the resistance of antibiotics and antimicrobial peptides. Here we report the crystal structures of Spr0694-0695 at 3.3 ? and Spr0693 at 3.0 ? resolution, revealing a MacAB-like efflux pump. The dimeric Spr0694-0695 adopts a non-canonical fold of ABC transporter, the transmembrane domain of which consists of eight tightly packed transmembrane helices with an insertion of extracellular domain between the first and second helices, whereas Spr0693 forms a nanotube channel docked onto the ABC transporter. Structural analyses combined with ATPase activity and antimicrobial susceptibility assays, enable us to propose a putative substrate-entrance tunnel with a lateral access controlled by a guard helix. Altogether, our findings provide structural insights and putative transport mechanism of a MacAB-like efflux pump in Gram-positive bacteria.
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389. |
Lacks SA,
Mannarelli BM,
Springhorn SS,
Greenberg B,
( 1986 ) Genetic basis of the complementary DpnI and DpnII restriction systems of S. pneumoniae: an intercellular cassette mechanism. PMID : 3019562 : DOI : 10.1016/0092-8674(86)90698-7 Abstract >>
Cells of S. pneumoniae contain either DpnI, a restriction endonuclease that cleaves only the methylated DNA sequence 5'-GmeATC-3', or DpnII, which cleaves the same sequence when not methylated. A chromosomal DNA segment containing DpnII genes was cloned in S. pneumoniae. Nucleotide sequencing of this segment revealed genes encoding the methylase and endonuclease and a third protein of unknown function. When the plasmid was introduced into DpnI cells, recombination during chromosomal facilitation of its establishment substituted genes encoding the DpnI endonuclease and another protein in place of the DpnII genes. DNA hybridization and sequencing showed that the DpnI and DpnII segments share homology on either side but not between themselves or with other regions of the chromosome. Thus, the complementary restriction systems are found on nonhomologous and mutually exclusive cassettes that can be inserted into a particular point in the chromosome of S. pneumoniae on the basis of neighboring homology.
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390. |
Oodate M,
Kimura K,
Banno H,
Yokoyama S,
Jin W,
Wachino JI,
Hasegawa Y,
Arakawa Y,
( 2018 ) Predominance of Serogroup 19 CC320/271 among Penicillin-Nonsusceptible Streptococcus pneumoniae Isolates after Introduction of the PCV7 Vaccine in Several Regions of Japan. PMID : 29093321 : DOI : 10.7883/yoken.JJID.2017.236 Abstract >>
Multidrug-resistant Streptococcus pneumoniae serogroup 19, including serotypes 19A and 19F, associated with clonal complex 320/271 (CC320/271), has been previously shown to be predominant in many countries after introduction of a 7-valent pneumococcal conjugate vaccine (PCV7). However, in Japan there has been no epidemiological research focused on penicillin-nonsusceptible isolates after this event. Therefore, we aimed to characterize penicillin-nonsusceptible S. pneumoniae (PNSSP; penicillin minimum inhibitory concentration [MIC] ? 4.0 �gg/ml) after the introduction of PCV7 in Japan. Throughout Japan, we collected 1,057 pneumococcal isolates from 2010 to 2014. We then evaluated MICs and performed serotyping, multilocus sequence typing, and sequencing of penicillin-binding protein genes in 51 isolates (penicillin MIC ? 2.0 �gg/ml). Twenty-three isolates (2.2%) showed penicillin nonsusceptibility (penicillin MIC ? 4.0 �gg/ml). Serotypes 19F (14 isolates, 60.9%) and 23F (4 isolates, 17.4%), which are covered by the vaccine, were predominant among PNSSP strains. Only 3 isolates belonged to nonvaccine serotype 19A. Among the PNSSP isolates, CC320/271 (16/23 strains, 69.6%) was the most prevalent clone. Moreover, CC320/271 clones showed high MIC values of a third-generation cephalosporin. Thus, we demonstrated clonal predominance of serogroup 19 CC320/271 with strong resistance to �]-lactams including a third-generation cephalosporin among PNSSP isolates.
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391. |
Wyllie AL,
Pannekoek Y,
Bovenkerk S,
van Engelsdorp Gastelaars J,
Ferwerda B,
van de Beek D,
Sanders EAM,
Trzci?ski K,
van der Ende A,
( 2017 ) Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species. PMID : 28931649 : DOI : 10.1098/rsob.170074 PMC : PMC5627049 Abstract >>
The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease (n = 101) and from carriage (n = 103), and on non-typeable pneumococci from asymptomatic individuals (n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae, targeting cpsA, lytA, piaB, ply, Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae, whereas assays targeting cpsA, ply, Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates.
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392. |
Gillis HD,
Demczuk WHB,
Griffith A,
Martin I,
Warhuus M,
Lang ALS,
ElSherif M,
McNeil SA,
LeBlanc JJ,
( 2018 ) PCR-based discrimination of emerging Streptococcus pneumoniae serotypes 22F and 33F. PMID : 29162393 : DOI : 10.1016/j.mimet.2017.11.017 Abstract >>
Serotyping of Streptococcus pneumoniae is important to monitor disease epidemiology and assess the impact of pneumococcal vaccines. Traditionally, the Quellung reaction used serotype-specific antibodies to classify S. pneumoniae based on differences in capsular antigens. More recently, PCR-based serotype deduction relying on serotype-specific capsule biosynthesis genes has been broadly applied for pneumococcal surveillance. However, PCR-based serotyping lacks discrimination for certain S. pneumoniae serotypes, including the differentiation of serotype 22F from 22A, and serotype 33F from 33A and 37. Serotypes 22F and 33F are emerging serotypes that are absent in the currently licensed 13-valent pneumococcal conjugate vaccine, but present in the new candidate 15-valent formulation. This study validated novel PCR reactions to detect and discriminate S. pneumoniae serotypes 22F and 33F. In order to differentiate S. pneumoniae serotypes 22F or 33F from genetically similar serotypes, two novel PCR reactions were designed and validated. The specificity of all PCR targets was evaluated using all 92 different S. pneumoniae serotypes, as well as 32 other streptococci. Reproducibility was evaluated using geographically and genetically diverse strains of S. pneumoniae serotypes 22F and 22A, or serotypes 33F, 33A, and 37 that were previously characterized by reputable reference laboratories. Overall, S. pneumoniae serotypes 22F and 33F could be accurately and reproducibly be detected and discriminated using PCR alone. Such a molecular serotyping approach provides a valuable diagnostic tool that is feasible in any molecular laboratory, to enable pneumococcal serotype surveillance and subsequent assessment of the impact of the new 15-valent candidate pneumococcal vaccine.
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393. |
González A,
Llull D,
Morales M,
García P,
García E,
( 2008 ) Mutations in the tacF gene of clinical strains and laboratory transformants of Streptococcus pneumoniae: impact on choline auxotrophy and growth rate. PMID : 18424523 : DOI : 10.1128/JB.01991-07 PMC : PMC2446756 Abstract >>
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394. |
( 2013 ) Update of pneumococcal PCR serotyping assay for detection of a commonly occurring type 19F wzy variant in Brazil. PMID : 23658255 : DOI : 10.1128/JCM.00743-13 PMC : PMC3697664 Abstract >>
N/A
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395. |
Geno KA,
Bush CA,
Wang M,
Jin C,
Nahm MH,
Yang J,
( 2017 ) WciG O-Acetyltransferase Functionality Differentiates Pneumococcal Serotypes 35C and 42. PMID : 28659323 : DOI : 10.1128/JCM.00822-17 PMC : PMC5648713 Abstract >>
Streptococcus pneumoniae expresses capsular polysaccharides (CPSs) to protect itself from opsonophagocytic killing. The genes responsible for capsules synthesized by the Wzy-dependent mechanism, which accounts for 96 of the 98 known pneumococcal capsule types, are in a chromosomal region known as the cps locus. The nucleotide sequence in this region has been determined for all serotypes. In contrast, not all CPS structures have been defined. The structure of the serotype 35C polysaccharide was recently reported, but the presence of O-acetyltransferase genes in the serotype 35C cps locus suggested that it could be incomplete, as the reported structure contains no O-acetylation. In addition, the genetic distinction of serotype 35C from the closely related serotype 42 was unclear, as their reported cps loci are nearly identical. To clarify these discrepancies, we obtained serotype 35C and 42 clinical and reference isolates and studied their serological and genetic properties, as well as the structures of CPSs purified from reference isolates. We demonstrated that the O-acetyltransferase WciG was functional in serotype 35C but nonfunctional in serotype 42 due to a deletion in wciG Serotype 35C was O-acetylated at the 5- and 6-positions of 3-�]-galactofuranose, as well as the 2-position of 6-�]-galactofuranose. However, serotype 42 has only O-acetylation at 3-�]-galactofuranose, an observation consistent with its loss of WciG functionality, which is associated with O-acetylation at the 2-position and subsequent reaction with typing antiserum 35a. These findings provide a comprehensive view of the genetic, biochemical structural, and serological bases of serotypes 35C and 42.
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396. |
García P,
García JL,
García E,
López R,
( 1986 ) Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli. PMID : 2875013 : DOI : 10.1016/0378-1119(86)90215-5 Abstract >>
Autolysins are enzymes that have several important biological functions and also seem to be responsible for the irreversible effects induced by the beta-lactam antibiotics. The pneumococcal autolysin gene (lyt) has been subcloned from the plasmid pGL30 [Garc?a et al., Mol. Gen. Genet. 201 (1985) 225-230] and we have found that the E form of the autolysin is synthesized in Escherichia coli using its own promoter. The high amount of autolysin obtained in the heterologous system when the lyt gene is present in different orientations in the recombinant plasmids studied supports the idea that the autolysin promoter could be a strong one. The nucleotide sequence of the HindIII fragment of pGL80 (1213 bp) containing the autolysin structural gene has been determined. A unique open reading frame (ORF) has been found, a consensus ribosome-binding site and -10 and -35 promoter-like sequences as well as A + T-rich regions farther upstream were also identified. The lyt ORF encodes a protein of 318 amino acid residues having a calculated Mr of 36,532, which agrees with previous size estimates based on electrophoretic migration [H?ltje and Tomasz, J. Biol. Chem. 251 (1976) 4199-4207; Briese and Hakenbeck, Eur. J. Biochem. 146 (1985) 417-427]. Our results also demonstrate that the lyt-4 marker represents the first example of a mutation in a structural gene of a bacterial autolysin. The polarity profile of the pneumococcal autolysin supports previous suggestions about the localization of this enzyme in the normal cell.
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397. |
Manna S,
Ortika BD,
Dunne EM,
Holt KE,
Kama M,
Russell FM,
Hinds J,
Satzke C,
( 2018 ) A novel genetic variant of Streptococcus pneumoniae serotype 11A discovered in Fiji. PMID : 28736074 : DOI : 10.1016/j.cmi.2017.06.031 PMC : PMC5869949 Abstract >>
As part of annual cross-sectional Streptococcus pneumoniae carriage surveys in Fiji (2012-2015), we detected pneumococci in over 100 nasopharyngeal swabs that serotyped as '11F-like' by microarray. We examined the genetic basis of this divergence in the 11F-like capsular polysaccharide (cps) locus compared to the reference 11F cps sequence. The impact of this diversity on capsule phenotype, and serotype results using genetic and serologic methods were determined. Genomic DNA from representative 11F-like S. pneumoniae isolates obtained from the nasopharynx of Fijian children was extracted and subject to whole genome sequencing. Genetic and phylogenetic analyses were used to identify genetic changes in the cps locus. Capsular phenotypes were evaluated using the Quellung reaction and latex agglutination. Compared to published 11F sequences, the wcwC and wcrL genes of the 11F-like cps locus are phylogenetically divergent, and the gct gene contains a single nucleotide insertion within a homopolymeric region. These changes within the DNA sequence of the 11F-like cps locus have modified the antigenic properties of the capsule, such that 11F-like isolates serotype as 11A by Quellung reaction and latex agglutination. This study demonstrates the ability of molecular serotyping by microarray to identify genetic variants of S. pneumoniae and highlights the potential for discrepant results between phenotypic and genotypic serotyping methods. We propose that 11F-like isolates are not a new serotype but rather are a novel genetic variant of serotype 11A. These findings have implications for invasive pneumococcal disease surveillance as well as studies investigating vaccine impact.
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398. |
( 1999 ) Activities of newer fluoroquinolones against Streptococcus pneumoniae clinical isolates including those with mutations in the gyrA, parC, and parE loci. PMID : 9925527 : PMC : PMC89072 Abstract >>
Resistance to fluoroquinolone (FQ) antibiotics in Streptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, > or = 8 micrograms/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE. The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 micrograms/ml) and trovafloxacin (MICs, 0.5 to 1 microgram/ml) were associated with either a mutation in parC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA, parC, and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC, is not predictive of pneumococcal resistance to these agents.
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399. |
( 2012 ) Bacteremic pneumonia caused by extensively drug-resistant Streptococcus pneumoniae. PMID : 23052301 : DOI : 10.1128/JCM.01642-12 PMC : PMC3502956 Abstract >>
The emergence of antimicrobial resistance threatens the successful treatment of pneumococcal infections. Here we report a case of bacteremic pneumonia caused by an extremely drug-resistant strain of Streptococcus pneumoniae, nonsusceptible to at least one agent in all classes but vancomycin and linezolid, posing an important new public health threat in our region.
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400. |
( 2013 ) Identification of an atypical zinc metalloproteinase, ZmpC, from an epidemic conjunctivitis-causing strain of Streptococcus pneumoniae. PMID : 23168398 : DOI : 10.1016/j.micpath.2012.11.006 PMC : PMC3578082 Abstract >>
Streptococcus pneumoniae is a pathogen associated with a range of invasive and noninvasive infections. Despite the identification of the majority of virulence factors expressed by S. pneumoniae, knowledge of the strategies used by this bacterium to trigger infections, especially those originating at wet-surfaced epithelia, remains limited. In this regard, we recently reported a mechanism used by a nonencapsulated, epidemic conjunctivitis-causing strain of S. pneumoniae (strain SP168) to gain access into ocular surface epithelial cells. Mechanistically, strain SP168 secretes a zinc metalloproteinase, encoded by a truncated zmpC gene, to cleave off the ectodomain of a vital defense component - the membrane mucin MUC16 - from the apical glycocalyx barrier of ocular surface epithelial cells and, thereby invades underlying epithelial cells. Here, we compare the truncated SP168 ZmpC to its highly conserved archetype from S. pneumoniae serotype 4 (TIGR4), which has been linked to pneumococcal virulence in previous studies. Comparative nucleotide sequence analyses revealed that the zmpC gene corresponding to strain SP168 has two stretches of DNA deleted near its 5' end. A third 3 bp in-frame deletion, resulting in the elimination of an alanine residue, was found towards the middle segment of the SP168 zmpC. Closer examination of the primary structure revealed that the SP168 ZmpC lacks the canonical LPXTG motif - a signature typical of several surface proteins of gram-positive bacteria and of other pneumococcal zinc metalloproteinases. Surprisingly, in vitro assays performed using recombinant forms of ZmpC indicated that the truncated SP168 ZmpC induces more cleavage of the MUC16 ectodomain than its TIGR4 counterpart. This feature may help explain, in part, why S. pneumoniae strain SP168 is better equipped at abrogating the MUC16 glycocalyx barrier en route to causing epidemic conjunctivitis.
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401. |
( 1999 ) Identification of a Streptococcus pneumoniae gene locus encoding proteins of an ABC phosphate transporter and a two-component regulatory system. PMID : 9973337 : PMC : PMC93488 Abstract >>
The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant phoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.
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( 1997 ) The adc locus, which affects competence for genetic transformation in Streptococcus pneumoniae, encodes an ABC transporter with a putative lipoprotein homologous to a family of streptococcal adhesins. PMID : 9765793 : DOI : 10.1016/S0923-2508(97)87643-7 Abstract >>
To identify new components involved in the phenomenon of transformation in Streptococcus pneumoniae, a library of potential mutants has been generated by random insertion of an erythromycin resistance gene. Transformation-deficient mutants were screened using an in situ colony transformation test. The adc locus, which was identified in this search, was cloned and sequenced. Sequence analysis revealed a putative operon of three ORFs (adcC, adcB and adcA) with homology to ATP-binding cassette (ABC) transport operons encoding streptococcal adhesins such as ScaA of S. gordonii and FimA of S. parasanguis. adcA can encode a lipoprotein of 313 amino acid residues containing a putative metal-binding site. The polypeptide shows about 30% sequence identity with ScaA and FimA. We discuss evidence which leads us to propose that AdcA, together with a set of 14 proteins including ScaA, FimA and homologous adhesins, defines a new family of external solute-binding proteins, cluster 9, specific for metals.
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( 1998 ) Association of a thr-371 substitution in a conserved amino acid motif of penicillin-binding protein 1A with penicillin resistance of Streptococcus pneumoniae. PMID : 9736547 : PMC : PMC105808 Abstract >>
We determined the nucleotide sequence between 1,903 and 3,097 bp of pbp1a, which encodes the transpeptidase domain of PBP 1A, from clinical isolates of penicillin-resistant Streptococcus pneumoniae (PRSP) serotypes 19 (n = 8), 6 (n = 9), 23 (n = 6), and 14 (n = 2) and two penicillin-susceptible S. pneumoniae (PSSP) isolates. These serotyped PRSP strains were isolated predominantly in Japan from 1993 through 1997. The 25 resistant strains were classified into five groups on the basis of the extent of sequence differences. Strains in groups I (n = 5; serotype 6), II (n = 3; serotype 19), and III (n = 12; different serotypes) had sequences highly homologous to the sequence of pbp1a of PRSP strains from South Africa, Spain, and the United States. The group IV strain (n = 1; serotype 14) had unique deletions from or insertions in the sequences. The sequences of group V strains (n = 4; serotypes 6 and 23) had relatively few differences from the sequences of the PSSP strains. For strains (n = 18) for which the threonine at codon 371 (Thr-371) in a conserved STMK motif of PBP 1A was substituted with an alanine or a serine (in addition to having altered pbp2x and pbp2b genes), penicillin MICs were >/= 1.0 microgram/ml. The PBPs 1A of these strains showed decreased affinities for [3H]benzylpenicillin and slightly faster mobilities on sodium dodecyl sulfate-polyacrylamide gels. In contrast, for strains (n = 4) without a substitution at Thr-371 in PBP 1A but with mutations in both pbp2x and pbp2b, penicillin MICs were 0.125 to 0.25 microgram/ml, and the affinities of their PBPs 1A were similar to that of PSSP PBPs 1A. Furthermore, for the Thr-371-substituted strains (n = 3) with altered pbp2x genes but native pbp2b genes, penicillin MICs were 0.125 to 0.25 microgram/ml. These results suggest that amino acid substitution of Thr-371 contributes to the development of penicillin resistance in PRSP strains with altered pbp2x and pbp2b genes.
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( 1998 ) A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. PMID : 9846740 : DOI : 10.1099/00221287-144-11-3049 Abstract >>
The population biology of Streptococcus pneumoniae is poorly understood. Most of the important issues could be addressed by the molecular characterization of large, well sampled populations from carriage and from the different manifestations of pneumococcal disease. The authors have therefore developed a pneumococcal multilocus sequence typing scheme and database by sequencing approximately 450 bp fragments of seven housekeeping loci from 295 isolates. The combination of alleles at the seven loci provided an allelic profile, or sequence type (ST), and the relatedness between isolates was obtained by constructing a dendrogram from the matrix of pairwise differences between STs. The typing scheme was validated using pneumococci of known genetic relatedness and could resolve >6 billion STs. Among 274 isolates from recent cases of invasive pneumococcal disease in eight countries, 143 STs were resolved, but 12 STs contained at least five isolates (range 5-21 isolates). The repeated recovery of indistinguishable isolates from invasive disease in different countries implies that these STs define strains with an increased capacity to cause invasive disease. The relationship between STs and serotypes suggested that, in the longer term, capsular genes have been distributed horizontally within the pneumococcal population, but in the short term, expansion of clones occurs with only occasional changes of serotype. The multilocus sequence typing scheme provides a powerful new approach to the characterization of pneumococci, since it provides molecular typing data that are electronically portable between laboratories, and which can be used to probe aspects of the population and evolutionary biology of these organisms. A Web site for the molecular characterization of pneumococci by MLST is available (http ://mlst.zoo.ox.ac.uk).
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( 1998 ) Identification of three major clones of multiply antibiotic-resistant Streptococcus pneumoniae in Taiwanese hospitals by multilocus sequence typing. PMID : 9817864 : PMC : PMC105231 Abstract >>
In this paper we demonstrate the advantages of a new molecular typing procedure, multilocus sequence typing, for the unambiguous characterization of penicillin-resistant pneumococci. The sequences of approximately 450-bp fragments of seven housekeeping genes were determined for 74 penicillin-resistant Taiwanese isolates of Streptococcus pneumoniae (MIC of penicillin > 0.5 microgram/ml). The combination of alleles at the seven loci defined an allelic profile for each strain, and a dendrogram, based on the pairwise mismatches in allelic profiles, grouped 86% of the isolates into one of three penicillin-resistant clones for which the MICs of penicillin were 1 to 2 microgram/ml. Isolates within each clone had identical alleles at all seven loci or differed at only a single locus, and the fingerprints of their pbp1A, pbp2B, and pbp2X genes were uniform. Isolates of the Taiwan-19F clone and the Taiwan-23F clone were resistant to penicillin, tetracycline, and erythromycin but were susceptible to chloramphenicol. A second serotype 23F clone and serotype 19F variants of this clone were resistant to penicillin, tetracycline, chloramphenicol, and, in some cases, erythromycin. Comparisons of the allelic profiles of the three major clones with those of reference isolates of the known penicillin-resistant clones showed that the Taiwan-19F and Taiwan-23F clones were previously undescribed, whereas the second serotype 23F clone was indistinguishable from the Spanish multidrug-resistant serotype 23F clone. Single isolates of the Spanish penicillin-resistant serotype 9V clone and the Spanish multidrug-resistant serotype 6B clone were also identified in the collection.
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( 1998 ) Penicillin tolerance genes of Streptococcus pneumoniae: the ABC-type manganese permease complex Psa. PMID : 9767595 : DOI : 10.1046/j.1365-2958.1998.01016.x Abstract >>
Downregulation of the major autolysin in Streptococcus pneumoniae leads to penicillin tolerance, a feature that is characterized by the ability to survive but not grow in the presence of antibiotic. Screening a library of mutants in pneumococcal surface proteins for the ability to survive 10x minimum inhibitory concentration (MIC) of penicillin revealed over 10 candidate tolerance genes. One such mutant contained an insertion in the known gene psaA, which is part of the psa locus. This locus encodes an ABC-type Mn permease complex. Sequence analysis of adjacent DNA extended the known genetic organization of the locus to include two new open reading frames (ORFs), psaB, which encodes an ATP-binding protein, and psaC, which encodes a hydrophobic transmembrane protein. Mutagenesis of psaB, psaC, psaA and downstream psaD resulted in penicillin tolerance. Defective adhesion and reduced transformation efficiency, as reported previously for a psaA- mutant, were phenotypes shared by psaB-, psaC- and psaD- knockout mutants. Western blot analysis demonstrated that the set of mutants expressed RecA, but none of them showed translation of the autolysin gene, which is located downstream of recA. The addition of manganese (Mn) failed to correct the abnormal physiology. These results suggest that this ABC-type Mn permease complex has a pleiotropic effect on pneumococcal physiology including adherence and autolysis. These are the first genes suggested as being involved in triggering autolysin. The results raise the possibility that loss of function of PsaA, by vaccine-induced antibody for instance, may promote penicillin tolerance.
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( 1998 ) Molecular characterization of the complete 23F capsular polysaccharide locus of Streptococcus pneumoniae. PMID : 9748469 : PMC : PMC107572 Abstract >>
The complete DNA sequence of the capsular locus 23F of Streptococcus pneumoniae is presented. The 18.6-kb cps23f locus is composed of 18 open reading frames flanked at the 5' and 3' ends by the genes dexB and aliA, an arrangement similar to those of some of the other identified cps loci.
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( 1998 ) Comparison of the PspA sequence from Streptococcus pneumoniae EF5668 to the previously identified PspA sequence from strain Rx1 and ability of PspA from EF5668 to elicit protection against pneumococci of different capsular types. PMID : 9746574 : PMC : PMC108585 Abstract >>
PspA (pneumococcal surface protein A) is a serologically varied virulence factor of Streptococcus pneumoniae. In mice, PspA has been shown to elicit an antibody response that protects against fatal challenge with encapsulated S. pneumoniae, and the protection-eliciting residues have been mapped to the alpha-helical N-terminal half of the protein. To date, a published DNA sequence for pspA is available only for S. pneumoniae Rx1, a laboratory strain. PspA/EF5668 (EF5668 indicates the strain of origin of the PspA) is serologically distinct from PspA/Rx1. Sequencing of the gene encoding PspA/EF5668 revealed 71% identity with that of PspA/Rx1. The greatest amount of divergence between the two proteins was seen in their alpha-helical portions, which are surface exposed and probably under selective pressure to diversify serologically. In spite of the diversity within the alpha-helical regions of PspAs, we have observed that recombinant PspA (rPspA)/EF5668, like rPspA/Rx1, can elicit cross-protection against pneumococci of different capsular and PspA serological types.
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409. |
( 1998 ) Fluoroquinolone resistance mutations in the parC, parE, and gyrA genes of clinical isolates of viridans group streptococci. PMID : 9797205 : PMC : PMC105945 Abstract >>
The nucleotide sequences of the quinolone resistance-determining regions (QRDRs) of the parC and gyrA genes from seven ciprofloxacin-resistant (Cpr) isolates of viridans group streptococci (two high-level Cpr Streptococcus oralis and five low-level Cpr Streptococcus mitis isolates) were determined and compared with those obtained from susceptible isolates. The nucleotide sequences of the QRDRs of the parE and gyrB genes from the five low-level Cpr S. mitis isolates and from the NCTC 12261 type strain were also analyzed. Four of these low-level Cpr isolates had changes affecting the subunits of DNA topoisomerase IV: three in Ser-79 (to Phe or Ile) of ParC and one in ParE at a position not previously described to be involved in quinolone resistance (Pro-424). One isolate did not show any mutation. The two high-level Cpr S. oralis isolates showed mutations affecting equivalent residue positions of ParC and GyrA, namely, Ser-79 to Phe and Ser-81 to Phe or Tyr, respectively. The parC mutations were able to transform Streptococcus pneumoniae to ciprofloxacin resistance, while the gyrA mutations transformed S. pneumoniae only when mutations in parC were present. These results suggest that DNA topoisomerase IV is a primary target of ciprofloxacin in viridans group streptococci, DNA gyrase being a secondary target.
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( 1999 ) Molecular characterization of a globally distributed lineage of serotype 12F Streptococcus pneumoniae causing invasive disease. PMID : 9878026 : DOI : 10.1086/314589 Abstract >>
These studies have identified a major genetic lineage of capsule serotype 12F Streptococcus pneumoniae, which has maintained two different types of the pneumococcal surface protein A (PspA) virulence factor and caused invasive disease in geographically disjoint locations. Twenty outbreak strains from a Texas jail and Maryland day care center and 16 reference strains from Texas, Maryland, Washington, Michigan, Oklahoma, Missouri, Alaska, and Australia were examined. Although the Texas and Maryland outbreak strains were indistinguishable by IS1167 and boxA genotyping procedures, all strains examined were members of a genetically similar lineage. The microevolutionary history of pspA differed from that of the overall genetic background of the strains. Taken together, these findings suggested that the Texas and Maryland outbreaks were caused by different clones of a major genetic lineage of serotype 12F pneumococci, within which at least one PspA has been acquired via localized genetic recombination.
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( 1998 ) Crystal structure of the DpnM DNA adenine methyltransferase from the DpnII restriction system of streptococcus pneumoniae bound to S-adenosylmethionine. PMID : 9862809 : Abstract >>
. Methyltransferases (Mtases) catalyze the transfer of methyl groups from S-adenosylmethionine (AdoMet) to a variety of small molecular and macromolecular substrates. These enzymes contain a characteristic alpha/beta structural fold. Four groups of DNA Mtases have been defined and representative structures have been determined for three groups. DpnM is a DNA Mtase that acts on adenine N6 in the sequence GATC; the enzyme represents group alpha DNA Mtases, for which no structures are known. . The structure of DpnM in complex with AdoMet was determined at 1.80 A resolution. The protein comprises a consensus Mtase fold with a helical cluster insert. DpnM binds AdoMet in a similar manner to most other Mtases and the enzyme contains a hollow that can accommodate DNA. The helical cluster supports a shelf within the hollow that may recognize the target sequence. Modeling studies indicate a potential site for binding the target adenine, everted from the DNA helix. Comparison of the DpnM structure and sequences of group alpha DNA Mtases indicates that the group is a genetically related family. Structural comparisons show DpnM to be most similar to a small-molecule Mtase and then to macromolecular Mtases, although several dehydrogenases show greater similarity than one DNA Mtase. . DpnM, and by extension the DpnM family or group alpha Mtases, contains the consensus fold and AdoMet-binding motifs found in most Mtases. Structural considerations suggest that macromolecular Mtases evolved from small-molecule Mtases, with different groups of DNA Mtases evolving independently. Mtases may have evolved from dehydrogenases. Comparison of these enzymes indicates that in protein evolution, the structural fold is most highly conserved, then function and lastly sequence.
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( 1999 ) Characterization of cryptic plasmids pDP1 and pSMB1 of Streptococcus pneumoniae. PMID : 9887308 : DOI : 10.1006/plas.1998.1364 Abstract >>
Cryptic plasmids pDP1 and pSMB1 from clinical strains of Streptococcus pneumoniae isolated 74 years apart were found to be essentially identical in their nucleotide sequence. pDP1, 3161 bp, contains five codirectional ORFs and presents all the general features of plasmids replicating by the rolling circle mechanism. The rep gene, 963 bp, is highly homologous to the rep gene of other streptococcal plasmids of the pC194 family.
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413. |
( 1998 ) Alterations in PBP 1A essential-for high-level penicillin resistance in Streptococcus pneumoniae. PMID : 9624469 : PMC : PMC105597 Abstract >>
High-level penicillin resistance in pneumococci is due to alterations in penicillin-binding proteins (PBPs) 2X, 2B, and 1A. We have sequenced the penicillin-binding domain of PBP 1A from penicillin-resistant South African pneumococcal isolates and have identified amino acid substitutions which are common to all the resistant isolates analyzed. Site-directed mutagenesis was then used to determine whether particular amino acid substitutions at specific positions in PBP 1A mediate penicillin resistance. PCR was used to isolate PBP 2X, 2B, and 1A genes from clinical isolate 8303 (penicillin MIC, 4 micrograms/ml). These wild-type PBP genes were cloned into pGEM-3Zf and were used as the transforming DNA. Susceptible strain R6 (MIC, 0.015 microgram/ml) was first transformed with PBP 2X and 2B DNA, resulting in PBP 2X/2B-R6 transformants for which MICs were 0.25 microgram/ml. When further transformed with PBP 1A DNA, 2X/2B/1A-R6 transformants for which MICs were 1.5 micrograms/ml were obtained. Site-directed mutagenesis of the PBP 1A gene from isolate 8303 was then used to reverse particular amino acid substitutions, followed by transformation of PBP 2X/2B-R6 transformants with the mutagenized PBP 1A DNA. For PBP 2X/2B/1A-R6 transformants, the introduction of the reversal of Thr-371 by Ser or Ala in PBP 1A decreased the MIC from 1.5 to 0.5 micrograms/ml, whereas the reversal of four consecutive amino acid substitutions (Thr-574 by Asn, Ser-575 by Thr, Gln-576 by Gly, and Phe-577 by Tyr) decreased the MIC from 1.5 to 0.375 micrograms/ml. These data reveal that amino acid residue 371 and residues 574 to 577 of PBP 1A are important positions in PBP 1A with respect to the interaction with penicillin and the development of resistance.
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( 1998 ) Characterization of the galU gene of Streptococcus pneumoniae encoding a uridine diphosphoglucose pyrophosphorylase: a gene essential for capsular polysaccharide biosynthesis. PMID : 9841918 : DOI : 10.1084/jem.188.11.2047 PMC : PMC2212384 Abstract >>
The galU gene of Streptococcus pneumoniae has been cloned and sequenced. Escherichia coli cells harboring the recombinant plasmid pMMG2 (galU) overproduced a protein that has been shown to correspond to a uridine 5'-triphosphate:glucose-1-phosphate uridylyltransferase (uridine diphosphoglucose [UDP-Glc] pyrophosphorylase) responsible for the synthesis of UDP-Glc, a key compound in the biosynthesis of polysaccharides. A gene very similar to the S. pneumoniae galU has been found in a partial nucleotide sequence of the Streptococcus pyogenes genome. Knockout galU mutants of type 1 pneumococci are unable to synthesize a detectable capsule. An identical result was found in type 3 S. pneumoniae cells in spite of the fact that these bacteria contain a type-specific gene (cap3C) that also encodes a UDP-Glc pyrophosphorylase. Since eukaryotic UDP-Glc pyrophosphorylases appear to be completely unrelated to their prokaryotic counterparts, we postulate that GalU may be an appropriate target for the search of new drugs to control the pathogenicity of bacteria like pneumococcus and S. pyogenes.
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415. |
( 1998 ) A conservative amino acid mutation in the chromosome-encoded dihydrofolate reductase confers trimethoprim resistance in Streptococcus pneumoniae. PMID : 9728538 : DOI : 10.1086/515371 Abstract >>
Multidrug-resistant Streptococcus pneumoniae strains have emerged over the past decade at an alarming rate. The molecular mechanism of trimethoprim resistance was investigated in 5 pneumococcal strains isolated in the Washington, DC, area from patients with invasive infections. Cloning and sequencing of the trimethoprim resistance determinant from these pneumococci indicated that an altered chromosome-encoded dihydrofolate reductase (DHFR) was responsible for the observed resistance. Comparison of DHFR sequences from pneumococcal strains with various susceptibilities to trimethoprim, together with site-directed mutagenesis, revealed that substitution of isoleucine-100 with a leucine residue resulted in trimethoprim resistance. Hydrogen bonding between the carbonyl oxygen of isoleucine-100 and the 4-amino group of trimethoprim is proposed to play a critical role in the inhibition of DHFR by trimethoprim. This enzyme-substrate model should facilitate the design of new antibacterial agents with improved activity against S. pneumoniae.
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( 1998 ) Unconventional organization of the division and cell wall gene cluster of Streptococcus pneumoniae. PMID : 9846742 : DOI : 10.1099/00221287-144-11-3069 Abstract >>
The genes responsible for cell wall biosynthesis and cell division (dcw genes) were identified and sequenced in Streptococcus pneumoniae. The genetic organization of the dcw cluster in Streptococcus pneumoniae differed significantly from the clusters of other bacteria reported to date. In particular, the genes corresponding to the 2 min region of the Escherichia coli chromosome were found distributed in three genetically separate regions of the Streptococcus pneumoniae chromosome. The first region contained the expected ftsA and ftsZ cell division genes at one end and pbp2b, ddl and murF at the other end. The murD, murG and divIB genes, always found located upstream of ftsA, were found in a second region separated from the first. A third region contained the yllC, yllD, pbp2x and mraY genes. The chromosomal region downstream of ftsZ was also sequenced and characterized. In Streptococcus pneumoniae this region contains four ORFs, all of unknown function, and an ORF encoding the Bacillus subtilis DivIVA homologue. The gene order and the organization of this region was found to be conserved in Staphylococcus aureus, Streptococcus pyogenes and Bacillus subtilis, raising the possibility that previously unidentified loci may also be involved in division.
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( 1998 ) Molecular characterization of PcpA: a novel choline-binding protein of Streptococcus pneumoniae. PMID : 9675866 : DOI : 10.1111/j.1574-6968.1998.tb13087.x Abstract >>
The gene pcpA that encodes a novel pneumococcal choline-binding protein has been cloned and characterized. Northern blot analysis revealed that pcpA is expressed during the exponential phase of growth of pneumococci as a monocistronic transcript of about 2.3 kb. The transcription start site has been located 132 bp upstream of the start codon and the proposed -35 and -10 boxes that are highly similar to those of the typical sigma 70 promoters from Escherichia coli. This gene encodes a putative 79 kDa protein that contains a typical C-terminal choline-binding domain (ChBD). The ChBD of PcpA is built up by 11 identical motifs of 20 amino acids plus a tail of 19 amino acids, which represents the longest ChBD that has been characterized so far. Interestingly, two tandem arrays of five characteristic amphipathic leucine reach repeats (LRRs) of 22-26 amino acids in length have been found in the N-terminal region of PcpA. Since LRRs have been proposed to be involved in protein-protein and protein-lipid interactions our findings suggests a role for PcpA in pneumococcal adhesion.
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418. |
( 1998 ) Molecular structure of the gene cluster responsible for the synthesis of the polysaccharide capsule of Streptococcus pneumoniae type 33F. PMID : 9838125 : DOI : 10.1016/s0167-4781(98)00213-9 Abstract >>
The organization and nucleotide sequence of the capsular gene cluster involved in the biosynthesis of the type 33F capsular polysaccharide of Streptococcus pneumoniae have been determined. The complete type 33F operon (cap33f) is composed of 14 potential open reading frames where the last ten genes are group-specific. Putative functions have been assigned to several gene products by sequence comparison with the proteins included in the databases. A functional promoter located immediately upstream from the first gene of the cap33f gene cluster has been demonstrated. A 20 kb DNA fragment containing the cap33f genes and the operon promoter was sufficient to transform a S. pneumoniae type 3 unencapsulated mutant to the type 33F capsule.
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419. |
( 1998 ) Identification of a DNA cytosine methyltransferase gene in conjugative transposon Tn5252. PMID : 9473447 : DOI : 10.1006/plas.1997.1316 Abstract >>
The nucleotide sequence of the 3.5-kb right junction fragment of the streptococcal conjugative transposon Tn5252 was obtained. The DNA fragment was found to carry four putative genes one of which displayed a high degree of similarity to prokaryotic 5C-cytosine methyltransferases carrying multiple sequence specificities. No cognate endonuclease gene was detected in the sequenced DNA. Purified methylase polypeptide synthesized in a T7 promoter-controlled overexpression system was found to lack methylase activity while the cell extracts of host cells containing the recombinant plasmid carrying the methylase gene were active. In vivo mutations in the methylase gene did not seem to affect the transferability of the element.
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420. |
( 1998 ) Capsule genetics in Streptococcus pneumoniae and a possible role for transposition in the generation of the type 3 locus. PMID : 9533721 : DOI : 10.1089/mdr.1998.4.11 Abstract >>
The capsule genes of Streptococcus pneumoniae have a cassette-like organization in which the type-specific biosynthetic genes are flanked by genes shared among the different capsular serotypes. This general organization has been identified in the capsule loci of all serotypes analyzed to date, but significant differences that may help explain novel capsule type formation are beginning to emerge. In particular, analysis of the type 3 locus has revealed its most striking feature to be a preponderance of partial genes that have homology to sequences involved in polysaccharide biosynthesis and transposition. The predicted proteins of cps3M, the most downstream type 3-specific gene, and tnpA and plpA, the non-type-specific flanking sequences downstream of cps3M, have homologies with phosphomutases, transposases, and peptide permeases, respectively. All three of these sequences are truncated when compared to their respective homologs. Mutation and transcription analyses of these partial sequences showed that none of these sequences is essential for type 3 polysaccharide synthesis but that all are transcribed. Partial sequences were also identified in the region upstream of the type 3-specific genes. The type 3 locus structure is conserved among independent type 3 isolates but similar deletions are not apparent in the common, non-type-specific flanking sequences in other capsular types. A role for transposition-mediated events in the generation of the type 3 locus, and possibly other pneumococcal capsule loci, is suggested by these findings.
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( 1998 ) Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae. PMID : 9466257 : DOI : 10.1046/j.1365-2958.1998.00658.x Abstract >>
Serotype 19F variants of the major Spanish multiresistant serotype 23F clone of Streptococcus pneumoniae have been proposed to have arisen by recombinational exchanges at the capsular biosynthetic locus. Members of the Spanish multiresistant serotype 23F clone and the serotype 19F variants were confirmed to be essentially identical in overall genotype, as they were indistinguishable by REP-PCR, and had identical sequences at three polymorphic housekeeping genes. Eight serotype 19F variants were studied and all had large recombinational replacements at the capsular biosynthetic locus. In all cases, one of the recombinational cross-over points appeared to be upstream of dexB, which flanks one end of the capsular locus, and in six of the variants the other cross-over point was downstream of aliA, which flanks the other end of the locus. In two strains a recombinational cross-over point between the introduced serotype 19F capsular region and that of the Spanish serotype 23F clone could be clearly identified, within cpsN in one strain and within cpsM in the other. The differences in the recombinational junctions and sequence polymorphisms within the introduced capsular genes, suggested that the eight serotype 19F variants emerged on at least four separate occasions. Changes in capsular type by recombination may therefore be relatively frequent in pneumococci and this has implications for the long-term efficacy of conjugate pneumococcal vaccines that will protect against only a limited number of serotypes.
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( 1998 ) Acquisition of five high-Mr penicillin-binding protein variants during transfer of high-level beta-lactam resistance from Streptococcus mitis to Streptococcus pneumoniae. PMID : 9537382 : PMC : PMC107097 Abstract >>
Penicillin-resistant isolates of Streptococcus pneumoniae generally contain mosaic genes encoding the low-affinity penicillin-binding proteins (PBPs) PBP2x, PBP2b, and PBP1a. We now present evidence that PBP2a and PBP1b also appear to be low-affinity variants and are encoded by distinct alleles in beta-lactam-resistant transformants of S. pneumoniae obtained with chromosomal donor DNA from a Streptococcus mitis isolate. Different lineages of beta-lactam-resistant pneumococcal transformants were analyzed, and transformants with low-affinity variants of all high-molecular-mass PBPs, PBP2x, -2a, -2b, -1a, and -1b, were isolated. The MICs of benzyl-penicillin, oxacillin, and cefotaxime for these transformants were up to 40, 100, and 50 microg/ml, respectively, close to the MICs for the S. mitis donor strain. Recruitment of low-affinity PBPs was accompanied by a decrease in cross-linked muropeptides as revealed by high-performance liquid chromatography of muramidase-digested cell walls, but no qualitative changes in muropeptide chemistry were detected. The growth rates of all transformants were identical to that of the parental S. pneumoniae strain. The results stress the potential for the acquisition by S. pneumoniae of high-level beta-lactam resistance by interspecies gene transfer.
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( 1998 ) Characterization of IS1515, a functional insertion sequence in Streptococcus pneumoniae. PMID : 9580131 : PMC : PMC107034 Abstract >>
We describe the characterization of a new insertion sequence, IS1515, identified in the genome of Streptococcus pneumoniae I41R, an unencapsulated mutant isolated many years ago (R. Austrian, H. P. Bernheimer, E. E. B. Smith, and G. T. Mills, J. Exp. Med. 110:585-602, 1959). A copy of this element located in the cap1EI41R gene was sequenced. The 871-bp-long IS1515 element possesses 12-bp perfect inverted repeats and generates a 3-bp target duplication upon insertion. The IS encodes a protein of 271 amino acid residues similar to the putative transposases of other insertion sequences, namely IS1381 from S. pneumoniae, ISL2 from Lactobacillus helveticus, IS702 from the cyanobacterium Calothrix sp. strain PCC 7601, and IS112 from Streptomyces albus G. IS1515 appears to be present in the genome of most type 1 pneumococci in a maximum of 13 copies, although it has also been found in the chromosome of pneumococcal isolates belonging to other serotypes. We have found that the unencapsulated phenotype of strain 141R is the result of both the presence of an IS1515 copy and a frameshift mutation in the cap1EI41R gene. Precise excision of the IS was observed in the type 1 encapsulated transformants isolated in experiments designed to repair the frameshift. These results reveal that IS1515 behaves quite differently from other previously described pneumococcal insertion sequences. Several copies of IS1515 were also able to excise and move to another locations in the chromosome of S. pneumoniae. To our knowledge, this is the first report of a functional IS in pneumococcus.
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( 1998 ) Competence-specific induction of recA is required for full recombination proficiency during transformation in Streptococcus pneumoniae. PMID : 9466264 : DOI : 10.1046/j.1365-2958.1998.00668.x Abstract >>
Transcriptional activation of the recA gene of Streptococcus pneumoniae was previously shown to occur at competence. A 5.7 kb recA-specific transcript that contained at least two additional genes, cinA and dinF, was identified. We now report the complete characterization of the recA operon and investigation of the role of the competence-specific induction of recA. The 5.7 kb competence-specific recA transcript is shown to include lytA, which encodes the pneumococcal autolysin, a protein previously shown to contribute to virulence of S. pneumoniae. Uncoupling (denoted Ind-) of recA and/or the downstream genes was achieved through the placement of transcription terminators within the operon, either upstream or downstream of recA. Prevention of the competence-specific induction of recA severely affected spontaneous transformation. Transformation efficiencies of recA+ (Ind-) and of wild-type cells were compared under various conditions and with different donor DNA. Chromosomal transformation was reduced 17-(chromosomal donor) to 45-fold (recombinant plasmid donor), depending on the donor DNA, and plasmid establishment was reduced 129-fold. Measurement of uptake of radioactively labelled donor DNA in transformed cells in parallel with scoring for transformants (chromosomal donor) revealed normal uptake, but a 21-fold reduction in recombination in a recA+ (Ind-) strain, indicating that the transformation defect was primarily in recombination. Strikingly enough, a much larger (460-fold) reduction in recombination was observed for the shortest homologous donor fragment used (878 nucleotides long). Possible interpretations of the observation that basal RecA appears unable to promote efficient recombination whatever the number and the length of donor fragments taken up are proposed. The role of recA induction is discussed in view of the potential contribution of transformation to genome plasticity in this pathogen.
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425. |
( 1998 ) Isolation and characterization of three Streptococcus pneumoniae transformation-specific loci by use of a lacZ reporter insertion vector. PMID : 9573156 : PMC : PMC107223 Abstract >>
Although more than a dozen new proteins are produced when Streptococcus pneumoniae cells become competent for genetic transformation, only a few of the corresponding genes have been identified to date. To find genes responsible for the production of competence-specific proteins, a random lacZ transcriptional fusion library was constructed in S. pneumoniae by using the insertional lacZ reporter vector pEVP3. Screening the library for clones with competence-specific beta-galactosidase (beta-Gal) production yielded three insertion mutants with induced beta-Gal levels of about 4, 10, and 40 Miller units. In all three clones, activation of the lacZ reporter correlated with competence and depended on competence-stimulating peptide. Chromosomal loci adjacent to the integrated vector were subcloned from the insertion mutants, and their nucleotide sequences were determined. Genes at two of the loci exhibited strong similarity to parts of Bacillus subtilis com operons. One locus contained open reading frames (ORFs) homologous to the comEA and comEC genes in B. subtilis but lacked a comEB homolog. A second locus contained four ORFs with homology to the B. subtilis comG gene ORFs 1 to 4, but comG gene ORFs 5 to 7 were replaced in S. pneumoniae with an ORF encoding a protein homologous to transport ATP-binding proteins. Genes at all three loci were confirmed to be required for transformation by mutagenesis using pEVP3 for insertion duplications or an erm cassette for gene disruptions.
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( 1997 ) Competence and virulence of Streptococcus pneumoniae: Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases. PMID : 9379902 : DOI : 10.1046/j.1365-2958.1997.5111879.x Abstract >>
The adcCBA putative operon of Streptococcus pneumoniae, an important human pathogen, was identified in a search for transformation-deficient mutants. It was found to exhibit homology to ATP-binding cassette (ABC) transport operons encoding streptococcal adhesins such as FimA of Streptococcus parasanguis and PsaA of S. pneumoniae. The latter was recently shown to be essential for virulence as judged by intranasal or intraperitoneal challenge of mice. We suggested previously that AdcA, together with a set of 14 proteins, including PsaA and homologous adhesins, defines a new family of external solute-binding proteins specific for metals. In this work, Northern analysis revealed the existence of two adcB-adcA specific transcripts originating within adcC or further upstream, consistent with the hypothesis that adc is an operon. Investigation of growth of adc and psaA mutants in synthetic medium revealed that the addition of Zn improved the growth rate of the former, whereas the latter exhibited an absolute requirement for added Mn. A psaA-adc double mutant turned out to be essentially non-viable unless both metals were added in the appropriate ratio. Taken together, these results suggest a previously undocumented requirement of S. pneumoniae for Zn and Mn. The addition of Zn also restored near-normal spontaneous transformation of adc mutant cells in standard transformation medium. Zn was found to be specifically required soon after contact of cells with the competence-stimulating peptide, revealing an unsuspected need for Zn in transformation of S. pneumoniae. The removal of Mn from standard transformation medium also resulted in transformation deficiency of psaA mutant cells. Taken together, these results lead us to propose that Adc is an ABC-type Zn permease, the first such protein complex identified in any organism, and that Psa is an ABC-type Mn permease complex.
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