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1. Escobar-Páramo  P, Giudicelli  C, Parsot  C, Denamur  E,     ( 2003 )

The evolutionary history of Shigella and enteroinvasive Escherichia coli revised.

Journal of molecular evolution 57 (2)
PMID : 14562958  :   DOI  :   10.1007/s00239-003-2460-3    
Abstract >>
In Shigella and enteroinvasive Escherichia coli (EIEC), the etiologic agents of shigellosis in humans, the determinants responsible for entry of bacteria into and dissemination within epithelial cells are encoded by a virulence plasmid. To understand the evolution of the association between the virulence plasmid and the chromosome, we performed a phylogenetic analysis using the sequences of four chromosomal genes (trpA, trpB, pabB, and putP) and three virulence plasmid genes (ipaB, ipaD, and icsA) of a collection of 51 Shigella and EIEC strains. The phylogenetic tree derived from chromosomal genes showed a typical "star" phylogeny, indicating a fast diversification of Shigella and EIEC groups. Phylogenetic groups obtained from the chromosomal and plasmidic genes were similar, suggesting that the virulence plasmid and the chromosome share similar evolutionary histories. The few incongruences between the trees could be attributed to exchanges of fragments of different plasmids and not to the transfer of an entire plasmid. This indicates that the virulence plasmid was not transferred between the different Shigella and EIEC groups. These data support a model of evolution in which the acquisition of the virulence plasmid in an ancestral E. coli strain preceded the diversification by radiation of all Shigella and EIEC groups, which led to highly diversified but highly specialized pathogenic groups.
KeywordMeSH Terms
Evolution, Molecular
Phylogeny
2. Lan  R, Stevenson  G, Reeves  PR,     ( 2003 )

Comparison of two major forms of the Shigella virulence plasmid pINV: positive selection is a major force driving the divergence.

Infection and immunity 71 (11)
PMID : 14573649  :   DOI  :   10.1128/iai.71.11.6298-6306.2003     PMC  :   PMC219609    
Abstract >>
All Shigella and enteroinvasive Escherichia coli (EIEC) strains carry a 230-kb virulence plasmid (pINV) which is essential for their invasiveness. There are two sequence forms, pINV A and pINV B, of the plasmid (R. Lan, B. Lumb, D. Ryan, and P. R. Reeves, Infect. Immun. 69:6303-6309, 2001), and the recently sequenced pINV plasmid from Shigella flexneri serotype 5 is a pINV B form. In this study we sequenced the majority of the coding region of the pINV A form from S. flexneri serotype 6 other than insertion sequence or related sequences and compared it with the pINV B form. More than half of the genes sequenced appear to be under positive selection based on their low ratio of synonymous to nonsynonymous substitutions. This high proportion of selected differences indicates that the two pINV forms have functional differences, and comparative studies of pathogenicity in different Shigella-EIEC strains could be informative. There are also genes absent in the S. flexneri serotype 6 plasmid, including the sepA gene encoding serine protease, the major secreted protein of S. flexneri serotype 2a, and the stbAB genes, which encode one of the two partition systems found in S. flexneri serotype 5. The incompatibility of the two pINV forms appears to be due to either small differences in the mvpAT postsegregational killing system or the presence of an unknown system in pINVA.
KeywordMeSH Terms
Plasmids
3. Brandon  LD, Goehring  N, Janakiraman  A, Yan  AW, Wu  T, Beckwith  J, Goldberg  MB,     ( 2003 )

IcsA, a polarly localized autotransporter with an atypical signal peptide, uses the Sec apparatus for secretion, although the Sec apparatus is circumferentially distributed.

Molecular microbiology 50 (1)
PMID : 14507362  :   DOI  :   10.1046/j.1365-2958.2003.03674.x    
Abstract >>
Asymmetric localization of proteins is essential to many biological functions of bacteria. Shigella IcsA, an outer membrane protein, is localized to the old pole of the bacillus, where it mediates assembly of a polarized actin tail during infection of mammalian cells. Actin tail assembly provides the propulsive force for intracellular movement and intercellular dissemination. Localization of IcsA to the pole is independent of the amino-terminal signal peptide (Charles, M., Perez, M., Kobil, J.H., and Goldberg, M.B., 2001, Proc Natl Acad Sci USA 98: 9871-9876) suggesting that IcsA targeting occurs in the bacterial cytoplasm and that its secretion across the cytoplasmic membrane occurs only at the pole. Here, we characterize the mechanism by which IcsA is secreted across the cytoplasmic membrane. We present evidence that IcsA requires the SecA ATPase and the SecYEG membrane channel (translocon) for secretion. Our data suggest that YidC is not required for IcsA secretion. Furthermore, we show that polar localization of IcsA is independent of SecA. Finally, we demonstrate that while IcsA requires the SecYEG translocon for secretion, components of this apparatus are uniformly distributed within the membrane. Based on these data, we propose a model for coordinate polar targeting and secretion of IcsA at the bacterial pole.
KeywordMeSH Terms
4. Casalino  M, Latella  MC, Prosseda  G, Colonna  B,     ( 2003 )

CadC is the preferential target of a convergent evolution driving enteroinvasive Escherichia coli toward a lysine decarboxylase-defective phenotype.

Infection and immunity 71 (10)
PMID : 14500464  :   DOI  :   10.1128/iai.71.10.5472-5479.2003     PMC  :   PMC201042    
Abstract >>
Enteroinvasive E. coli (EIEC), like Shigella, is the etiological agent of bacillary dysentery, a particularly severe syndrome in children in developing countries. All EIEC strains share with Shigella the inability to synthesize lysine decarboxylase (the LDC phenotype). The lack of this function is considered a pathoadaptive mutation whose emergence was necessary to obtain the full expression of invasiveness. Cadaverine, the product of lysine decarboxylation, is a small polyamine which interferes mainly with the inflammatory process induced by dysenteric bacteria. Genes coding for lysine decarboxylase and its transporter constitute a single operon (cadBA) and are expressed at low pH under the positive control of CadC. This regulator is an inner membrane protein that is able to sense pH variation and to respond by transcriptionally activating the cadBA genes. In this study we show that, unlike in Shigella, mutations affecting the cad locus in the EIEC strains we have analyzed are not followed by a novel gene arrangement and that the LCD(-) phenotype is dependent mainly on inactivation of the cadC gene. Introduction of a functional CadC restores cadaverine expression in all EIEC strains harboring either an IS2 element or a defective cadC promoter. Comparative analysis between the cad regions of S. flexneri and EIEC suggests that the LDC(-) phenotype has been attained by different strategies within the E. coli species.
KeywordMeSH Terms
5. Truniger  V, Boos  W, Sweet  G,     ( 1992 )

Molecular analysis of the glpFKX regions of Escherichia coli and Shigella flexneri.

Journal of bacteriology 174 (21)
PMID : 1400248  :   DOI  :   10.1128/jb.174.21.6981-6991.1992     PMC  :   PMC207378    
Abstract >>
We have identified a new gene, glpX, belonging to the glp regulon of Escherichia coli, located directly downstream of the glpK gene. The transcription of glpX is inducible with glycerol and sn-glycerol-3-phosphate and is constitutive in a glpR mutant. glpX is the third gene in the glpFKX operon. The function of GlpX remains unknown. GlpX has an apparent molecular weight of 40,000 on sodium dodecyl sulfate-polyacrylamide gels. In addition to determining the E. coli glpX sequence, we also sequenced the corresponding glpFKX region originating from Shigella flexneri, which after transfer into E. coli was instrumental in elucidating the function of glpF in glycerol transport (D. P. Richey and E. C. C. Lin, J. Bacteriol. 112:784-790, 1972). Sequencing of the glpFKX region of this hybrid strain revealed an amber mutation instead of the tryptophan 215 codon in glpF. The most striking difference between the E. coli and S. flexneri DNA was found directly behind glpK, where two repetitive (REP) sequences were present in S. flexneri, but not in the E. coli sequence. The presence or absence of these REP sequences had no effect on transport or on growth on glycerol. Not including the REP sequence-containing region, only 1.1% of a total of 2,167 bp sequenced was different in the two sequences. Comparison of the sequence with those in the EMBL data library revealed a 99% identity between the last third of glpX and the first part of a gene called mvrA. We show that the cloned mvrA gene (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988) originated from the 88-min region of the Escherichia coli chromosome and not, as reported, from the 7-min region and that the gene product identified as MvrA is in fact encoded by a gene distal to glpX.
KeywordMeSH Terms
Aquaporins
Bacterial Proteins
Escherichia coli Proteins
Ferredoxin-NADP Reductase
Fructose-Bisphosphatase
6. Nakata  N, Sasakawa  C, Okada  N, Tobe  T, Fukuda  I, Suzuki  T, Komatsu  K, Yoshikawa  M,     ( 1992 )

Identification and characterization of virK, a virulence-associated large plasmid gene essential for intercellular spreading of Shigella flexneri.

Molecular microbiology 6 (16)
PMID : 1406277  :   DOI  :   10.1111/j.1365-2958.1992.tb01413.x    
Abstract >>
Seven virulence loci have been identified by Tn5 insertion mutagenesis on the large 230 kb plasmid (pMYSH6000) of Shigella flexneri 2a. In this study, we used Tn10 insertion mutagenesis and identified a novel virulence locus on pMYSH6000 responsible for bacterial spread. Characterization of the invading bacteria of the Tn10 insertion mutants in the epithelial cells revealed that the bacteria were capable of at least some intracellular spreading but not intercellular spreading. Immunoblot analysis of lysates of the Tn10 insertion mutants with a VirG-specific antipeptide antibody revealed diminished levels of the 116 kDa VirG protein. The virG mRNA in the mutants, however, was expressed at the same level as that in the wild type. The DNA region required for the virulence phenotype was localized to a 1.6 kb DNA sequence in the SalI-K fragment on the plasmid, and thus the locus was designated virK. Expression of virK in Escherichia coli using a T7 RNA polymerase-dependent promoter system yielded a 36 kDa protein. The nucleotide sequence of 1642 bp encoding VirK function was determined, and an open reading frame encoding 316 amino acid residues was shown to encode the VirK protein. The virK region was highly conserved among the large virulence plasmids of shigellae and enteroinvasive Escherichia coli. These results suggest that VirK function is an essential virulence determinant for shigellae involved in the expression of virG gene product at post-transcriptional level.
KeywordMeSH Terms
Plasmids
7. Hromockyj  AE, Tucker  SC, Maurelli  AT,     ( 1992 )

Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1(Tyr)).

Molecular microbiology 6 (15)
PMID : 1406252  :   DOI  :   10.1111/j.1365-2958.1992.tb01385.x    
Abstract >>
virR is the central regulatory locus required for coordinate temperature-regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI-AccI DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique HpaI restriction site resulted in a loss of VirR+ activity. The virR ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone-like protein, H-NS. Based on the predicted amino acid sequence of E. coli H-NS, only a single conservative base-pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNA(Tyr). These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genetic Complementation Test
Temperature
Virulence Factors
8. Tobe  T, Sasakawa  C, Okada  N, Honma  Y, Yoshikawa  M,     ( 1992 )

vacB, a novel chromosomal gene required for expression of virulence genes on the large plasmid of Shigella flexneri.

Journal of bacteriology 174 (20)
PMID : 1400189  :   DOI  :   10.1128/jb.174.20.6359-6367.1992     PMC  :   PMC207582    
Abstract >>
Shigellae, the causative agents of bacillary dysentery, are capable of adhering to and invading epithelial cells and spreading into adjacent cells. A chromosomal mutant of Shigella flexneri 2a YSH6000 with reduced invasive capacity was isolated by Tn5 insertion mutagenesis. The linkage of the mutant phenotype to the Tn5 insertion was determined by P1 phage transduction. The site of the Tn5 insertion was assigned to a NotI chromosomal restriction map, confirming that the virulence-associated locus, designated vacB, is a new locus on the chromosome. In the vacB mutant, production of the four plasmid-encoded virulence antigens, IpaB, -C, and -D and VirG, decreased to a low level compared with that in the wild type. In contrast, levels of transcription of the operons for virG, ipa, region-3.4, region-5, virF, and virB on the large plasmid, as determined by Northern dot blotting, were unaffected in the vacB mutant. Furthermore, transcriptional activation of the ipa operon by exploiting a tac promoter could not restore the vacB mutant to production of the same levels of the IpaB, -C, and -D proteins as those in the wild type, indicating that the vacB locus is involved in expression of the vir genes on the large plasmid at the posttranscriptional level. Cloning followed by nucleotide sequencing of the vacB region showed it to contain a 2,280-bp open reading frame encoding an 86.9-kDa protein located 669 bp downstream from the 3' end of the open reading frame for the purA gene. Disruption of the vacB gene of other serotypes of Shigella spp. and enteroinvasive Escherichia coli (EIEC) resulted in reduced expression of virulence phenotypes, indicating that the vacB gene encodes a novel type of virulence-associated gene required for the full expression of the virulence phenotype of Shigella spp. and EIEC.
KeywordMeSH Terms
Escherichia coli Proteins
Exoribonucleases
9. Hamlett  NV, Landale  EC, Davis  BH, Summers  AO,     ( 1992 )

Roles of the Tn21 merT, merP, and merC gene products in mercury resistance and mercury binding.

Journal of bacteriology 174 (20)
PMID : 1328156  :   DOI  :   10.1128/jb.174.20.6377-6385.1992     PMC  :   PMC207586    
Abstract >>
The mercury resistance (mer) operon of the gram-negative transposon Tn21 encodes not only a mercuric reductase and regulatory genes but also two inner membrane proteins (MerT and MerC) and a periplasmic protein (MerP). Although the merT, merP, and merC genes have been implicated in Hg(II) transport, the individual roles of these genes have not been established. We created in vitro precise deletion and frameshift mutations that eliminated each of the genes singly and in combination. Our results show that both merT and merP are required for Hg(II) binding but that merC is not. Both merT and merP are required for full expression of Hg(II) resistance, but loss of merP is less deleterious than loss of merT. Furthermore, mutations eliminating both merT and merP decrease resistance more than the single mutations do. In contrast, mutating merC had no effect on Hg(II) resistance. Both the merT and merP mutations increase the threshold Hg(II) concentration for induction of merA-lacZ transcriptional fusions and cause an increase in the maximal expression level. In contrast, the merC mutation had little effect on the threshold inducing concentration of Hg(II) but decreased the level of expression. Our results show that merT and merP alone are sufficient to specify a mercury transport system. The role of merC remains obscure.
KeywordMeSH Terms
Cation Transport Proteins
10. Allaoui  A, Sansonetti  PJ, Parsot  C,     ( 1992 )

MxiJ, a lipoprotein involved in secretion of Shigella Ipa invasins, is homologous to YscJ, a secretion factor of the Yersinia Yop proteins.

Journal of bacteriology 174 (23)
PMID : 1332940  :   DOI  :   10.1128/jb.174.23.7661-7669.1992     PMC  :   PMC207479    
Abstract >>
Shigella flexneri causes bacillary dysentery by invading epithelial cells of the colonic mucosa. The invasion process requires the synthesis and secretion of the virulence plasmid-encoded Ipa proteins. Using TnphoA mutagenesis, we have identified two virulence plasmid genes, mxiJ and mxiM, that encode proteins exported by the general export pathway. Analysis of the MxiJ and MxiM deduced amino acid sequences suggested that mxiJ and mxiM might encode lipoproteins, which was confirmed by [3H]palmitate labeling of MxiJ:PhoA and MxiM:PhoA fusion proteins. A mxiJ mutant was unable to invade HeLa cells, to induce the formation of plaques on confluent monolayers of HeLa cells, and to provoke keratoconjunctivitis in guinea pigs. In addition, secretion of seven polypeptides, including IpaA, IpaB, and IpaC, was abolished in the mxiJ mutant. Sequence comparisons indicated that MxiJ and MxiH, which is encoded by a gene located upstream from mxiJ, are homologous to the Yersinia enterocolitica YscJ and YscF proteins, respectively.
KeywordMeSH Terms
Adhesins, Bacterial
Bacterial Outer Membrane Proteins
11. Venkatesan  MM, Buysse  JM, Oaks  EV,     ( 1992 )

Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus.

Journal of bacteriology 174 (6)
PMID : 1312536  :   DOI  :   10.1128/jb.174.6.1990-2001.1992     PMC  :   PMC205806    
Abstract >>
An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases.
KeywordMeSH Terms
12. Ogawa  M, Suzuki  T, Tatsuno  I, Abe  H, Sasakawa  C,     ( 2003 )

IcsB, secreted via the type III secretion system, is chaperoned by IpgA and required at the post-invasion stage of Shigella pathogenicity.

Molecular microbiology 48 (4)
PMID : 12753186  :   DOI  :   10.1046/j.1365-2958.2003.03489.x    
Abstract >>
Shigella deliver a subset of effector proteins such as IpaA, IpaB and IpaC via the type III secretion system (TTSS) into host cells during the infection of colonic epithelial cells. Many bacterial effectors including some from Shigella require specific chaperones for protection from degradation and targeting to the TTSS. In this study, we have investigated the role of the icsB gene located upstream of the ipaBCDA operon in Shigella infection because the role of IcsB as a virulence factor remains unknown. Here, we found that the IcsB protein is secreted via the TTSS of Shigella in vitro and in vivo. We show that IpgA protein encoded by ipgA, the gene immediately downstream of icsB, serves as the chaperone required for the stabilization and secretion of IcsB. We have shown that IcsB binds to IpgA in bacterial cytosol and the binding site is in the middle of the IcsB protein. Intriguingly, although its significance in Shigella pathogenicity is as yet unclear, the icsB gene can be read-through into the ipgA gene to create a translational fusion protein. Furthermore, the contribution of IcsB to the pathogenicity of Shigella was demonstrated by plaque-forming assay and the Sereny test. The ability of the icsB mutant to form plaques was greatly reduced compared with that of the wild type in MDCK cell monolayers. Furthermore, when guinea pig eyes were infected with a non-polar icsB mutant, the bacteria failed to provoke keratoconjunctivitis. These results suggest that IcsB is secreted via the TTSS, chaperoned by IpgA, and required at the post-invasion stage of Shigella pathogenicity
KeywordMeSH Terms
Shigella
13. Wei  J, Goldberg  MB, Burland  V, Venkatesan  MM, Deng  W, Fournier  G, Mayhew  GF, Plunkett  G, Rose  DJ, Darling  A, Mau  B, Perna  NT, Payne  SM, Runyen-Janecky  LJ, Zhou  S, Schwartz  DC, Blattner  FR,     ( 2003 )

Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T.

Infection and immunity 71 (5)
PMID : 12704152  :   DOI  :   10.1128/iai.71.5.2775-2786.2003     PMC  :   PMC153260    
Abstract >>
We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organism's distinctive lifestyle that have yet to be explained.
KeywordMeSH Terms
Genome, Bacterial
Genomics
14. Runyen-Janecky  LJ, Reeves  SA, Gonzales  EG, Payne  SM,     ( 2003 )

Contribution of the Shigella flexneri Sit, Iuc, and Feo iron acquisition systems to iron acquisition in vitro and in cultured cells.

Infection and immunity 71 (4)
PMID : 12654809  :   DOI  :   10.1128/iai.71.4.1919-1928.2003     PMC  :   PMC152062    
Abstract >>
Shigella flexneri possesses multiple iron acquisition systems, including proteins involved in the synthesis and uptake of siderophores and the Feo system for ferrous iron utilization. We identified an additional S. flexneri putative iron transport gene, sitA, in a screen for S. flexneri genes that are induced in the eukaryotic intracellular environment. sitA was present in all Shigella species and in most enteroinvasive Escherichia coli strains but not in any other E. coli isolates tested. The sit locus consists of four genes encoding a potential ABC transport system. The deduced amino acid sequence of the S. flexneri sit locus was homologous to the Salmonella enterica serovar Typhimurium Sit and Yersinia pestis Yfe systems, which mediate both manganese and iron transport. The S. flexneri sit promoter was repressed by either iron or manganese, and the iron repression was partially dependent upon Fur. A sitA::cam mutation was constructed in S. flexneri. The sitA mutant showed reduced growth, relative to the wild type, in Luria broth containing an iron chelator but formed wild-type plaques on Henle cell monolayers, indicating that the sitA mutant was able to acquire iron and/or manganese in the host cell. However, mutants defective in two of these iron acquisition systems (sitA iucD, sitA feoB, and feoB iucD) formed slightly smaller plaques on Henle cell monolayers. A strain carrying mutations in sitA, feoB, and iucD did not form plaques on Henle cell monolayers.
KeywordMeSH Terms
15. Hartman  AB, Essiet  II, Isenbarger  DW, Lindler  LE,     ( 2003 )

Epidemiology of tetracycline resistance determinants in Shigella spp. and enteroinvasive Escherichia coli: characterization and dissemination of tet(A)-1.

Journal of clinical microbiology 41 (3)
PMID : 12624025  :   DOI  :   10.1128/jcm.41.3.1023-1032.2003     PMC  :   PMC150258    
Abstract >>
To make a comprehensive study of tetracycline resistance determinant distribution in the genus Shigella, a collection of 577 clinical isolates of Shigella spp. and enteroinvasive Escherichia coli (EIEC) from a variety of geographical locations was screened to identify tetracycline-resistant strains. The 459 tetracycline-resistant isolates identified were then screened by PCR analysis to determine the distribution in these strains of tetracycline efflux resistance determinants belonging to classes A to E, G, and H that have been identified in gram-negative bacteria. Only classes A to D were represented in these strains. Although Tet B was the predominant determinant in all geographical locations, there were geographical and species differences in the distribution of resistance determinants. An allele of tet(A), designated tet(A)-1, was identified and sequenced, and the 8.6-kb plasmid containing determinant Tet A-1, designated pSSTA-1, was found to have homologies to portions of a Salmonella enterica cryptic plasmid and the broad-host-range resistance plasmid RSF1010. This allele and pSSTA-1 were used as epidemiological markers to monitor clonal and horizontal transmission of determinant Tet A-1. An analysis of serotype, distribution of tetracycline resistance determinants, and resistance profiles indicated that both clonal spread and horizontal transfer had contributed to the spread of specific tetracycline resistance determinants in these populations and demonstrated the use of these parameters as an epidemiological tool to follow the transmission of determinants and strains.
KeywordMeSH Terms
16. Waterman  SR, Small  PL,     ( 2003 )

The glutamate-dependent acid resistance system of Escherichia coli and Shigella flexneri is inhibited in vitro by L-trans-pyrrolidine-2,4-dicarboxylic acid.

FEMS microbiology letters 224 (1)
PMID : 12855178  :   DOI  :   10.1016/S0378-1097(03)00427-0    
Abstract >>
Strains of Escherichia coli K-12, O157:H7, and Shigella flexneri grown to stationary phase in complex unbuffered media can survive for several hours at pH 2.5. This stationary-phase acid resistance phenotype is dependent upon the alternate sigma factor sigmas and the supplementation of either glutamate or glutamine in the acidified media used for acid challenge. Acid resistance under these defined conditions can be inhibited by the glutamate analog L-trans-pyrrolidine-2,4-dicarboxylic acid which blocks uptake of glutamate/glutamine by selective inhibition. The gadC gene, encoding an inner membrane antiporter essential for the expression of acid resistance, could not be detected in other family members of the Enterobacteriacae.
KeywordMeSH Terms
Bacterial Proteins
Escherichia coli Proteins
17. Partridge  SR, Hall  RM,     ( 2003 )

In34, a complex In5 family class 1 integron containing orf513 and dfrA10.

Antimicrobial agents and chemotherapy 47 (1)
PMID : 12499211  :   DOI  :   10.1128/aac.47.1.342-349.2003     PMC  :   PMC149023    
Abstract >>
A complex class 1 integron, In34, found in a conjugative plasmid from a multidrug-resistant Klebsiella pneumoniae strain isolated in 1997 at a hospital in Sydney, Australia, was shown to have a backbone related to that of In2, which belongs to the In5 family. In In34, the aadB gene cassette replaces the aadA1a cassette in In2, and two additional resistance genes, dfrA10 and aphA1, that are not part of a gene cassette are present. The aphA1 gene is in a Tn4352-like transposon that is located in the tniA gene. The dfrA10 gene lies adjacent to a 2,154-bp DNA segment, known as the common region, that contains an open reading frame predicting a product of 513 amino acids (Orf513). Orf513 is 66 and 55% identical to the products of two further open reading frames that, like the common region, are found adjacent to antibiotic resistance genes. A 27-bp conserved sequence was found at one end of each type of common region. The loss of dfrA10 due to homologous recombination between flanking direct repeats and incorporation of the excised circle by homologous recombination were demonstrated. Part of In34 is identical to the sequenced portion of In7, which is from a multidrug-resistant Escherichia coli strain that had been isolated 19 years earlier in the same hospital. In34 and In7 are in plasmids that contain the same six resistance genes conferring resistance to ampicillin, chloramphenicol, gentamicin, kanamycin, neomycin, tobramycin, trimethoprim, and sulfonamides, but the plasmid backbones appear to be unrelated, suggesting that translocation of a multiple-drug-resistance-determining region as well as horizontal transfer may have occurred.
KeywordMeSH Terms
18. Mavris  M, Sansonetti  PJ, Parsot  C,     ( 2002 )

Identification of the cis-acting site involved in activation of promoters regulated by activity of the type III secretion apparatus in Shigella flexneri.

Journal of bacteriology 184 (24)
PMID : 12446624  :   DOI  :   10.1128/jb.184.24.6751-6759.2002     PMC  :   PMC135465    
Abstract >>
Bacteria of Shigella spp. use a virulence plasmid-encoded type III secretion (TTS) system to invade the colonic epithelium in humans. The activity of the TTS apparatus is tightly regulated in the wild-type strain and is induced upon contact of bacteria with epithelial cells, whereas it is deregulated, i.e., constitutively active, in some mutants. Under conditions of deregulated secretion, approximately 20 proteins are secreted, including VirA, OspB to OspG, and at least three members of the IpaH family, all of which are encoded by the virulence plasmid. Conditions inducing or deregulating the activity of secretion also induce the transcription of virA and four ipaH genes. The transcription of virA and ipaH9.8 requires both MxiE, a transcriptional activator of the AraC family, and IpgC, the chaperone of IpaB and IpaC, acting as a coactivator. Using reporter plasmids containing lacZ transcriptional fusions, we showed that the ipaH7.8. ipa4.5. ospC1, and ospF promoters are activated under conditions of deregulated secretion and that both MxiE and IpgC are necessary and sufficient for their activation in both Shigella flexneri and Escherichia coli. Promoter mapping and deletion analysis of the ipaH9.8. virA, and ospC1 promoters identified a 17-bp motif, the MxiE box, which overlaps the -35 region and is essential for the activation of these promoters. The presence of eight MxiE boxes on the virulence plasmid suggests that 11 genes encoding secreted proteins may be regulated by the activity of secretion. We also present evidence that at least one ipaH gene that is carried by the chromosome is controlled by MxiE and IpgC.
KeywordMeSH Terms
Antigens, Bacterial
DNA-Binding Proteins
Gene Expression Regulation, Bacterial
Promoter Regions, Genetic
Transcription Factors
19. Jin  Q, Yuan  Z, Xu  J, Wang  Y, Shen  Y, Lu  W, Wang  J, Liu  H, Yang  J, Yang  F, Zhang  X, Zhang  J, Yang  G, Wu  H, Qu  D, Dong  J, Sun  L, Xue  Y, Zhao  A, Gao  Y, Zhu  J, Kan  B, Ding  K, Chen  S, Cheng  H, Yao  Z, He  B, Chen  R, Ma  D, Qiang  B, Wen  Y, Hou  Y, Yu  J,     ( 2002 )

Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157.

Nucleic acids research 30 (20)
PMID : 12384590  :   DOI  :   10.1093/nar/gkf566     PMC  :   PMC137130    
Abstract >>
We have sequenced the genome of Shigella flexneri serotype 2a, the most prevalent species and serotype that causes bacillary dysentery or shigellosis in man. The whole genome is composed of a 4 607 203 bp chromosome and a 221 618 bp virulence plasmid, designated pCP301. While the plasmid shows minor divergence from that sequenced in serotype 5a, striking characteristics of the chromosome have been revealed. The S.flexneri chromosome has, astonishingly, 314 IS elements, more than 7-fold over those possessed by its close relatives, the non-pathogenic K12 strain and enterohemorrhagic O157:H7 strain of Escherichia coli. There are 13 translocations and inversions compared with the E.coli sequences, all involve a segment larger than 5 kb, and most are associated with deletions or acquired DNA sequences, of which several are likely to be bacteriophage-transmitted pathogenicity islands. Furthermore, S.flexneri, resembling another human-restricted enteric pathogen, Salmonella typhi, also has hundreds of pseudogenes compared with the E.coli strains. All of these could be subjected to investigations towards novel preventative and treatment strategies against shigellosis.
KeywordMeSH Terms
Genome, Bacterial
20. Niebuhr  K, Giuriato  S, Pedron  T, Philpott  DJ, Gaits  F, Sable  J, Sheetz  MP, Parsot  C, Sansonetti  PJ, Payrastre  B,     ( 2002 )

Conversion of PtdIns(4,5)P(2) into PtdIns(5)P by the S.flexneri effector IpgD reorganizes host cell morphology.

The EMBO journal 21 (19)
PMID : 12356723  :   DOI  :   10.1093/emboj/cdf522     PMC  :   PMC129044    
Abstract >>
Phosphoinositides play a central role in the control of several cellular events including actin cytoskeleton organization. Here we show that, upon infection of epithelial cells with the Gram-negative pathogen Shigella flexneri, the virulence factor IpgD is translocated directly into eukaryotic cells and acts as a potent inositol 4-phosphatase that specifically dephosphorylates phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] into phosphatidylinositol 5-monophosphate [PtdIns(5)P] that then accumulates. Transfection experiments indicate that the transformation of PtdIns(4,5)P(2) into PtdIns(5)P by IpgD is responsible for dramatic morphological changes of the host cell, leading to a decrease in membrane tether force associated with membrane blebbing and actin filament remodelling. These data provide the molecular basis for a new mechanism employed by a pathogenic bacterium to promote membrane ruffling at the entry site.
KeywordMeSH Terms
21. Kane  CD, Schuch  R, Day  WA, Maurelli  AT,     ( 2002 )

MxiE regulates intracellular expression of factors secreted by the Shigella flexneri 2a type III secretion system.

Journal of bacteriology 184 (16)
PMID : 12142411  :   DOI  :   10.1128/jb.184.16.4409-4419.2002     PMC  :   PMC135254    
Abstract >>
The mxi-spa locus on the virulence plasmid of Shigella flexneri encodes components of the type III secretion system. mxiE, a gene within this locus, encodes a protein that is homologous to the AraC/XylS family of transcriptional regulators, but currently its role in pathogenesis remains undefined. We characterized the virulence phenotype of a nonpolar mxiE mutant and found that this mutant retained the ability to invade mammalian cells in tissue culture and secrete Ipas (type III effectors required for host cell invasion), although it was less efficient than wild-type Shigella at cell-to-cell spread. Despite its invasive properties in culture, the mxiE mutant was completely avirulent in an animal model. Potential targets for MxiE activation were identified by using promoter-green fluorescent protein fusions, and gene expression was examined under various growth conditions. Six MxiE-regulated genes were discovered: ospB, ospC1, ospE2, ospF, virA, and ipaH(9.8). Notably, activation of these genes only occurred within the intracellular environment of the host and not during growth at 37 degrees C in liquid culture. Interestingly, all of the MxiE-regulated proteins previously have been shown to be secreted through the type III secretion system and are putative virulence factors. Our findings suggest that some of these Osp proteins may be involved in postinvasion events related to virulence. Since bacterial pathogens adapt to multiple environments during the course of infecting a host, we propose that Shigella evolved a mechanism to take advantage of a unique intracellular cue, which is mediated through MxiE, to express proteins when the organism reaches the eukaryotic cytosol.
KeywordMeSH Terms
Antigens, Bacterial
DNA-Binding Proteins
Lipoproteins
Transcription Factors
Virulence Factors
22. Fukushima  M, Kakinuma  K, Kawaguchi  R,     ( 2002 )

Phylogenetic analysis of Salmonella, Shigella, and Escherichia coli strains on the basis of the gyrB gene sequence.

Journal of clinical microbiology 40 (8)
PMID : 12149329  :   DOI  :   10.1128/jcm.40.8.2779-2785.2002     PMC  :   PMC120687    
Abstract >>
Phylogenetic analysis of about 200 strains of Salmonella, Shigella, and Escherichia coli was carried out using the nucleotide sequence of the gene for DNA gyrase B (gyrB), which was determined by directly sequencing PCR fragments. The results establish a new phylogenetic tree for the classification of Salmonella, Shigella, and Escherichia coli in which Salmonella forms a cluster separate from but closely related to Shigella and E. coli. In comparison with 16S rRNA analysis, the gyrB sequences indicated a greater evolutionary divergence for the bacteria. Thus, in screening for the presence of bacteria, the gyrB gene might be a useful tool for differentiating between closely related species of bacteria such as Shigella spp. and E. coli. At present, 16S rRNA sequence analysis is an accurate and rapid method for identifying most unknown bacteria to the genus level because the highly conserved 16S rRNA region is easy to amplify; however, analysis of the more variable gyrB sequence region can identify unknown bacteria to the species level. In summary, we have shown that gyrB sequence analysis is a useful alternative to 16S rRNA analysis for constructing the phylogenetic relationships of bacteria, in particular for the classification of closely related bacterial species.
KeywordMeSH Terms
Phylogeny
23. Yoshida  S, Katayama  E, Kuwae  A, Mimuro  H, Suzuki  T, Sasakawa  C,     ( 2002 )

Shigella deliver an effector protein to trigger host microtubule destabilization, which promotes Rac1 activity and efficient bacterial internalization.

The EMBO journal 21 (12)
PMID : 12065406  :   DOI  :   10.1093/emboj/cdf319     PMC  :   PMC126072    
Abstract >>
Shigella deliver a subset of effectors into the host cell via the type III secretion system, that stimulate host cell signal pathways to modulate the actin dynamics required for invasion of epithelial cells. Here we show that one of the Shigella effectors, called VirA, can interact with tubulin to promote microtubule (MT) destabilization, and elicit protrusions of membrane ruffling. Under in vitro conditions, VirA inhibited polymerization of tubulin and stimulated MT destabilization. Upon microinjection of VirA into HeLa cells, a localized membrane ruffling was induced rapidly. Overexpression of VirA in host cells caused MT destruction and protruding membrane ruffles which were absent when VirA was co-expressed with a dominant-negative Rac1 mutant. Indeed, Shigella but not the virA mutant stimulated Rac1, including the formation of membrane ruffles in infected cells. Importantly, the MT structure beneath the protruding ruffling was destroyed. Furthermore, drug-induced MT growth in HeLa cells greatly enhanced the Shigella entry. These results indicate that VirA is a novel type of bacterial effector capable of inducing membrane ruffling through the stimulation of MT destabilization.
KeywordMeSH Terms
Virulence Factors
24. Wong  RS, Chow  AW,     ( 2002 )

Identification of enteric pathogens by heat shock protein 60 kDa (HSP60) gene sequences.

FEMS microbiology letters 206 (1)
PMID : 11786265  :   DOI  :   10.1111/j.1574-6968.2002.tb10994.x    
Abstract >>
A highly specific and reproducible approach for the simultaneous detection of enteric pathogenic bacteria was developed using bacterial hsp60 gene and molecular biological tools. A single pair of universal primers was derived from the highly conserved sequence of hsp60 genes encompassing a 600-bp hypervariable region. PCR amplification followed by either dot blot hybridization or restriction enzyme digestion performed on 38 enteric bacteria indicated that this approach could differentiate not only different genera such as Campylobacter, Yersinia and Vibrio, but also species that are closely related genetically, such as between C. jejuni and C. coli, or between Salmonella and Shigella or Escherichia coli.
KeywordMeSH Terms
Amino Acid Sequence
25. Sundin  GW,     ( 2002 )

Distinct recent lineages of the strA- strB streptomycin-resistance genes in clinical and environmental bacteria.

Current microbiology 45 (1)
PMID : 12029529  :   DOI  :   10.1007/s00284-001-0100-y    
Abstract >>
We report the linkage of the strA-strB streptomycin-resistance genes with Class 1 integron sequences on pSTR1, a 75-kb multiple antibiotic-resistance plasmid from Shigella flexneri. strA-strB had previously been detected only within Tn 5393, a Tn 3-family transposon, and on small nonconjugative broad-host-range plasmids such as RSF1010. The geographic range of Tn 5393 was also extended to Pseudomonas spp. isolated from apple trees in New Zealand and soil in the USA. Comparative sequence analyses indicated that strA-strB from Tn 5393 and nonconjugative plasmids constitute distinct recent lineages with strA-strB from pSTR1 intermediate between the other two. The carriage of strA-strB within an integron, a transposon, and on broad-host-range plasmids has facilitated the world-wide dissemination of this determinant among at least 21 bacterial genera.
KeywordMeSH Terms
Genes, Bacterial
26. Mavris  M, Page  AL, Tournebize  R, Demers  B, Sansonetti  P, Parsot  C,     ( 2002 )

Regulation of transcription by the activity of the Shigella flexneri type III secretion apparatus.

Molecular microbiology 43 (6)
PMID : 11971264  :   DOI  :   10.1046/j.1365-2958.2002.02836.x    
Abstract >>
The virulence plasmid-encoded type III secretion system of Shigella flexneri consists of the Mxi-Spa secretion apparatus, secreted proteins IpaA-D and IpgD involved in entry of bacteria into epithelial cells,cytoplasmic chaperones IpgC and IpgE and 15 other secreted proteins of unknown function, including VirA and members of the IpaH family. The activity of the Mxi-Spa apparatus is regulated by external signals, and transcription of virA and IpaH genes is specifically induced in conditions of active secretion. We present genetic evidence that regulation of these genes involves both MxiE, the transcriptional activator of the AraC family encoded by the mxi operon, and IpgC, the chaperone for IpaB and IpaC. We also show that together MxiE and IpgC are sufficient to activatevirA and IpaH 9.8 promoters in Escherichia coli. InS. flexneri, increasing the expression of IpgC led to a concomitant increase in IpaH production in conditions of non-secretion. This suggests that the activity of secretion is sensed by the presence of free IpgC, which acts as a coactivator to allow MxiE to activate transcription at its target promoters.
KeywordMeSH Terms
Antigens, Bacterial
DNA-Binding Proteins
Gene Expression Regulation, Bacterial
Transcription Factors
Transcription, Genetic
Virulence Factors
27. Yokoigawa  K, Hirasawa  R, Ueno  H, Okubo  Y, Umesako  S, Soda  K,     ( 2001 )

Gene cloning and characterization of alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei.

Biochemical and biophysical research communications 288 (3)
PMID : 11676496  :   DOI  :   10.1006/bbrc.2001.5817    
Abstract >>
Alanine racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei were cloned and expressed in Escherichia coli JM109. All genes encoded a polypeptide of 359 amino acids, and showed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two residues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homogeneity has an apparent molecular mass about 43,000 by SDS-gel electrophoresis, and about 46,000 by gel filtration. However, all enzymes showed an apparent molecular mass about 60,000 by gel filtration in the presence of a substrate, 0.1 M l-alanine. These results suggest that the Shigella alr enzymes having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymological properties, and showed lower catalytic activities (about 210 units/mg) than those of homodimeric alanine racemases reported.
KeywordMeSH Terms
28. Li  MS, Kroll  JS, Yu  J,     ( 2001 )

Influence of the yihE gene of Shigella flexneri on global gene expression: on analysis using DNA arrays.

Biochemical and biophysical research communications 288 (1)
PMID : 11594757  :   DOI  :   10.1006/bbrc.2001.5734    
Abstract >>
Inactivation of dsbA (disulfide bond formation), either by an insertion (Sh4, dsbA::kan) or by alteration of the active site (Sh42, dsbA33G), renders Shigella flexneri avirulent. However, Sh4 and Sh42 behave differently in many ways in vitro and in vivo. A gene of unknown function, yihE, up-stream and cotranscribed with dsbA, is thought to differentiate Sh4 and Sh42 as the kan insertion may result in a truncated unstable yihE-dsbA mRNA in Sh4. To gain insight into the function of yihE, DNA array hybridization was performed to study the genomic expression in Sh4, Sh42, and a newly constructed yihE mutant (Sh54). Compared to the wild-type, M90TS, Sh4, and Sh54 demonstrated significantly changed transcription levels of about 100 genes, of which many involved in energy metabolism and stress response were down- and up-regulated, respectively. In contrast, Sh42 showed altered transcription levels of only 20 genes. The results argue that yihE is principally responsible for the changed genomic expression in Sh4 and Sh54. Given the fact that the transcription of yihE-dsbA is regulated by the CpxRA two-component signal transduction system, yihE is probably involved in the extracytoplasmic stress response in a manor deserving further studies.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
29. Mogull  SA, Runyen-Janecky  LJ, Hong  M, Payne  SM,     ( 2001 )

dksA is required for intercellular spread of Shigella flexneri via an RpoS-independent mechanism.

Infection and immunity 69 (9)
PMID : 11500451  :   DOI  :   10.1128/iai.69.9.5742-5751.2001     PMC  :   PMC98691    
Abstract >>
Pathogenesis of Shigella flexneri is dependent on the ability of the bacterium to invade and spread within epithelial cells. In this study, we identified dksA as a gene necessary for intercellular spread in, but not invasion of, cultured cells. The S. flexneri dksA mutant exhibited sensitivity to acid and oxidative stress, in part due to an effect of DksA on production of RpoS. However, an S. flexneri rpoS mutant formed plaques on tissue culture monolayers, thus excluding DksA regulation of RpoS as the mechanism responsible for the inability of the dksA mutant to spread intercellularly. Intracellular analysis of the dksA mutant indicates that it survived and divided within the Henle cell cytoplasm, but the dksA mutant cells were elongated, and some exhibited filamentation in the intracellular environment. Some of the S. flexneri dksA mutant cells showed aberrant localization of virulence protein IcsA, which may inhibit spread between epithelial cells.
KeywordMeSH Terms
Escherichia coli Proteins
30. Charles  M, Pérez  M, Kobil  JH, Goldberg  MB,     ( 2001 )

Polar targeting of Shigella virulence factor IcsA in Enterobacteriacae and Vibrio.

Proceedings of the National Academy of Sciences of the United States of America 98 (17)
PMID : 11481451  :   DOI  :   10.1073/pnas.171310498     PMC  :   PMC55545    
Abstract >>
Asymmetric localization is key to the proper function of certain prokaryotic proteins important to virulence, chemotaxis, cell division, development, motility, and adhesion. Shigella IcsA is localized to the old pole of the bacterium, where it mediates assembly of an actin tail inside infected mammalian cells. IcsA (VirG) is essential to Shigella intracellular motility and virulence. We used translational fusions between portions of IcsA and the green fluorescent protein (GFP) to determine the regions of IcsA that are necessary and sufficient for its targeting to the bacterial old pole. An IcsA-GFP fusion that lacks a signal peptide localized to the old pole, indicating that signal peptide-mediated secretion is not required for polar localization. Two regions within IcsA were required for localization of an IcsA-GFP fusion to the old pole. Further characterization of these regions indicated that amino acids 1-104 and 507-620 were each independently sufficient for polar localization. Finally, when expressed in Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Vibrio cholerae, each of the two targeting regions localized to the pole, indicating that the mechanism of polar targeting used by IcsA is present generally among Enterobacteriacae and Vibrio.
KeywordMeSH Terms
31. Toyotome  T, Suzuki  T, Kuwae  A, Nonaka  T, Fukuda  H, Imajoh-Ohmi  S, Toyofuku  T, Hori  M, Sasakawa  C,     ( 2001 )

Shigella protein IpaH(9.8) is secreted from bacteria within mammalian cells and transported to the nucleus.

The Journal of biological chemistry 276 (34)
PMID : 11418613  :   DOI  :   10.1074/jbc.M101882200    
Abstract >>
Various pathogenic bacteria such as Shigella deliver effector proteins into mammalian cells via the type III secretion system. The delivered Shigella effectors have been shown to variously affect host functions required for efficient bacterial internalization into the cells. In the present study, we investigated the IpaH proteins for their ability to be secreted via the type III secretion system and their fate in mammalian cells. Upon incubation in a medium containing Congo red, the bacteria secrete IpaH into the medium, but secretion of IpaH occurs later than that of IpaBCD. Immunofluorescence microscopy indicated that IpaH(9.8) is secreted from intracellular bacteria and transported into the nucleus. On microinjection of the protein, intracellular IpaH(9.8) is accumulated at one place around the nucleus and transported into the nucleus. This movement seems to be dependent on the microtubule network, since nuclear accumulation of IpaH(9.8) is inhibited in cells treated with microtubule-destabilizing agents. In nuclear import assay, IpaH(9.8) was efficiently transported into the nucleus, which was completely blocked by treatment with wheat germ agglutinin. The nuclear transport of IpaH(9.8) does not depend on host cytosolic factors but is partially dependent on ATP/GTP, suggesting that, like beta-catenin, IpaH(9.8) secreted from intracellular Shigella can be transported into the nucleus.
KeywordMeSH Terms
Antigens, Bacterial
32. Fortineau  N, Naas  T, Gaillot  O, Nordmann  P,     ( 2001 )

SHV-type extended-spectrum beta-lactamase in a Shigella flexneri clinical isolate.

The Journal of antimicrobial chemotherapy 47 (5)
PMID : 11328785  :   DOI  :   10.1093/jac/47.5.685    
Abstract >>
A Shigella flexneri isolate resistant to oxyimino-cephalosporins was recovered from a stool sample of a 16 month-old Algerian child hospitalized in Paris, France. This isolate harboured an SHV-2 beta-lactamase gene located on a c. 80 kb self-transferable plasmid. This is the first report of an Ambler class A extended-spectrum beta-lactamase from Shigella spp.
KeywordMeSH Terms
33. Smajs  D, Weinstock  GM,     ( 2001 )

The iron- and temperature-regulated cjrBC genes of Shigella and enteroinvasive Escherichia coli strains code for colicin Js uptake.

Journal of bacteriology 183 (13)
PMID : 11395459  :   DOI  :   10.1128/JB.183.13.3958-3966.2001     PMC  :   PMC95278    
Abstract >>
A cosmid library of DNA from colicin Js-sensitive enteroinvasive Escherichia coli (EIEC) strain O164 was made in colicin Js-resistant strain E. coli VCS257, and colicin Js-sensitive clones were identified. Sensitivity to colicin Js was associated with the carriage of a three-gene operon upstream of and partially overlapping senB. The open reading frames were designated cjrABC (for colicin Js receptor), coding for proteins of 291, 258, and 753 amino acids, respectively. Tn7 insertions in any of them led to complete resistance to colicin Js. A near-consensus Fur box was found upstream of cjrA, suggesting regulation of the cjr operon by iron levels. CjrA protein was homologous to iron-regulated Pseudomonas aeruginosa protein PhuW, whose function is unknown; CjrB was homologous to the TonB protein from Pseudomonas putida; and CjrC was homologous to a putative outer membrane siderophore receptor from Campylobacter jejuni. Cloning experiments showed that the cjrB and cjrC genes are sufficient for colicin Js sensitivity. Uptake of colicin Js into sensitive bacteria was dependent on the ExbB protein but not on the E. coli K-12 TonB and TolA, -B, and -Q proteins. Sensitivity to colicin Js is positively regulated by temperature via the VirB protein and negatively controlled by the iron source through the Fur protein. Among EIEC strains, two types of colicin Js-sensitive phenotypes were identified that differed in sensitivity to colicin Js by 1 order of magnitude. The difference in sensitivity to colicin Js is not due to differences between the sequences of the CjrB and CjrC proteins.
KeywordMeSH Terms
Escherichia coli Proteins
Virulence Factors
34. Adams  MM, Allison  GE, Verma  NK,     ( 2001 )

Type IV O antigen modification genes in the genome of Shigella flexneri NCTC 8296.

Microbiology (Reading, England) 147 (Pt 4)
PMID : 11283281  :   DOI  :   10.1099/00221287-147-4-851    
Abstract >>
The genes encoding type IV O antigen glucosylation were characterized from both Escherichia coli and Shigella flexneri. The putative O antigen modification genes from E. coli, o120 o306 o443, were PCR-amplified and introduced into S. flexneri serotype Y strain SFL124. Immunogold labelling and phage sensitivity indicated the presence of both serotype Y and serotype 4a O antigens on the cell surface of the resulting recombinant SFL124 strains, suggesting that only partial serotype conversion was conferred by the E. coli genes. The type IV O antigen modification genes were then isolated and characterized from S. flexneri serotype 4a strain NCTC 8296. A 3.8 kb chromosomal fragment conferred complete conversion to serotype 4a when introduced into SFL124. Sequence analysis of the fragment revealed the presence of three genes, gtrA(IV) gtrB(IV) gtrIV(Sf). DNAs homologous to bacteriophage int and attP were located upstream of gtrA(IV), suggesting that this region of the NCTC 8296 genome may have originated from a bacteriophage; however, a serotype-converting phage could not be induced from this strain nor from other strains used in this study. Comparison of the GtrIV(Sf) and GtrIV(Ec) (o443) proteins revealed that they are 41% identical and 63% similar, which is the highest degree of similarity reported among the S. flexneri O antigen glucosyltransferases.
KeywordMeSH Terms
Genome, Bacterial
35. Venkatesan  MM, Goldberg  MB, Rose  DJ, Grotbeck  EJ, Burland  V, Blattner  FR,     ( 2001 )

Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri.

Infection and immunity 69 (5)
PMID : 11292750  :   DOI  :   10.1128/IAI.69.5.3271-3285.2001     PMC  :   PMC98286    
Abstract >>
The complete sequence analysis of the 210-kb Shigella flexneri 5a virulence plasmid was determined. Shigella spp. cause dysentery and diarrhea by invasion and spread through the colonic mucosa. Most of the known Shigella virulence determinants are encoded on a large plasmid that is unique to virulent strains of Shigella and enteroinvasive Escherichia coli; these known genes account for approximately 30 to 35% of the virulence plasmid. In the complete sequence of the virulence plasmid, 286 open reading frames (ORFs) were identified. An astonishing 153 (53%) of these were related to known and putative insertion sequence (IS) elements; no known bacterial plasmid has previously been described with such a high proportion of IS elements. Four new IS elements were identified. Fifty putative proteins show no significant homology to proteins of known function; of these, 18 have a G+C content of less than 40%, typical of known virulence genes on the plasmid. These 18 constitute potentially unknown virulence genes. Two alleles of shet2 and five alleles of ipaH were also identified on the plasmid. Thus, the plasmid sequence suggests a remarkable history of IS-mediated acquisition of DNA across bacterial species. The complete sequence will permit targeted characterization of potential new Shigella virulence determinants.
KeywordMeSH Terms
Plasmids
36. Robb  CW, Orihuela  CJ, Ekkelenkamp  MB, Niesel  DW,     ( 2001 )

Identification and characterization of an in vivo regulated D15/Oma87 homologue in Shigella flexneri using differential display polymerase chain reaction.

Gene 262 (1��2��)
PMID : 11179681  :   DOI  :   10.1016/s0378-1119(00)00537-0    
Abstract >>
Shigella genes expressed during infection likely contribute to adaptation and virulence in the host. Using differential display PCR (DDPCR), a cDNA fragment from Shigella flexneri serotype 5 that showed enhanced expression in a murine model was identified, cloned and sequenced. Enhanced expression was verified by RNA dot blot. The full-length gene was cloned using PCR and sequenced. The complete gene sequence was BLAST searched against GenBank, and exhibited strong homology to genes encoding Haemophilus influenzae D15 and Pasteurella multocida Oma87 protective outer membrane antigens. The S. flexneri gene putatively encodes a approximately 90-kDa protein and was termed oma90. The deduced amino acid sequence from oma90 was analyzed and compared to the D15/Oma87 antigens. Additionally, oma90 mapped to a cluster of orthologous groups, and probably contains an ancient conserved domain. The chromosomal organization of oma90 was similar to that for H. influenzae and P. multocida as well as for other known homologues. Northern blot revealed that the oma90 transcript encoded only oma90. This report represents the first description of a S. flexneri gene identified based on enhanced expression in the host. Furthermore, we report the first evidence demonstrating in vivo regulation of a member of the d15/oma87 gene family.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
37. Al-Hasani  K, Rajakumar  K, Bulach  D, Robins-Browne  R, Adler  B, Sakellaris  H,     ( 2001 )

Genetic organization of the she pathogenicity island in Shigella flexneri 2a.

Microbial pathogenesis 30 (1)
PMID : 11162180  :   DOI  :   10.1006/mpat.2000.0404    
Abstract >>
In this study we report the complete nucleotide sequence and genetic organization of the she pathogenicity island (PAI) of Shigella flexneri 2a strain YSH6000T. The 46 603 bp she PAI is situated adjacent to the 3' terminus of the pheV tRNA gene and includes an imperfect direct repeat of the 3'-terminal 22 bp of the pheV gene at the right boundary of the PAI. The she PAI carries a bacteriophage P4-like integrase gene within the pheV -proximal boundary of the PAI, intact and truncated mobile genetic elements, plasmid-related sequences, open reading frames exhibiting high sequence similarity to those found on the locus of enterocyte effacement (LEE) PAI of enterohemorrhagic Escherichia coli (EHEC), and the SHI-2 PAI of S. flexneri and several other open reading frames of unknown function. The she PAI also encodes two autotransporter proteins, including SigA, a cytopathic protease that contributes to intestinal fluid accumulation and Pic, a protease with mucinase, and hemagglutinin activities. In addition, an open reading frame (orf) termed sap, has high sequence similarity to the gene encoding Antigen 43, a surface-located autotransporter protein of E. coli. The ShET1 enterotoxin genes, associated predominantly with S. flexneri 2a strains, are also located on the she PAI.
KeywordMeSH Terms
38. Tran Van Nhieu  G, Bourdet-Sicard  R, Duménil  G, Blocker  A, Sansonetti  PJ,     ( 2000 )

Bacterial signals and cell responses during Shigella entry into epithelial cells.

Cellular microbiology 2 (3)
PMID : 11207575  :  
Abstract >>
Shigella invades epithelial cells by inducing cytoskeletal reorganization localized at the site of bacterial-host cell interaction. During entry, the Shigella type III secretion apparatus allows the insertion of a pore that contains the IpaB and IpaC proteins into cell membranes. Insertion of this complex is thought to allow translocation of the carboxy-terminus moiety of IpaC, but also of other Shigella effectors, such as IpaA, into the cell cytosol. IpaC triggers actin polymerization and the formation of filopodial and lamellipodial extensions dependent on the Cdc42 and Rac GTPases. IpaA, on the other hand, binds to the focal adhesion protein vinculin and induces depolymerization of actin filaments. IpaA and the GTPase Rho are not required for actin polymerization at the site of bacterial contact with the cell membrane, but allow the transformation of the IpaC-induced extensions into a structure that is productive for bacterial entry. Rho is required for the recruitment at entry foci of ezrin, a cytoskeletal linker required for Shigella entry, and also of the Src tyrosine kinase. The Src tyrosine kinase activity, which is required for Shigella-induced actin polymerization, also appears to be involved in a negative regulatory loop that downregulates Rho at the site of entry.
KeywordMeSH Terms
39. Buchrieser  C, Glaser  P, Rusniok  C, Nedjari  H, D'Hauteville  H, Kunst  F, Sansonetti  P, Parsot  C,     ( 2000 )

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri.

Molecular microbiology 38 (4)
PMID : 11115111  :   DOI  :   10.1046/j.1365-2958.2000.02179.x    
Abstract >>
Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi-Spa type III secretion apparatus and the secreted IpaA-D proteins, all of which are encoded by a virulence plasmid. In wild-type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N-terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.
KeywordMeSH Terms
40. Chau  PY, Yuen  KY, Lo  JY, Siu  LK,     ( 2000 )

beta-lactamases in Shigella flexneri isolates from Hong Kong and Shanghai and a novel OXA-1-like beta-lactamase, OXA-30.

Antimicrobial agents and chemotherapy 44 (8)
PMID : 10898672  :   DOI  :   10.1128/aac.44.8.2034-2038.2000     PMC  :   PMC90010    
Abstract >>
Ninety-one ampicillin-resistant Shigella flexneri strains from Hong Kong and Shanghai were studied for production of beta-lactamases. TEM-1-like and OXA-1-like enzymes were identified in 21 and 79% of the strains, respectively, by isoelectric focusing (IEF). No difference in the pattern of beta-lactamase production was found between strains from Hong Kong and Shanghai. Four ribotypes were detected. Over 88% of OXA-producing strains had the same ribotype. All TEM-1-like strains harbored a plasmid which hybridized positively with the bla(TEM) probe. Total DNA from OXA-1-like strains failed to hybridize or only hybridized weakly with an OXA probe. The OXA resistance was not transferable. OXA-1-like enzymes exhibited substrate and inhibition profiles similar to that of OXA-1 and were shown to have a pI of 7.3 by further IEF using a narrow-range ampholine gel. The gene encoding the OXA-1-like enzyme from one isolate (CH-07) was cloned, sequenced, and found to differ from bla(OXA-1) at codon 131 (AGA-->GGA; Arg to Gly), resulting in the novel designation OXA-30. The predominance of OXA-type enzymes in ampicillin-resistant S. flexneri suggests host preference for specific beta-lactamases.
KeywordMeSH Terms
41. Niebuhr  K, Jouihri  N, Allaoui  A, Gounon  P, Sansonetti  PJ, Parsot  C,     ( 2000 )

IpgD, a protein secreted by the type III secretion machinery of Shigella flexneri, is chaperoned by IpgE and implicated in entry focus formation.

Molecular microbiology 38 (1)
PMID : 11029686  :   DOI  :   10.1046/j.1365-2958.2000.02041.x    
Abstract >>
Invasion of epithelial cells by Shigella flexneri involves entry and intercellular dissemination. Entry of bacteria into non-phagocytic cells requires the IpaA-D proteins that are secreted by the Mxi-Spa type III secretion machinery. Type III secretion systems are found in several Gram-negative pathogens and serve to inject bacterial effector proteins directly into the cytoplasm of host cells. In this study, we have analysed the IpgD protein of S. flexneri, the gene of which is located on the virulence plasmid at the 5' end of the mxi-spa locus. We have shown that IpgD (i) is stored in the bacterial cytoplasm in association with a specific chaperone, IpgE; (ii) is secreted by the Mxi-Spa type III secretion system in amounts similar to those of the IpaA-D proteins; (iii) is associated with IpaA in the extracellular medium; and (iv) is involved in the modulation of the host cell response after contact of the bacterium with epithelial cells. This suggests that IpgD is an effector that might be injected into host cells to manipulate cellular processes during infection.
KeywordMeSH Terms
Membrane Fusion
42. Moreno  F, del Castillo  FJ,     ( 2000 )

Characterization of the genes encoding the SheA haemolysin in Escherichia coli O157:H7 and Shigella flexneri 2a.

Research in microbiology 151 (3)
PMID : 10865950  :  
Abstract >>
N/A
KeywordMeSH Terms
Escherichia coli Proteins
43. Sewitz  S, Chalmers  R,     ( 2000 )

Complete nucleotide sequence of Tn10.

Journal of bacteriology 182 (10)
PMID : 10781570  :   DOI  :   10.1128/jb.182.10.2970-2972.2000     PMC  :   PMC102010    
Abstract >>
The complete nucleotide sequence of Tn10 has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis. The glutamate permease was fully functional when expressed, but Tn10 did not protect Escherichia coli from the toxic effects of various metals.
KeywordMeSH Terms
Amino Acid Transport Systems, Acidic
DNA Transposable Elements
DNA, Bacterial
44. Rajakumar  K, Grant  T, Sakellaris  H, Henderson  IR, Al-Hasani  K,     ( 2000 )

The sigA gene which is borne on the she pathogenicity island of Shigella flexneri 2a encodes an exported cytopathic protease involved in intestinal fluid accumulation.

Infection and immunity 68 (5)
PMID : 10768931  :   DOI  :   10.1128/iai.68.5.2457-2463.2000     PMC  :   PMC97446    
Abstract >>
In this study, the sigA gene situated on the she pathogenicity island of Shigella flexneri 2a was cloned and characterized. Sequence analysis showed that sigA encodes a 139.6-kDa protein which belongs to the SPATE (serine protease autotransporters of Enterobacteriaceae) subfamily of autotransporter proteins. The demonstration that SigA is autonomously secreted from the cell to yield a 103-kDa processed form and possesses a conserved C-terminal domain for export from the cell were consistent with the autotransporter pathway of secretion. Functional analysis showed that SigA is a secreted temperature-regulated serine protease capable of degrading casein. SigA was cytopathic for HEp-2 cells, suggesting that it may be a cell-altering toxin with a role in the pathogenesis of Shigella infections. SigA was at least partly responsible for the ability of S. flexneri to stimulate fluid accumulation in ligated rabbit ileal loops.
KeywordMeSH Terms
45. Atkins  A, Jamieson  SJ, Bullough  PA, Stillman  TJ, Wallace  AJ,     ( 2000 )

E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy.

Cell 100 (2)
PMID : 10660049  :   DOI  :   10.1016/s0092-8674(00)81564-0    
Abstract >>
Hemolysin E (HlyE) is a novel pore-forming toxin of Escherichia coli, Salmonella typhi, and Shigella flexneri. Here we report the X-ray crystal structure of the water-soluble form of E. coli HlyE at 2.0 A resolution and the visualization of the lipid-associated form of the toxin in projection at low resolution by electron microscopy. The crystal structure reveals HlyE to be the first member of a new family of toxin structures, consisting of an elaborated helical bundle some 100 A long. The electron micrographs show how HlyE oligomerizes in the presence of lipid to form transmembrane pores. Taken together, the data from these two structural techniques allow us to propose a simple model for the structure of the pore and for membrane interaction.
KeywordMeSH Terms
Escherichia coli Proteins
46. Rüdiger  M, Jockusch  BM, Gounon  P, Bourdet-Sicard  R,     ( 1999 )

Binding of the Shigella protein IpaA to vinculin induces F-actin depolymerization.

The EMBO journal 18 (21)
PMID : 10545097  :   DOI  :   10.1093/emboj/18.21.5853     PMC  :   PMC1171651    
Abstract >>
Shigella flexneri, the causative agent of bacillary dysentery, enters into epithelial cells by a macropinocytic process. IpaA, a Shigella protein secreted upon cell contact, binds to the focal adhesion protein vinculin and is required for efficient bacterial uptake. IpaA was shown here to bind with high affinity to the N-terminal residues 1-265 of vinculin. Using co-sedimentation and solid-phase assays, we demonstrated that binding of IpaA to vinculin strongly increases the association of vinculin with F-actin. We also characterized a depolymerizing activity on actin filaments associated with the vinculin-IpaA complex both in vitro and in microinjected cells. We propose that the conformational change of vinculin induced by IpaA binding allows interaction of the vinculin-IpaA complex with F-actin and subsequent depolymerization of actin filaments.
KeywordMeSH Terms
47. Li  R, Pantaloni  D, Laurent  V, Loisel  TP, Egile  C,     ( 1999 )

Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility.

The Journal of cell biology 146 (6)
PMID : 10491394  :   DOI  :   10.1083/jcb.146.6.1319     PMC  :   PMC2156126    
Abstract >>
To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Using a combination of bacterial motility assays in platelet extracts with Escherichia coli expressing the Shigella IcsA protein and in vitro analysis of reconstituted systems from purified proteins, we show here that the bacterial protein IcsA binds N-WASP and activates it in a Cdc42-like fashion. Dramatic stimulation of actin assembly is linked to the formation of a ternary IcsA-N-WASP-Arp2/3 complex, which nucleates actin polymerization. The Arp2/3 complex is essential in initiation of actin assembly and Shigella movement, as previously observed for Listeria monocytogenes. Activation of N-WASP by IcsA unmasks two domains acting together in insertional actin polymerization. The isolated COOH-terminal domain of N-WASP containing a verprolin-homology region, a cofilin-homology sequence, and an acidic terminal segment (VCA) interacts with G-actin in a unique profilin-like functional fashion. Hence, when N-WASP is activated, its COOH-terminal domain feeds barbed end growth of filaments and lowers the critical concentration at the bacterial surface. On the other hand, the NH(2)-terminal domain of N-WASP interacts with F-actin, mediating the attachment of the actin tail to the bacterium surface. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions.
KeywordMeSH Terms
Contractile Proteins
Cytoskeletal Proteins
48. Czeczulin  J, Eslava  C, Henderson  IR,     ( 1999 )

Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli.

Infection and immunity 67 (11)
PMID : 10531204  :   PMC  :   PMC96930    
Abstract >>
We have identified and characterized a secreted protein, designated Pic, which is encoded on the chromosomes of enteroaggregative Escherichia coli (EAEC) 042 and Shigella flexneri 2457T. The product of the pic gene is synthesized as a 146.5-kDa precursor molecule which is processed at the N and C termini during secretion, allowing the release of a mature protein (109.8 kDa) into the culture supernatant. The deduced amino acid sequence of Pic shows high homology to autotransporter proteins, particularly a subgroup termed the SPATEs (serine protease autotransporters of the Enterobacteriaceae). Present in all members of this subgroup is a motif similar to the active sites of certain serine proteases. Pic catalyzes gelatin degradation, which can be abolished by disruption of the predicted proteolytic active site. Functional analysis of the Pic protein implicates this factor in mucinase activity, serum resistance, and hemagglutination. Our data suggest that Pic may be a multifunctional protein involved in enteric pathogenesis.
KeywordMeSH Terms
49. Cardozo  TJ, Zychlinsky  A, Groisman  EA,     ( 1999 )

The selC-associated SHI-2 pathogenicity island of Shigella flexneri.

Molecular microbiology 33 (1)
PMID : 10411725  :   DOI  :   10.1046/j.1365-2958.1999.01449.x    
Abstract >>
Pathogenicity islands are chromosomal gene clusters, often located adjacent to tRNA genes, that encode virulence factors present in pathogenic organisms but absent or sporadically found in related non-pathogenic species. The selC tRNA locus is the site of integration of different pathogenicity islands in uropathogenic Escherichia coli, enterohaemorrhagic E. coli and Salmonella enterica. We show here that the selC locus of Shigella flexneri, the aetiological agent of bacterial dysentery, also contains a pathogenicity island. This pathogenicity island, designated SHI-2 (Shigella island 2), occupies 23.8 kb downstream of selC and contains genes encoding the aerobactin iron acquisition siderophore system, colicin V immunity and several novel proteins. Remnants of multiple mobile genetic elements are present in SHI-2. SHI-2-hybridizing sequences were detected in all S. flexneri strains tested and parts of the island were also found in other Shigella species. SHI-2 may allow Shigella survival in stressful environments, such as those encountered during infection.
KeywordMeSH Terms
Colicins
Genes, Bacterial
50. Reeves  SA, Torres  AG, Payne  SM,     ( 1999 )

The aerobactin iron transport system genes in Shigella flexneri are present within a pathogenicity island.

Molecular microbiology 33 (1)
PMID : 10411724  :   DOI  :   10.1046/j.1365-2958.1999.01448.x    
Abstract >>
Genes encoding the synthesis and transport of aerobactin, a hydroxamate siderophore associated with increased virulence of enteric bacteria, were mapped within a pathogenicity island in Shigella flexneri. The island, designated SHI-2 for Shigella pathogenicity island 2, was located downstream of selC, the site of insertion of pathogenicity islands in several other enteric pathogens. DNA sequence analysis revealed the presence of multiple insertion sequences upstream and downstream of the aerobactin genes and an integrase gene that was nearly identical to an int gene found in Escherichia coli O157:H7. SHI-2 sequences adjacent to selC were similar to sequences at the junction between selC and pathogenicity islands found in E. coli O157:H7 and in enteropathogenic E. coli, but the junctions between the island and downstream yic genes were variable. SHI-2 also encoded immunity to the normally plasmid-encoded colicins I and V, suggesting a common origin for the aerobactin genes in both S. flexneri and E. coli pColV. Polymerase chain reaction and Southern hybridization data indicate that SHI-2 is present in the same location in Shigella sonnei, but the aerobactin genes are not located within SHI-2 in Shigella boydii or enteroinvasive E. coli. Shigella dysenteriae type 1 strains do not produce aerobactin but do contain sequences downstream of selC that are homologous to SHI-2. The presence of the aerobactin genes on plasmids in E. coli pColV and Salmonella, on a pathogenicity island in S. flexneri and S. sonnei and in a different chromosomal location in S. boydii and some E. coli suggests that these virulence-enhancing genes are mobile, and they may constitute an island within an island in S. flexneri.
KeywordMeSH Terms
Colicins
Genes, Bacterial
51. Forst  S,     ( 1999 )

Identification of a conserved N-terminal sequence involved in transmembrane signal transduction in EnvZ.

Journal of bacteriology 181 (17)
PMID : 10464234  :   PMC  :   PMC94069    
Abstract >>
To determine whether N-terminal sequences are involved in the transmembrane signaling mechanism of EnvZ, the nucleotide sequences of envZ genes from several enteric bacteria were determined. Comparative analysis revealed that the amino acid sequence between Pro41 and Glu53 was highly conserved. To further analyze the role of the conserved sequence, envZ of Escherichia coli was subjected to random PCR mutagenesis and mutant alleles that produced a high-osmolarity phenotype, in which ompF was repressed, were isolated. The mutations identified clustered within, as well as adjacent to, the Pro41-to-Glu53 sequence. These findings suggest that the conserved Pro41-to-Glu53 sequence is involved in the signal transduction mechanism of EnvZ.
KeywordMeSH Terms
Conserved Sequence
Escherichia coli Proteins
Multienzyme Complexes
Signal Transduction
52. Allison  G, Whittle  B, Verma  NK,     ( 1999 )

Serotype 1a O-antigen modification: molecular characterization of the genes involved and their novel organization in the Shigella flexneri chromosome.

Journal of bacteriology 181 (15)
PMID : 10419979  :   PMC  :   PMC103612    
Abstract >>
The factors responsible for serotype 1a O-antigen modification in Shigella flexneri were localized to a 5.8-kb chromosomal HindIII fragment of serotype 1a strain Y53. The entire 5.8-kb fragment and regions up- and downstream of it (10.6-kb total) were sequenced. A putative three-gene operon, which showed homology with other serotype conversion genes, was identified and shown to confer serotype 1a O-antigen modification. The serotype conversion genes were flanked on either side by phage DNA. Multiple insertion sequence (IS) elements were located within and upstream of the phage DNA in a composite transposon-like structure. Host DNA homologous to the dsdC and the thrW proA genes was located upstream of the IS elements and downstream of the phage DNA, respectively. The sequence analysis indicates that the organization of the 10.6-kb region of the Y53 chromosome is unique and suggests that the serotype conversion genes were originally brought into the host by a bacteriophage. Several features of this region are also characteristic of pathogenicity islands.
KeywordMeSH Terms
Chromosomes, Bacterial
53. Steinhauer  J, Goldberg  MB, Varga  AW,     ( 1999 )

The unipolar Shigella surface protein IcsA is targeted directly to the bacterial old pole: IcsP cleavage of IcsA occurs over the entire bacterial surface.

Molecular microbiology 32 (2)
PMID : 10231492  :   DOI  :   10.1046/j.1365-2958.1999.01356.x    
Abstract >>
Shigella flexneri is an intracellular pathogen that is able to move within the cytoplasm of infected cells by the continual assembly of actin onto one pole of the bacterium. IcsA, an outer membrane protein, is localized to the old pole of the bacterium and is both necessary and sufficient for actin assembly. IcsA is slowly cleaved from the bacterial surface by the protease IcsP (SopA). Absence of IcsP leads to an alteration in the distribution of surface IcsA, such that the polar cap is maintained and some IcsA is distributed along the lateral walls of the bacillus. The mechanism of unipolar localization of IcsA and the role of IcsP in its unipolar localization are incompletely understood. Here, we demonstrate that cleavage of IcsA occurs exclusively in the outer membrane and that IcsP is localized to the outer membrane. In addition, we show that IcsA at the old pole is susceptible to cleavage by IcsP and that native IcsP is active at the pole. Taken together, these data indicate that IcsP cleaves IcsA over the entire bacterial surface. Finally, we show that, immediately after induction from a tightly regulated promoter, IcsA is expressed exclusively at the old pole in both the icsP- icsA- and the icsA- background. These data demonstrate that unipolar localization of IcsA results from its direct targeting to the pole, followed by its diffusion laterally in the outer membrane.
KeywordMeSH Terms
54. Prunier  AL, Schuch  R, Fernández  RE, Maurelli  AT,     ( 2007 )

Genetic structure of the nadA and nadB antivirulence loci in Shigella spp.

Journal of bacteriology 189 (17)
PMID : 17586625  :   DOI  :   10.1128/JB.00525-07     PMC  :   PMC1951923    
Abstract >>
Comparison of nadA and nadB in 14 Shigella strains and enteroinvasive Escherichia coli versus E. coli showed that at least one locus is altered in all strains. These observations explain the characteristic nicotinic acid auxotrophy of Shigella organisms and are consistent with the previously identified antivirulence nature of these genes for these pathogens.
KeywordMeSH Terms
55. Runyen-Janecky  LJ, Hong  M,     ( 1999 )

The virulence plasmid-encoded impCAB operon enhances survival and induced mutagenesis in Shigella flexneri after exposure to UV radiation.

Infection and immunity 67 (3)
PMID : 10024589  :   PMC  :   PMC96475    
Abstract >>
Upon exposure to UV radiation, Shigella flexneri SA100 displayed survival and mutation frequencies comparable to those of Escherichia coli AB1157, which contains a functional UmuDC error-prone DNA repair system. Survival of SA100 after UV irradiation was associated with the presence of the 220-kb virulence plasmid, pVP. This plasmid encodes homologues of ImpA and ImpB, which comprise an error-prone DNA repair system encoded on plasmid TP110 that was initially identified in Salmonella typhimurium, and ImpC, encoded upstream of ImpA and ImpB. Although the impB gene was present in representatives of all four species of Shigella, not all isolates tested contained the gene. Shigella isolates that lacked impB were more sensitive to UV radiation than isolates that contained impB. The nucleotide sequence of a 2.4-kb DNA fragment containing the imp operon from S. flexneri SA100 pVP was 96% identical to the imp operon from the plasmid TP110. An SA100 derivative with a mutation in the impB gene had reduced survival following UV irradiation and less UV-induced mutagenesis relative to the parental strain. We also found that S. flexneri contained a chromosomally encoded umuDC operon; however, the umuDC promoter was not induced by exposure to UV radiation. This suggests that the imp operon but not the umuDC operon contributes to survival and induced mutagenesis in S. flexneri following exposure to UV radiation.
KeywordMeSH Terms
Escherichia coli Proteins
Mutagenesis
Operon
Plasmids
Serine Endopeptidases
56. Iwai  H, Kim  M, Yoshikawa  Y, Ashida  H, Ogawa  M, Fujita  Y, Muller  D, Kirikae  T, Jackson  PK, Kotani  S, Sasakawa  C,     ( 2007 )

A bacterial effector targets Mad2L2, an APC inhibitor, to modulate host cell cycling.

Cell 130 (4)
PMID : 17719540  :   DOI  :   10.1016/j.cell.2007.06.043     DOI  :   10.1016/j.cell.2007.06.043    
Abstract >>
The gut epithelium self-renews every several days, providing an important innate defense system that limits bacterial colonization. Nevertheless, many bacterial pathogens, including Shigella, efficiently colonize the intestinal epithelium. Here, we show that the Shigella effector IpaB, when delivered into epithelial cells, causes cell-cycle arrest by targeting Mad2L2, an anaphase-promoting complex/cyclosome (APC) inhibitor. Cyclin B1 ubiquitination assays revealed that APC undergoes unscheduled activation due to IpaB interaction with the APC inhibitor Mad2L2. Synchronized HeLa cells infected with Shigella failed to accumulate Cyclin B1, Cdc20, and Plk1, causing cell-cycle arrest at the G2/M phase in an IpaB/Mad2L2-dependent manner. IpaB/Mad2L2-dependent cell-cycle arrest by Shigella infection was also demonstrated in rabbit intestinal crypt progenitors, and the IpaB-mediated arrest contributed to efficient colonization of the host cells. These results strongly indicate that Shigella employ special tactics to influence epithelial renewal in order to promote bacterial colonization of intestinal epithelium.
KeywordMeSH Terms
57. Hu  LF, Li  JB, Ye  Y, Li  X,     ( 2007 )

Mutations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in clinical strains of fluoroquinolone-resistant Shigella in Anhui, China.

Journal of microbiology (Seoul, Korea) 45 (2)
PMID : 17483803  :  
Abstract >>
In this research 26 Shigella isolates were examined by PCR and direct nucleotide sequencing for genetic alterations in the quinolone-resistance determining regions (QRDRs). We tested for the presence of qnr genes by PCR in 91 strains, but no qnr genes were found. The results did show, however, some novel mutations at codon 83 of gyrA (Ser-->Ile) and codon 64 of parC (Ala64-->Cys, Ala64-->Asp), which were related to fluroquinolone resistance.
KeywordMeSH Terms
Mutation
58. Young  JM, Park  DC,     ( 2007 )

Relationships of plant pathogenic enterobacteria based on partial atpD, carA, and recA as individual and concatenated nucleotide and peptide sequences.

Systematic and applied microbiology 30 (5)
PMID : 17451899  :   DOI  :   10.1016/j.syapm.2007.03.002    
Abstract >>
Relationships of the genera in the Enterobacteriaceae containing plant pathogenic species: Brenneria, Dickeya, Enterobacter, Erwinia, Pantoea, Pectobacterium, and Samsonia, were investigated by comparison of their nucleotide and peptide sequences of atpD, carA, recA, and the concatenated sequences. Erwinia spp. and Pantoea spp., with Pectobacterium cypripedii, formed a group distinct from other pathogenic taxa. Pectobacterium, Brenneria, Dickeya, and Samsonia formed a contiguous clade. Samsonia was usually concurrent with Pectobacterium. Most Brenneria were also close to Pectobacterium, suggesting that these three taxa might be better represented as a single genus. Brenneria quercina was not closely associated with other members of this genus and may represent a separate genus. The sequences representing Dickeya were distinct, further supporting the generic status of the taxon. Plant pathogenic Enterobacter spp. display such sequence variability that few definite conclusions as to their specific placement could be made. These data highlight the difficulty of drawing reliable and robust taxonomic conclusions based on comparative analysis of sequence data without some independent criterion to calibrate a scale for diversity.
KeywordMeSH Terms
59. Li  H, Xu  H, Zhou  Y, Zhang  J, Long  C, Li  S, Chen  S, Zhou  JM, Shao  F,     ( 2007 )

The phosphothreonine lyase activity of a bacterial type III effector family.

Science (New York, N.Y.) 315 (5814)
PMID : 17303758  :   DOI  :   10.1126/science.1138960    
Abstract >>
Pathogenic bacteria use the type III secretion system to deliver effector proteins into host cells to modulate the host signaling pathways. In this study, the Shigella type III effector OspF was shown to inactivate mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated kinases 1 and 2 (Erk1/2), c-Jun N-terminal kinase, and p38]. OspF irreversibly removed phosphate groups from the phosphothreonine but not from the phosphotyrosine residue in the activation loop of MAPKs. Mass spectrometry revealed a mass loss of 98 daltons in p-Erk2, due to the abstraction of the alpha proton concomitant with cleavage of the C-OP bond in the phosphothreonine residue. This unexpected enzymatic activity, termed phosphothreonine lyase, appeared specific for MAPKs and was shared by other OspF family members.
KeywordMeSH Terms
MAP Kinase Signaling System
60. Pham  HN, Ohkusu  K, Mishima  N, Noda  M, Monir Shah  M, Sun  X, Hayashi  M, Ezaki  T,     ( 2007 )

Phylogeny and species identification of the family Enterobacteriaceae based on dnaJ sequences.

Diagnostic microbiology and infectious disease 58 (2)
PMID : 17368802  :   DOI  :   10.1016/j.diagmicrobio.2006.12.019    
Abstract >>
Phylogenetic relations within the family Enterobacteriaceae were analyzed using partial dnaJ sequences of 165 strains belonging to 93 species from 27 enterobacterial genera. The dnaJ phylogeny was in relative agreement with that constructed by 16S rDNA sequences, but more monophyletic groups were obtained from the dnaJ tree than from the 16S rDNA tree. The degree of divergence of the dnaJ gene was approximately 6 times greater than that of 16S rDNA. Also, the dnaJ gene showed the most discriminatory power in comparison with tuf and atpD genes, facilitating clear differentiation of any 2 enterobacterial species by dnaJ sequence analysis. The application of dnaJ sequences to the identification was confirmed by assigning 72 clinical isolates to the correct enterobacterial species. Our data indicate that analysis of the dnaJ gene sequences can be used as a powerful marker for phylogenetic study and identification at the species level of the family Enterobacteriaceae.
KeywordMeSH Terms
HSP40 Heat-Shock Proteins
Phylogeny
61. Kramer  RW, Slagowski  NL, Eze  NA, Giddings  KS, Morrison  MF, Siggers  KA, Starnbach  MN, Lesser  CF,     ( 2007 )

Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation.

PLoS pathogens 3 (2)
PMID : 17305427  :   DOI  :   10.1371/journal.ppat.0030021     PMC  :   PMC1797620    
Abstract >>
Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.
KeywordMeSH Terms
Genome, Fungal
Immunity, Innate
62. Arbibe  L, Kim  DW, Batsche  E, Pedron  T, Mateescu  B, Muchardt  C, Parsot  C, Sansonetti  PJ,     ( 2007 )

An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses.

Nature immunology 8 (1)
PMID : 17159983  :   DOI  :   10.1038/ni1423    
Abstract >>
Phosphorylation of histone H3 at Ser10 increases chromatin accessibility to transcription factor NF-kappaB on a subset of genes involved in immune responses. Here we report that a bacterial pathogen abrogated phosphorylation of histone H3 to 'shape' the transcriptional responses of infected host cells. We identify the Shigella flexneri protein effector OspF as a dually specific phosphatase that dephosphorylated mitogen-activated protein kinases in the nucleus, thus preventing histone H3 phosphorylation at Ser10 in a gene-specific way. That activity of OspF enabled shigella to block the activation of a subset of NF-kappaB-responsive genes, leading to compromised recruitment of polymorphonuclear leukocytes to infected tissues. S. flexneri has thus evolved the capacity to precisely modulate host cell epigenetic 'information' as a strategy for repressing innate immunity.
KeywordMeSH Terms
Transcription, Genetic
63. Ahmed  AM, Furuta  K, Shimomura  K, Kasama  Y, Shimamoto  T,     ( 2006 )

Genetic characterization of multidrug resistance in Shigella spp. from Japan.

Journal of medical microbiology 55 (Pt 12)
PMID : 17108272  :   DOI  :   10.1099/jmm.0.46725-0    
Abstract >>
This study characterized the genetic basis of antimicrobial resistance of a number of Shigella spp. isolated from humans from 2000 to 2004 in Hiroshima prefecture, Japan. A total of 26 isolates of Shigella spp. were included in this study. Antimicrobial susceptibility tests revealed high levels of resistance, especially to ampicillin, streptomycin, trimethoprim, tetracycline, nalidixic acid and ciprofloxacin. PCR and DNA sequencing were used for screening and characterization of antibiotic-resistance determinants. PCR sequencing analysis revealed the presence of only one type of class 1 integron in one isolate of Shigella sonnei. This class 1 integron was 1904 bp and contained two gene cassettes: a probable esterase/lipase (estX) and aadA1, which confers resistance to streptomycin and spectinomycin. Two types of class 2 integron were identified in this study. One was the classic type (2158 bp) and carried the three conserved resistance gene cassettes of the class 2 integron, dfrA1, sat1 and aadA1, which confer resistance to trimethoprim, streptothricin and streptomycin/spectinomycin, respectively. This type was detected in both Shigella sonnei (14 isolates) and Shigella flexneri (five isolates). The other type was shorter (1313 bp) and carried only two gene cassettes, dfrA1 and sat1. This integron was detected in a single isolate of Shigella sonnei. PFGE patterns showed limited diversity within clusters of the same species. Furthermore, an extended-spectrum beta-lactamase gene, bla(OXA-30), which confers resistance to ampicillin, was characterized in all isolates of Shigella flexneri except the oldest strain, which was isolated in 2000. Southern blot hybridization and conjugation experiments showed that bla(OXA-30) was located in the chromosome.
KeywordMeSH Terms
64. Yang  J, Nie  H, Chen  L, Zhang  X, Yang  F, Xu  X, Zhu  Y, Yu  J, Jin  Q,     ( 2007 )

Revisiting the molecular evolutionary history of Shigella spp.

Journal of molecular evolution 64 (1)
PMID : 17160643  :   DOI  :   10.1007/s00239-006-0052-8    
Abstract >>
The theory that Shigella is derived from multiple independent origins of Escherichia coli (Pupo et al. 2000) has been challenged by recent findings that the virulence plasmids (VPs) and the chromosomes share a similar evolutionary history (Escobar-Paramo et al. 2003), which suggests that an ancestral VP entered an E. coli strain only once, which gave rise to Shigella spp. In an attempt to resolve these conflicting theories, we constructed three phylogenetic trees in this study: a robust chromosomal tree using 23 housekeeping genes from 46 strains of Shigella and enteroinvasive E. coli (EIEC), a chromosomal tree using 4 housekeeping genes from 19 EcoR strains and 46 Shigella/EIEC strains, and a VP tree using 5 genes outside of the VP cell-entry region from 38 Shigella/EIEC strains. Both chromosomal trees group Shigella into three main clusters and five outliers, and strongly suggest that Shigella has multiple origins within E. coli. Most strikingly, the VP tree shows that the VPs from two main Shigella clusters, C1 and C2, are more closely related, which contradicts the chromosomal trees that place C2 and C3 next to each other but C1 at a distance. Additionally, we have identified a complete tra operon of the F-plasmid in the genome sequence of an EIEC strain and found that two other EIEC strains are also likely to possess a complete tra operon. All lines of evidence support an alternative multiorigin theory that transferable diverse ancestral VPs entered diverse origins of E. coli multiple times during a prolonged period of time, resulting in Shigella species with diverse genomes but similar pathogenic properties.
KeywordMeSH Terms
Evolution, Molecular
Phylogeny
65. Yoshida  S, Handa  Y, Suzuki  T, Ogawa  M, Suzuki  M, Tamai  A, Abe  A, Katayama  E, Sasakawa  C,     ( 2006 )

Microtubule-severing activity of Shigella is pivotal for intercellular spreading.

Science (New York, N.Y.) 314 (5801)
PMID : 17095701  :   DOI  :   10.1126/science.1133174    
Abstract >>
Some pathogenic bacteria actually invade the cytoplasm of their target host cells. Invasive bacteria acquire the propulsive force to move by recruiting actin and inducing its polymerization. Here we show that Shigella movement within the cytoplasm was severely hindered by microtubules and that the bacteria destroyed surrounding microtubules by secreting VirA by means of the type III secretion system. Degradation of microtubules by VirA was dependent on its alpha-tubulin-specific cysteine protease-like activity. virA mutants did not move within the host cytoplasm and failed to move into adjacent cells.
KeywordMeSH Terms
66. Izard  T, Tran Van Nhieu  G, Bois  PR,     ( 2006 )

Shigella applies molecular mimicry to subvert vinculin and invade host cells.

The Journal of cell biology 175 (3)
PMID : 17088427  :   DOI  :   10.1083/jcb.200605091     PMC  :   PMC2064523     DOI  :   10.1083/jcb.200605091     PMC  :   PMC2064523    
Abstract >>
Shigella flexneri, the causative agent of bacillary dysentery, injects invasin proteins through a type III secretion apparatus upon contacting the host cell, which triggers pathogen internalization. The invasin IpaA is essential for S. flexneri pathogenesis and binds to the cytoskeletal protein vinculin to facilitate host cell entry. We report that IpaA harbors two vinculin-binding sites (VBSs) within its C-terminal domain that bind to and activate vinculin in a mutually exclusive fashion. Only the highest affinity C-terminal IpaA VBS is necessary for efficient entry and cell-cell spread of S. flexneri, whereas the lower affinity VBS appears to contribute to vinculin recruitment at entry foci of the pathogen. Finally, the crystal structures of vinculin in complex with the VBSs of IpaA reveal the mechanism by which IpaA subverts vinculin's functions, where S. flexneri utilizes a remarkable level of molecular mimicry of the talin-vinculin interaction to activate vinculin. Mimicry of vinculin's interactions may therefore be a general mechanism applied by pathogens to infect the host cell.
KeywordMeSH Terms
Molecular Mimicry
Molecular Mimicry
67. Johnson  S, Roversi  P, Espina  M, Olive  A, Deane  JE, Birket  S, Field  T, Picking  WD, Blocker  AJ, Galyov  EE, Picking  WL, Lea  SM,     ( 2007 )

Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD.

The Journal of biological chemistry 282 (6)
PMID : 17077085  :   DOI  :   10.1074/jbc.M607945200     PMC  :   PMC1894746    
Abstract >>
Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.
KeywordMeSH Terms
68. Xiong  Z, Tang  X, Yang  F, Zhang  X, Yang  J, Chen  L, Nie  H, Yan  Y, Jiang  Y, Wang  J, Xue  Y, Xu  X, Zhu  Y, Dong  J, An  L, Wang  X, Jin  Q,     ( 2006 )

Comparison of the virulence plasmid genomes of two strains of Shigella which lost the ability to bind Congo red.

Science in China. Series C, Life sciences 49 (2)
PMID : 16704117  :  
Abstract >>
We determined and analyzed the Shigella flexneri serotype 5 (pSF5) and S. dysenteriae serotype 1 (pSD1) virulence plasmid genomes. The total length of pSF5 is 136513 bp, including 165 open reading frames (ORFs). Of these ORFs, 133 were identified and 32 of those had no significant homology to proteins with known functions. The length of pSD1 is 182545 bp, including 224 ORFs, of which we identified 181. The remaining 43 ORFs were not significantly homologous to proteins with known functions. The insertion sequence (IS) elements are 53787 bp in pSF5, and 49616 bp in pSD1, which represents 39.4% and 27.1% of the genome, respectively. There are 22 IS element types in pSF5 and pSD1, among which we report ISEc8 and ISSbo6 for the first time in the Shigella virulence plasmid. Compared to pCP301, there are a large number of deleted genes and gene inversions in both pSF5 and pSD1. The ipa-mxi-spa locus in pSF5 is completely absent, and the genes related to the O-antigen biosynthesis are partially missing. In contrast, the above genes in pSD1 are integral, with the exception of virF. The whole genome analysis of the two plasmids shows that the loss of genes related to gene invasion or regulation also obliterates the ability of pPF5 and pSD1 to bind Congo red (Crb). Whether these genes determine the Crb function requires continued investigation.
KeywordMeSH Terms
69. Zurawski  DV, Mitsuhata  C, Mumy  KL, McCormick  BA, Maurelli  AT,     ( 2006 )

OspF and OspC1 are Shigella flexneri type III secretion system effectors that are required for postinvasion aspects of virulence.

Infection and immunity 74 (10)
PMID : 16988276  :   DOI  :   10.1128/IAI.00594-06     PMC  :   PMC1594884    
Abstract >>
Shigella flexneri is the causative agent of dysentery, and its pathogenesis is mediated by a type III secretion system (T3SS). S. flexneri secretes effector proteins into the eukaryotic cell via the T3SS, and these proteins usurp host cellular functions to the benefit of the bacteria. OspF and OspC1 are known to be secreted by S. flexneri, but their functions are unknown. We transformed S. flexneri with a plasmid that expresses a two-hemagglutinin tag (2HA) in frame with OspF or OspC1 and verified that these proteins are secreted in a T3SS-dependent manner. Immunofluorescence of HeLa cells infected with S. flexneri expressing OspF-2HA or OspC1-2HA revealed that both proteins localize in the nucleus and cytoplasm of host cells. To elucidate the function of these T3SS effectors, we constructed DeltaospF and DeltaospC1 deletion mutants by allelic exchange. We found that DeltaospF and DeltaospC1 mutants invade host cells and form plaques in confluent monolayers similar to wild-type S. flexneri. However, in the polymorphonuclear (PMN) cell migration assay, a decrease in neutrophil migration was observed for both mutants in comparison to the migration of wild-type bacteria. Moreover, infection of polarized T84 intestinal cells infected with DeltaospF and DeltaospC1 mutants resulted in decreased phosphorylation of extracellular signal-regulated kinase 1/2 in comparison to that of T84 cells infected with wild-type S. flexneri. To date, these are the first examples of T3SS effectors implicated in mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway activation. Ultimately, OspF and OspC1 are essential for PMN transepithelial migration, a phenotype associated with increased inflammation and bacterial access to the submucosa, which are fundamental aspects of S. flexneri pathogenesis.
KeywordMeSH Terms
Dysentery, Bacillary
70. Hartman  AB, Venkatesan  M, Oaks  EV, Buysse  JM,     ( 1990 )

Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri.

Journal of bacteriology 172 (4)
PMID : 1690703  :   DOI  :   10.1128/jb.172.4.1905-1915.1990     PMC  :   PMC208685    
Abstract >>
A lambda gt11 expression library of Tn5-tagged invasion plasmid pWR110 (from Shigella flexneri serotype 5, strain M90T-W) contained a set of recombinants encoding a 60-kilodalton protein (designated IpaH) recognized by rabbit antisera raised against S. flexneri invasion plasmid antigens (J. M. Buysse, C. K. Stover, E. V. Oaks, M. M. Venkatesan, and D. J. Kopecko, J. Bacteriol. 169:2561-2569, 1987). Southern blot analysis of wild-type S. flexneri serotype 5 invasion plasmid DNA (pWR100) digested with various combinations of five restriction enzymes and hybridized with defined ipaH probes showed complex hybridization patterns resulting from multiple copies of the ipaH gene on pWR100. DNA sequence analysis of a 2.9-kilobase (kb) EcoRI fragment directing IpaH antigen synthesis in plasmid recombinant pWR390 revealed an open reading frame coding for a 532-amino-acid protein (60.8 kilodaltons); this size matched well with the estimated size of IpaH determined by Western blot analysis of M90T-W cells and maxicell analysis of Escherichia coli HB101(pWR390) transformants. Examination of the amino acid sequence of IpaH revealed a hydrophilic protein with six evenly spaced 14-residue (L-X2-L-P-X-L-P-X2-L-X2-L) repeat motifs in the amino-terminal end of the molecule. Southern blot analysis of HindIII-digested pWR100 DNA probed with defined segments of the pWR390 2.9-kb insert demonstrated that the multiple band hybridization pattern resulted from repeats of a significant portion of the ipaH structural gene in five distinct HindIII fragments (9.8, 7.8, 4.5, 2.5, and 1.4 kb). Affinity-purified IpaH antibody, used to monitor the expression of the antigen in M90T-W cells grown at 30 and 37 degrees C, showed that IpaH synthesis was not regulated by growth temperature.
KeywordMeSH Terms
Plasmids
71. Osipiuk  J, Maltseva  N, Dementieva  I, Clancy  S, Collart  F, Joachimiak  A,     ( 2006 )

Structure of YidB protein from Shigella flexneri shows a new fold with homeodomain motif.

Proteins 65 (2)
PMID : 16927377  :   DOI  :   10.1002/prot.21054     PMC  :   PMC2885951    
Abstract >>
N/A
KeywordMeSH Terms
Protein Folding
72. Kim  Y, Maltseva  N, Dementieva  I, Collart  F, Holzle  D, Joachimiak  A,     ( 2006 )

Crystal structure of hypothetical protein YfiH from Shigella flexneri at 2 A resolution.

Proteins 63 (4)
PMID : 16498617  :   DOI  :   10.1002/prot.20589     PMC  :   PMC2792012    
Abstract >>
N/A
KeywordMeSH Terms
73. Okuda  J, Toyotome  T, Kataoka  N, Ohno  M, Abe  H, Shimura  Y, Seyedarabi  A, Pickersgill  R, Sasakawa  C,     ( 2005 )

Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses.

Biochemical and biophysical research communications 333 (2)
PMID : 15950937  :   DOI  :   10.1016/j.bbrc.2005.05.145    
Abstract >>
Shigella effectors injected into the host cell via the type III secretion system are involved in various aspects of infection. Here, we show that one of the effectors, IpaH9.8, plays a role in modulating inflammatory responses to Shigella infection. In murine lung infection model, DeltaipaH9.8 mutant caused more severe inflammatory responses with increased pro-inflammatory cytokine production levels than did wild-type Shigella, which resulted in a 30-fold decrease in bacterial colonization. Binding assays revealed that IpaH9.8 has a specific affinity to U2AF(35), a mammalian splicing factor, which interferes with U2AF(35)-dependent splicing as assayed for IgM pre-mRNA. Reducing the U2AF(35) level in HeLa cells and infecting HeLa cells with wild-type caused a decrease in the expression of the il-8, RANTES, GM-CSF, and il-1beta genes as examined by RT-PCR. The results indicate that IpaH9.8 plays a role in Shigella infection to optimize the host inflammatory responses, thus facilitating bacterial colonization within the host epithelial cells.
KeywordMeSH Terms
74. Roberts  F, Jennison  AV, Verma  NK,     ( 2005 )

The Shigella flexneri serotype Y vaccine candidate SFL124 originated from a serotype 2a background.

FEMS immunology and medical microbiology 45 (2)
PMID : 15963704  :   DOI  :   10.1016/j.femsim.2005.05.002    
Abstract >>
Shigella flexneri is endemic in most developing countries and responsible for the highest mortality rate among the Shigella species. The attenuated serotype Y S. flexneri strain SFL124 has been used as the parental strain for the development of recombinant vaccines expressing multiple O-antigen structures. During the development of one such multivalent vaccine, a region of gtrII homology was found in SFL124. Sequencing and analysis of this region revealed the presence of an insertion element interrupted serotype 2a serotype-conversion locus in the serotype Y vaccine strain SFL124. The data presented suggests that SFL124 has derived from a serotype 2a background.
KeywordMeSH Terms
75. Andrews  GP, Maurelli  AT,     ( 1992 )

mxiA of Shigella flexneri 2a, which facilitates export of invasion plasmid antigens, encodes a homolog of the low-calcium-response protein, LcrD, of Yersinia pestis.

Infection and immunity 60 (8)
PMID : 1639496  :   PMC  :   PMC257313    
Abstract >>
The plasmid-encoded invasion plasmid antigen (Ipa) export accessory locus of Shigella flexneri 2a, mxiA, was cloned, and the complete DNA sequence of the gene was determined. The mixA open reading frame was found to encode a polypeptide of 74.03 kDa with a pI of 5.02. A hydropathy analysis of the predicted protein revealed a hydrophilic C terminus and an extremely hydrophobic N terminus without a cleavable signal sequence but with several potential membrane-spanning regions. While a homology search did not reveal any significant relatedness of the mxiA DNA sequence to any known bacterial gene sequences, the derived amino acid sequence of MxiA was found to be highly homologous (68%) to the sequence of the protein encoded by the low-calcium-response locus, lcrD, of Yersinia pestis. The lcrD encodes an inner membrane regulatory protein that has an N-terminal membrane anchor and that is implicated in facilitating the export of Y. pestis outer membrane proteins (G. V. Plano, S. S. Barve, and S. C. Straley, J. Bacteriol. 173:7293-7303, 1991). Congo red binding, HeLa cell invasion, and Ipa excretion were restored in two avirulent mxiA fusion mutants when they were transformed with a cloned copy of the mxiA gene. Furthermore, the expression of the cloned mxiA gene was independent of any vector-specified promoter, suggesting that the transcription of mxiA is driven by its own promoter in this clone. In contrast, the overexpression of mxiA that resulted when it was placed under the control of the lac promoter was found to be deleterious in Escherichia coli. We conclude that mxiA is a homolog of the Y. pestis lcrD locus and may function similarly in S. flexneri, either by directly affecting the excretion of virulence factors or by regulating the expression of export accessory genes.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
Plasmids
Sequence Homology, Nucleic Acid
76. Andres  P, Petroni  A, Faccone  D, Pasterán  F, Melano  R, Rapoport  M, Martínez  M, Culasso  C, Di Bella  A, Irigoyen  B, Mulki  J, Procopio  A, von Specht  M, Galas  M,     ( 2005 )

Extended-spectrum beta-lactamases in Shigella flexneri from Argentina: first report of TOHO-1 outside Japan.

International journal of antimicrobial agents 25 (6)
PMID : 15878653  :   DOI  :   10.1016/j.ijantimicag.2005.02.016    
Abstract >>
A 9-year nation-wide survey of the presence of extended-spectrum beta-lactamases (ESBLs) in Shigella flexneri is described. Ten of 9033 (0.1%) isolates produced ESBLs, which were characterized by isoelectric focusing, PCR and DNA sequencing. These were CTX-M-2 (five isolates), TOHO-1 (one isolate), SHV-2 (two isolates) and PER-2 (two isolates, the first report in S. flexneri world wide). The emergence of each ESBL type in S. flexneri was not restricted to a particular region of Argentina. TOHO-1 showed a more basic isoelectric point (8.4) than that previously found (7.8) and its encoding gene (bla(TOHO-1a)) harboured a silent change, G825A, relative to the reported bla(TOHO-1). All the ESBL-encoding genes were transferred to Escherichia coli by conjugation. PFGE analysis indicated that the 10 ESBL-producing S. flexneri isolates were subtypes of a unique clone.
KeywordMeSH Terms
77. Tominaga  A, Lan  R, Reeves  PR,     ( 2005 )

Evolutionary changes of the flhDC flagellar master operon in Shigella strains.

Journal of bacteriology 187 (12)
PMID : 15937193  :   DOI  :   10.1128/JB.187.12.4295-4302.2005     PMC  :   PMC1151726    
Abstract >>
Shigella strains are nonmotile. The master operon of flagellar synthesis, flhDC, was analyzed for genetic damage in 46 Shigella strains representing all known serotypes. In 11 strains (B1, B3, B6, B8, B10, B18, D5, F1B, D10, F3A, and F3C) the flhDC operon was completely deleted. PCR and sequence analysis of the flhDC region of the remaining 35 strains revealed many insertions or deletions associated with insertion sequences, and the majority of the strains were found to be defective in their flhDC genes. As these genes also play a role in regulation of non-flagellar genes, the loss may have other consequences or be driven by selection pressures other than those against flagellar motility. It has been suggested that Shigella strains fall mostly into three clusters within Escherichia coli, with five outlier strains, four of which are also within E. coli (G. M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000). The distribution of genetic changes in the flhDC region correlated very well with the three clusters and outlier strains found using housekeeping gene DNA sequences, enabling us to follow the sequence of mutational change in the flhDC locus. Two cluster 2 strains were found to have unique flhDC sequences, which are most probably due to recombination during the exchange of the adjacent O-antigen gene clusters.
KeywordMeSH Terms
78. Dorman  CJ,     ( 1992 )

The VirF protein from Shigella flexneri is a member of the AraC transcription factor superfamily and is highly homologous to Rns, a positive regulator of virulence genes in enterotoxigenic Escherichia coli.

Molecular microbiology 6 (11)
PMID : 1625585  :   DOI  :   10.1111/j.1365-2958.1992.tb00879.x    
Abstract >>
N/A
KeywordMeSH Terms
79. Kim  DW, Lenzen  G, Page  AL, Legrain  P, Sansonetti  PJ, Parsot  C,     ( 2005 )

The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes.

Proceedings of the National Academy of Sciences of the United States of America 102 (39)
PMID : 16162672  :   DOI  :   10.1073/pnas.0504466102     PMC  :   PMC1236552    
Abstract >>
Bacteria of Shigella spp. are responsible for shigellosis in humans. They use a type III secretion system to inject effector proteins into host cells and induce their entry into epithelial cells or trigger apoptosis in macrophages. We present evidence that the effector OspG is a protein kinase that binds various ubiquitinylated ubiquitin-conjugating enzymes, including UbcH5, which belongs to the stem cell factor SCF(beta-TrCP) complex promoting ubiquitination of phosphorylated inhibitor of NF-kappaB type alpha (phospho-IkappaBalpha). Transfection experiments indicated that OspG can prevent phospho-IkappaBalpha degradation and NF-kappaB activation induced by TNF-alpha stimulation. Infection of epithelial cells by the S. flexneri wild-type strain, but not an ospG mutant, led to accumulation of phospho-IkappaBalpha, consistent with OspG inhibiting SCF(beta-TrCP) activity. Upon infection of ileal loops in rabbits, the ospG mutant induced a stronger inflammatory response than the wild-type strain. This finding indicates that OspG negatively controls the host innate response induced by S. flexneri upon invasion of the epithelium.
KeywordMeSH Terms
80. Le Gall  T, Mavris  M, Martino  MC, Bernardini  ML, Denamur  E, Parsot  C,     ( 2005 )

Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri.

Microbiology (Reading, England) 151 (Pt 3)
PMID : 15758240  :   DOI  :   10.1099/mic.0.27639-0    
Abstract >>
Proteins directly involved in entry and dissemination of Shigella flexneri into epithelial cells are encoded by a virulence plasmid of 200 kb. A 30-kb region (designated the entry region) of this plasmid encodes components of a type III secretion (TTS) apparatus, substrates of this apparatus and their dedicated chaperones. During growth of bacteria in broth, expression of these genes is induced at 37 degrees C and the TTS apparatus is assembled in the bacterial envelope but is not active. Secretion is activated upon contact of bacteria with host cells and is deregulated in an ipaB mutant. The plasmid encodes four transcriptional regulators, VirF, VirB, MxiE and Orf81. VirF controls transcription of virB, whose product is required for transcription of entry region genes. MxiE, with the chaperone IpgC acting as a co-activator, controls expression of several effectors that are induced under conditions of secretion. Genes under the control of Orf81 are not known. The aim of this study was to define further the repertoires of virulence plasmid genes that are under the control of (i) the growth temperature, (ii) each of the known virulence plasmid-encoded transcriptional regulators (VirF, VirB, MxiE and Orf81) and (iii) the activity of the TTS apparatus. Using a macroarray analysis, the expression profiles of 71 plasmid genes were compared in the wild-type strain grown at 37 and 30 degrees C and in virF, virB, mxiE, ipaB, ipaB mxiE and orf81 mutants grown at 37 degrees C. Many genes were found to be under the control of VirB and indirectly of VirF. No alteration of expression of any gene was detected in the orf81 mutant. Expression of 13 genes was increased in the secretion-deregulated ipaB mutant in an MxiE-dependent manner. On the basis of their expression profile, substrates of the TTS apparatus can be classified into three categories: (i) those that are controlled by VirB, (ii) those that are controlled by MxiE and (iii) those that are controlled by both VirB and MxiE. The differential regulation of expression of TTS effectors in response to the TTS apparatus activity suggests that different effectors might be required at different times following contact of bacteria with host cells.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
81. Picking  WL, Nishioka  H, Hearn  PD, Baxter  MA, Harrington  AT, Blocker  A, Picking  WD,     ( 2005 )

IpaD of Shigella flexneri is independently required for regulation of Ipa protein secretion and efficient insertion of IpaB and IpaC into host membranes.

Infection and immunity 73 (3)
PMID : 15731041  :   DOI  :   10.1128/IAI.73.3.1432-1440.2005     PMC  :   PMC1064949     DOI  :   10.1128/IAI.73.3.1432-1440.2005     PMC  :   PMC1064949    
Abstract >>
Shigella flexneri causes human dysentery after invading the cells of the colonic epithelium. The best-studied effectors of Shigella entry into colonocytes are the invasion plasmid antigens IpaC and IpaB. These proteins are exported via a type III secretion system (TTSS) to form a pore in the host membrane that may allow the translocation of other effectors into the host cytoplasm. TTSS-mediated secretion of IpaD is also required for translocation pore formation, bacterial invasion, and virulence, but the mechanistic role of this protein is unclear. IpaD is also known to be involved in controlling Ipa protein secretion, but here it is shown that this activity can be separated from its requirement for cellular invasion. Amino acids 40 to 120 of IpaD are not essential for IpaD-dependent invasion; however, deletions in this region still lead to constitutive IpaB/IpaC secretion. Meanwhile, a central deletion causes only a partial loss of control of Ipa secretion but completely eliminates IpaD's invasion function, indicating that IpaD's role in invasion is not a direct outcome of its ability to control Ipa secretion. As shigellae expressing ipaD N-terminal deletion mutations have reduced contact-mediated hemolysis activity and are less efficient at introducing IpaB and IpaC into erythrocyte membranes, it is possible that IpaD is responsible for insertion of IpaB/IpaC pores into target cell membranes. While efficient insertion of IpaB/IpaC pores is needed for optimal invasion efficiency, it may be especially important for Ipa-dependent membrane disruption and thus for efficient vacuolar escape and intercellular spread.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Gene Expression Regulation, Bacterial
82. Pandey  DP, Gerdes  K,     ( 2005 )

Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes.

Nucleic acids research 33 (3)
PMID : 15718296  :   DOI  :   10.1093/nar/gki201     PMC  :   PMC549392    
Abstract >>
Prokaryotic chromosomes code for toxin-antitoxin (TA) loci, often in multiple copies. In E.coli, experimental evidence indicates that TA loci are stress-response elements that help cells survive unfavorable growth conditions. The first gene in a TA operon codes for an antitoxin that combines with and neutralizes a regulatory 'toxin', encoded by the second gene. RelE and MazF toxins are regulators of translation that cleave mRNA and function, in interplay with tmRNA, in quality control of gene expression. Here, we present the results from an exhaustive search for TA loci in 126 completely sequenced prokaryotic genomes (16 archaea and 110 bacteria). We identified 671 TA loci belonging to the seven known TA gene families. Surprisingly, obligate intracellular organisms were devoid of TA loci, whereas free-living slowly growing prokaryotes had particularly many (38 in Mycobacterium tuberculosis and 43 in Nitrosomonas europaea). In many cases, TA loci were clustered and closely linked to mobile genetic elements. In the most extreme of these cases, all 13 TA loci of Vibrio cholerae were bona fide integron elements located in the V.cholerae mega-integron. These observations strongly suggest that TA loci are mobile cassettes that move frequently within and between chromosomes and also lend support to the hypothesis that TA loci function as stress-response elements beneficial to free-living prokaryotes.
KeywordMeSH Terms
Genes, Archaeal
Genes, Bacterial
83. Hata  M, Suzuki  M, Matsumoto  M, Takahashi  M, Sato  K, Ibe  S, Sakae  K,     ( 2005 )

Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri 2b.

Antimicrobial agents and chemotherapy 49 (2)
PMID : 15673773  :   DOI  :   10.1128/AAC.49.2.801-803.2005     PMC  :   PMC547361    
Abstract >>
A novel gene for quinolone resistance was cloned from a transferable plasmid carried by a clinical isolate of Shigella flexneri 2b that was resistant to fluoroquinolones. The plasmid conferred low-level resistance to quinolones on Escherichia coli HB101. The protein encoded by the gene showed 59% amino acid identity with Qnr.
KeywordMeSH Terms
84. d'Hauteville  H, Sansonetti  PJ,     ( 1992 )

Phosphorylation of IcsA by cAMP-dependent protein kinase and its effect on intracellular spread of Shigella flexneri.

Molecular microbiology 6 (7)
PMID : 1602963  :   DOI  :   10.1111/j.1365-2958.1992.tb01534.x    
Abstract >>
Shigella flexneri, a Gram-negative bacillus belonging to the family Enterobacteriaceae, causes bacillary dysentery in humans by invading colonic epithelial cells. Processes by which epithelial cells, which are not professional phagocytes, may limit the spread of the invading microorganisms are poorly understood. This paper shows that IcsA (VirG), a 120 kDa bacterial outer membrane protein responsible for intracellular and cell-to-cell spread through polymerization of actin, is a major substrate for phosphorylation by cyclic-dependent protein kinases. Site-directed mutagenesis of a sequence encoding phosphorylation consensus motif SSRRASS, located at residues 754-760, almost completely abolished the ability of this protein to be phosphorylated by protein kinase A. Such mutants expressed a 'super lcs' phenotype, characterized by an increased capacity to spread from cell-to-cell during the first three hours of infection in the HeLa cell infection assay. These data suggest that host-cell phosphorylation of key virulence proteins located on the bacterial surface may represent a significant host defence mechanism during the invasion process.
KeywordMeSH Terms
DNA-Binding Proteins
Transcription Factors
85. Li  B, Brown  EW, D'Agostino  C, Leclerc  JE, Cebula  TA,     ( 2005 )

Structure and distribution of the phosphoprotein phosphatase genes, prpA and prpB, among Shigella subgroups.

Microbiology (Reading, England) 151 (Pt 8)
PMID : 16079345  :   DOI  :   10.1099/mic.0.27990-0    
Abstract >>
Phosphoprotein phosphatases encoded by the prpA and prpB genes function in signal transduction pathways for degradation of misfolded proteins in the extracytoplasmic compartments of Escherichia coli. In order to trace the evolution of prp genes and assess their roles in other enteric pathogens, the structure and distribution of these genes among closely related Shigella subgroups were studied. PCR amplification, probe hybridization studies and DNA sequencing were used to determine the prp genotypes of 58 strains from the four Shigella subgroups, Dysenteriae, Boydii, Sonnei and Flexneri. It was found that the prp alleles among Shigella subgroups were extremely susceptible to gene inactivation and that the mutations involved in prp allele inactivation were varied. They included IS insertions, gene replacement by an IS element, a small deletion within the gene or large deletion engulfing the entire gene region, and base substitutions that generated premature termination codons. As a result, of 58 strains studied, only eight (14 %) possessed intact prpA and prpB genes. Of the Shigella strains examined, 76 % (44/58) showed at least one of the prp alleles inactivated by one or more IS elements, including IS1, IS4, IS600 and IS629. Phylogenetic analysis revealed that IS elements have been independently acquired in multiple lineages of Shigella, suggesting that loss of functional alleles has been advantageous during Shigella strain evolution.
KeywordMeSH Terms
Signal Transduction
86. Zhu  DY, Zhu  YQ, Huang  RH, Xiang  Y, Yang  N, Lu  HX, Li  GP, Jin  Q, Wang  DC,     ( 2005 )

Crystal structure of the copper homeostasis protein (CutCm) from Shigella flexneri at 1.7 A resolution: the first structure of a new sequence family of TIM barrels.

Proteins 58 (3)
PMID : 15624211  :   DOI  :   10.1002/prot.20362    
Abstract >>
N/A
KeywordMeSH Terms
87. Close  SM, Kado  CI,     ( 1992 )

A gene near the plasmid pSa origin of replication encodes a nuclease.

Molecular microbiology 6 (4)
PMID : 1560781  :   DOI  :   10.1111/j.1365-2958.1992.tb01497.x    
Abstract >>
We have cloned and sequenced a gene (nuc) from the IncW plasmid pSa which shows amino acid sequence similarity to staphylococcal nuclease (EC 3.1.4.7) and to the parB locus of plasmid RP4. The 525 bp open reading frame encodes a 174-amino-acid potential polypeptide of 19.7 kDa. Expression of the gene was confirmed using an in vitro transcription-translation assay which produced a protein of identical size. Nuclease activity was demonstrated using DNA as the substrate in toluidine blue-DNA agar plates. The deduced amino acid sequence revealed a signal sequence, and TnphoA insertion within the open reading frame indicated that a portion of the protein is transported across the bacterial cell membrane.
KeywordMeSH Terms
Endonucleases
88. Ogawa  M, Yoshimori  T, Suzuki  T, Sagara  H, Mizushima  N, Sasakawa  C,     ( 2005 )

Escape of intracellular Shigella from autophagy.

Science (New York, N.Y.) 307 (5710)
PMID : 15576571  :   DOI  :   10.1126/science.1106036    
Abstract >>
The degradation of undesirable cellular components or organelles, including invading microbes, by autophagy is crucial for cell survival. Here, Shigella, an invasive bacteria, was found to be able to escape autophagy by secreting IcsB by means of the type III secretion system. Mutant bacteria lacking IcsB were trapped by autophagy during multiplication within the host cells. IcsB did not directly inhibit autophagy. Rather, Shigella VirG, a protein required for intracellular actin-based motility, induced autophagy by binding to the autophagy protein, Atg5. In nonmutant Shigella, this binding is competitively inhibited by IcsB binding to VirG.
KeywordMeSH Terms
Autophagy
89. Chen  H, Ponniah  G, Salonen  N, Blum  P,     ( 2004 )

Culture-independent analysis of fecal enterobacteria in environmental samples by single-cell mRNA profiling.

Applied and environmental microbiology 70 (8)
PMID : 15294770  :   DOI  :   10.1128/AEM.70.8.4432-4439.2004     PMC  :   PMC492453    
Abstract >>
A culture-independent method called mRNA profiling has been developed for the analysis of fecal enterobacteria and their physiological status in environmental samples. This taxon-specific approach determines the single-cell content of selected gene transcripts whose abundance is either directly or inversely proportional to growth state. Fluorescence in situ hybridization using fluorochrome-labeled oligonucleotide probes was used to measure the cellular concentration of fis and dps mRNA. Relative levels of these transcripts provided a measure of cell growth state and the ability to enumerate fecal enterobacterial cell number. Orthologs were cloned by inverse PCR from several major enterobacterial genera, and probes specific for fecal enterobacteria were designed using multiple DNA sequence alignments. Probe specificity was determined experimentally using pure and mixed cultures of the major enterobacterial genera as well as secondary treated wastewater samples seeded with pure culture inocula. Analysis of the fecal enterobacterial community resident in unseeded secondary treated wastewater detected fluctuations in transcript abundance that were commensurate with incubation time and nutrient availability and demonstrated the utility of the method using environmental samples. mRNA profiling provides a new strategy to improve wastewater disinfection efficiency by accelerating water quality analysis.
KeywordMeSH Terms
Water Microbiology
90. van Eerde  A, Hamiaux  C, Pérez  J, Parsot  C, Dijkstra  BW,     ( 2004 )

Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity.

EMBO reports 5 (5)
PMID : 15088068  :   DOI  :   10.1038/sj.embor.7400144     PMC  :   PMC1299055    
Abstract >>
Type III secretion (TTS) systems are used by many Gram-negative pathogens to inject virulence proteins into the cells of their hosts. Several of these virulence effectors require TTS chaperones that maintain them in a secretion-competent state. Whereas most chaperones bind only one effector, Spa15 from the human pathogen Shigella flexneri and homologous chaperones bind several seemingly unrelated effectors, and were proposed to form a special subgroup. Its 1.8 A crystal structure confirms this specific classification, showing that Spa15 has the same fold as other TTS effector chaperones, but forms a different dimer. The presence of hydrophobic sites on the Spa15 surface suggests that the different Spa15 effectors all possess similar structural elements that can bind these sites. Furthermore, the Spa15 structure reveals larger structural differences between class I chaperones than previously anticipated, which does not support the hypothesis that chaperone-effector complexes are structurally conserved and function as three-dimensional secretion signals.
KeywordMeSH Terms
Protein Structure, Tertiary
91. Edwards-Jones  B, Langford  PR, Kroll  JS, Yu  J,     ( 2004 )

The role of the Shigella flexneri yihE gene in LPS synthesis and virulence.

Microbiology (Reading, England) 150 (Pt 4)
PMID : 15073317  :   DOI  :   10.1099/mic.0.26840-0    
Abstract >>
Previously, the authors have shown that inactivation of Shigella flexneri yihE, a gene of unknown function upstream of dsbA, which encodes a periplasmic disulphide catalyst, results in a global change of gene expression. Among the severely down-regulated genes are galETKM, suggesting that the yihE mutant, Sh54, may inefficiently produce the UDP-glucose and UDP-galactose required for LPS synthesis. This paper demonstrates that LPS synthesis in Sh54 is impaired. As a result, Sh54 is unable to polymerize host cell actin, due to aberrant localization of IcsA, or to cause keratoconjunctivitis in guinea pigs. Furthermore, Sh54 is more sensitive to some antimicrobial agents, and exhibits epithelial cytotoxicity characteristic of neither wild-type nor dsbA mutants. Supplying galETK in trans restores LPS synthesis and corrects all the defects. Hence, it is clear that the Shigella yihE gene is important not only in regulating global gene expression, as shown previously, but also in virulence through LPS synthesis via regulating the expression of the galETK operon.
KeywordMeSH Terms
92. Allaoui  A, Mounier  J, Prévost  MC, Sansonetti  PJ, Parsot  C,     ( 1992 )

icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread.

Molecular microbiology 6 (12)
PMID : 1495389  :   DOI  :   10.1111/j.1365-2958.1992.tb00885.x    
Abstract >>
Shigella flexneri causes bacillary dysentery by invading epithelial cells of the colonic mucosa. We have characterized the icsB gene which is located on the virulence plasmid pWR100. After inactivation of icsB, the mutant strain remained invasive, but formed abnormally small plaques on HeLa cell monolayers, colonized only the peripheral cells of Caco-2 islets, and was unable to provoke a keratoconjunctivitis in guinea-pigs. Examination of infected HeLa cells showed that the icsB mutant was able to lyse the phagocytic vacuole and to form protrusions at the surface of infected cells, but, unlike the wild type, remained trapped in protrusions surrounded by two membranes. These results indicate that IcsB is involved in the lysis of the protrusions, a step necessary for intercellular spread.
KeywordMeSH Terms
93. Prosseda  G, Falconi  M, Giangrossi  M, Gualerzi  CO, Micheli  G, Colonna  B,     ( 2004 )

The virF promoter in Shigella: more than just a curved DNA stretch.

Molecular microbiology 51 (2)
PMID : 14756791  :   DOI  :   10.1046/j.1365-2958.2003.03848.x    
Abstract >>
In the human enteropathogen Shigella transcription of virF, the primary regulator of the invasion functions, is strictly temperature-dependent and is antagonistically mediated by H-NS and FIS, which bind to specific sites on the virF promoter. Here we report on the relevance of DNA geometry to the thermoregulation of virF and demonstrate that the virF promoter hosts a major DNA bend halfway between two H-NS sites. The bent region has been mutagenized in vitro to mimic temperature-induced changes of DNA curvature. Functional analysis of curvature mutants and of promoter constructs in which the two H-NS sites are phased-out by a half-helix turn reveals that modifying the spatial relationships between these sites severely affects the interaction of H-NS with the virF promoter, as well as its in vivo and in vitro temperature-dependent activity. The role of promoter curvature as thermosensor is also compatible with the present observation that, with increasing temperature, the virF bending centre moves downstream at a rate having its maximum around the transition temperature, abruptly unmasking a binding site for the transcriptional activator FIS.
KeywordMeSH Terms
94. Wing  HJ, Yan  AW, Goldman  SR, Goldberg  MB,     ( 2004 )

Regulation of IcsP, the outer membrane protease of the Shigella actin tail assembly protein IcsA, by virulence plasmid regulators VirF and VirB.

Journal of bacteriology 186 (3)
PMID : 14729695  :   DOI  :   10.1128/jb.186.3.699-705.2004     PMC  :   PMC321486    
Abstract >>
The Shigella outer membrane protease IcsP removes the actin assembly protein IcsA from the bacterial surface, and consequently modulates Shigella actin-based motility and cell-to-cell spread. Here, we demonstrate that IcsP expression is undetectable in mutants lacking either of two transcriptional activators, VirF and VirB. In wild-type Shigella spp., virB expression is entirely dependent on VirF; therefore, to circumvent this regulatory cascade, we independently expressed VirF or VirB in Shigella strains lacking both activators and measured both IcsP levels and transcription from the icsP promoter. Our results show that VirB significantly enhanced icsP transcription, even in the absence of VirF. In contrast, when VirF was induced in the absence of VirB, VirF had variable effects. The regulation of icsP is distinctly different from the regulation of the gene encoding its major substrate, icsA, which is activated by VirF and not VirB. We propose that the different pathways regulating icsA and icsP may be critical to the modulation of IcsA-mediated actin-based motility by IcsP.
KeywordMeSH Terms
Plasmids
95. Yang  J, Wang  J, Chen  L, Yu  J, Dong  J, Yao  ZJ, Shen  Y, Jin  Q, Chen  R,     ( 2003 )

Identification and characterization of simple sequence repeats in the genomes of Shigella species.

Gene 322 (N/A)
PMID : 14644500  :   DOI  :   10.1016/j.gene.2003.09.017    
Abstract >>
A variety of simple sequence repeats (SSRs) have been identified in the genome of Shigella flexneri serotype 2a (strain Sf301), an enteric pathogen that causes bacillary dysentery in man. The distribution of SSRs, with unit length ranging from 1 to 9 nucleotides, was biased in different regions of the genome. The tri-, tetra- and hexanucleotide SSRs prevailed in the coding regions while the mono- and dinucleotide SSRs were more common in the noncoding regions. Many intergenic SSRs are less than 30 bp away from the downstream open reading frames (ORFs), suggesting a potential role in transcriptional regulation. To study polymorphism of SSRs, we compared 17 coding-region SSRs from strain Sf301 with the corresponding sequences from 23 other strains of four Shigella species. Five chromosomal loci were found to be polymorphic, of which those from S. flexneri strains were most variable. Particularly interesting is the C5-1 locus in the coding sequence of the hcaD gene encoding a subunit of ferredoxin reductase. Depending on the insertion of variable numbers of the unit sequence (CGCAG), the Shigella hcaD genes can encode truncated products due to premature stop codons or frame shifts, or products with extended core alpha helices that leads to radical alterations in the predicted tertiary structure. Hence, SSRs may serve as genotyping markers for epidemiological investigations, and may offer insights into evolutionary adaptation of the pathogens.
KeywordMeSH Terms
Genome, Bacterial
96. Gueneau de Novoa  P, Williams  KP,     ( 2004 )

The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts.

Nucleic acids research 32 (Database issue)
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.
KeywordMeSH Terms
Databases, Nucleic Acid
Evolution, Molecular
Internet
97. Venkatesan  MM, Buysse  JM,     ( 1990 )

Nucleotide sequence of invasion plasmid antigen gene ipaA from Shigella flexneri 5.

Nucleic acids research 18 (6)
PMID : 2183200  :   DOI  :   10.1093/nar/18.6.1648     PMC  :   PMC330554    
Abstract >>
N/A
KeywordMeSH Terms
Bacterial Proteins
98. Winther  KS, Gerdes  K,     ( 2011 )

Enteric virulence associated protein VapC inhibits translation by cleavage of initiator tRNA.

Proceedings of the National Academy of Sciences of the United States of America 108 (18)
PMID : 21502523  :   DOI  :   10.1073/pnas.1019587108     PMC  :   PMC3088637    
Abstract >>
Eukaryotic PIN (PilT N-terminal) domain proteins are ribonucleases involved in quality control, metabolism and maturation of mRNA and rRNA. The majority of prokaryotic PIN-domain proteins are encoded by the abundant vapBC toxin--antitoxin loci and inhibit translation by an unknown mechanism. Here we show that enteric VapCs are site-specific endonucleases that cleave tRNA(fMet) in the anticodon stem-loop between nucleotides +38 and +39 in vivo and in vitro. Consistently, VapC inhibited translation in vivo and in vitro. Translation-reactions could be reactivated by the addition of VapB and extra charged tRNA(fMet). Similarly, ectopic production of tRNA(fMet) counteracted VapC in vivo. Thus, tRNA(fMet) is the only cellular target of VapC. Depletion of tRNA(fMet) by vapC induction was bacteriostatic and stimulated ectopic translation initiation at elongator codons. Moreover, addition of chloramphenicol to cells carrying vapBC induced VapC activity. Thus, by cleavage of tRNA(fMet), VapC simultaneously may regulate global cellular translation and reprogram translation initiation.
KeywordMeSH Terms
99. Zhao  H, Sequeira  RD, Galeva  NA, Tang  L,     ( 2011 )

The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion.

Virology 409 (2)
PMID : 21071053  :   DOI  :   10.1016/j.virol.2010.10.030     PMC  :   PMC3053050    
Abstract >>
Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379-394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus may be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.
KeywordMeSH Terms
100. Zhu  JY, Duan  GC, Yang  HY, Fan  QT, Xi  YL,     ( 2011 )

Atypical class 1 integron coexists with class 1 and class 2 integrons in multi-drug resistant Shigella flexneri isolates from China.

Current microbiology 62 (3)
PMID : 20976456  :   DOI  :   10.1007/s00284-010-9790-3    
Abstract >>
The antimicrobial resistance and the character of integrons were determined in 58 Shigella flexneri strains isolated from China. All isolates were multi-drug resistant and found to carry integrons of class 1 (94.8%), class 2 (100%), or both (94.8%). No intI3 was detected. The typical class 1 integrons were found in conjugative plasmids and could be transferred to the recipient E. coli DH5�\. The gene cassettes of typical class 1 integrons dfrA17-aadA5 and dfrA12-orfF-aadA2 were detected in 54 strains (93.1%) and 1 strain, respectively. Atypical class 1 integrons located on the chromosome with gene cassettes bla (oxa-30)-aadA1 were detected in 55 isolates (94.8%). All the intI2 positive isolates carried gene cassettes dfrA1-sat1-aadA1. To our knowledge, this is the first report that atypical and typical class 1 integrons coexisted with class 2 integron in multi-drug resistant S. flexneri strains.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
Integrons
101. Seyedarabi  A, Sullivan  JA, Sasakawa  C, Pickersgill  RW,     ( 2010 )

A disulfide driven domain swap switches off the activity of Shigella IpaH9.8 E3 ligase.

FEBS letters 584 (19)
PMID : 20831869  :   DOI  :   10.1016/j.febslet.2010.09.006    
Abstract >>
We show that the monomeric form of Shigella IpaH9.8 E3 ligase catalyses the ubiquitination of human U2AF35 in vitro, providing a molecular mechanism for the observed in vivo effect. We further discover that under non-reducing conditions IpaH9.8 undergoes a domain swap driven by the formation of a disulfide bridge involving the catalytic cysteine and that this dimer is unable to catalyse the ubiquitination of U2AF35. The crystal structure of the domain-swapped dimer is presented. The redox inactivation of IpaH9.8 could be a mechanism of regulating the activity of the IpaH9.8 E3 ligase in response to cell damage so that the host cell in which the bacteria resides is maintained in a benign state suitable for bacterial survival.
KeywordMeSH Terms
102. Klink  BU, Barden  S, Heidler  TV, Borchers  C, Ladwein  M, Stradal  TE, Rottner  K, Heinz  DW,     ( 2010 )

Structure of Shigella IpgB2 in complex with human RhoA: implications for the mechanism of bacterial guanine nucleotide exchange factor mimicry.

The Journal of biological chemistry 285 (22)
PMID : 20363740  :   DOI  :   10.1074/jbc.M110.107953     PMC  :   PMC2878080    
Abstract >>
A common theme in bacterial pathogenesis is the manipulation of eukaryotic cells by targeting the cytoskeleton. This is in most cases achieved either by modifying actin, or indirectly via activation of key regulators controlling actin dynamics such as Rho-GTPases. A novel group of bacterial virulence factors termed the WXXXE family has emerged as guanine nucleotide exchange factors (GEFs) for these GTPases. The precise mechanism of nucleotide exchange, however, has remained unclear. Here we report the structure of the WXXXE-protein IpgB2 from Shigella flexneri and its complex with human RhoA. We unambiguously identify IpgB2 as a bacterial RhoA-GEF and dissect the molecular mechanism of GDP release, an essential prerequisite for GTP binding. Our observations uncover that IpgB2 induces conformational changes on RhoA mimicking DbI- but not DOCK family GEFs. We also show that dissociation of the GDP.Mg(2+) complex is preceded by the displacement of the metal ion to the alpha-phosphate of the nucleotide, diminishing its affinity to the GTPase. These data refine our understanding of the mode of action not only of WXXXE GEFs but also of mammalian GEFs of the DH/PH family.
KeywordMeSH Terms
103. Arbing  MA, Handelman  SK, Kuzin  AP, Verdon  G, Wang  C, Su  M, Rothenbacher  FP, Abashidze  M, Liu  M, Hurley  JM, Xiao  R, Acton  T, Inouye  M, Montelione  GT, Woychik  NA, Hunt  JF,     ( 2010 )

Crystal structures of Phd-Doc, HigA, and YeeU establish multiple evolutionary links between microbial growth-regulating toxin-antitoxin systems.

Structure (London, England : 1993) 18 (8)
PMID : 20696400  :   DOI  :   10.1016/j.str.2010.04.018    
Abstract >>
Bacterial toxin-antitoxin (TA) systems serve a variety of physiological functions including regulation of cell growth and maintenance of foreign genetic elements. Sequence analyses suggest that TA families are linked by complex evolutionary relationships reflecting likely swapping of functional domains between different TA families. Our crystal structures of Phd-Doc from bacteriophage P1, the HigA antitoxin from Escherichia coli CFT073, and YeeU of the YeeUWV systems from E. coli K12 and Shigella flexneri confirm this inference and reveal additional, unanticipated structural relationships. The growth-regulating Doc toxin exhibits structural similarity to secreted virulence factors that are toxic for eukaryotic target cells. The Phd antitoxin possesses the same fold as both the YefM and NE2111 antitoxins that inhibit structurally unrelated toxins. YeeU, which has an antitoxin-like activity that represses toxin expression, is structurally similar to the ribosome-interacting toxins YoeB and RelE. These observations suggest extensive functional exchanges have occurred between TA systems during bacterial evolution.
KeywordMeSH Terms
Evolution, Molecular
Models, Molecular
Protein Conformation
104. Ashida  H, Kim  M, Schmidt-Supprian  M, Ma  A, Ogawa  M, Sasakawa  C,     ( 2010 )

A bacterial E3 ubiquitin ligase IpaH9.8 targets NEMO/IKKgamma to dampen the host NF-kappaB-mediated inflammatory response.

Nature cell biology 12 (1)
PMID : 20010814  :   DOI  :   10.1038/ncb2006     PMC  :   PMC3107189    
Abstract >>
NF-kappaB (nuclear factor kappaB) has a pivotal role in many cellular processes, including the inflammatory and immune responses and, therefore, its activation is tightly regulated by the IKK (IkappaB kinase) complex and by IkappaBalpha degradation. When Shigella bacteria multiply within epithelial cells they release peptidoglycans, which are recognized by Nod1 and stimulate the NF-kappaB pathway, thus leading to a severe inflammatory response. Here, we show that IpaH9.8, a Shigella effector possessing E3 ligase activity, dampens the NF-kappaB-mediated inflammatory response to the bacterial infection in a unique way. IpaH9.8 interacts with NEMO/IKKgamma and ABIN-1, a ubiquitin-binding adaptor protein, promoting ABIN-1-dependent polyubiquitylation of NEMO. Consequently, polyubiquitylated NEMO undergoes proteasome-dependent degradation, which perturbs NF-kappaB activation. As NEMO is essential for NF-kappaB activation, we propose that the polyubiquitylation and degradation of NEMO during Shigella infection is a new bacterial strategy to modulate host inflammatory responses.
KeywordMeSH Terms
105. Ye  C, Lan  R, Xia  S, Zhang  J, Sun  Q, Zhang  S, Jing  H, Wang  L, Li  Z, Zhou  Z, Zhao  A, Cui  Z, Cao  J, Jin  D, Huang  L, Wang  Y, Luo  X, Bai  X, Wang  Y, Wang  P, Xu  Q, Xu  J,     ( 2010 )

Emergence of a new multidrug-resistant serotype X variant in an epidemic clone of Shigella flexneri.

Journal of clinical microbiology 48 (2)
PMID : 19955273  :   DOI  :   10.1128/JCM.00614-09     PMC  :   PMC2815595    
Abstract >>
Shigella spp. are the causative agent of shigellosis with Shigella flexneri serotype 2a being the most prevalent in developing countries. Epidemiological surveillance in China found that a new serotype of S. flexneri appeared in 2001 and replaced serotype 2a in 2003 as the most prevalent serotype in Henan Province. The new serotype also became the dominant serotype in 7 of the 10 other provinces under surveillance in China by 2007. The serotype was identified as a variant of serotype X. It differs from serotype X by agglutination to the monovalent anti-IV type antiserum and the group antigen-specific monoclonal antibody MASF IV-I. Genome sequencing of a serotype X variant isolate, 2002017, showed that it acquired a Shigella serotype conversion island, also as an SfX bacteriophage, containing gtr genes for type X-specific glucosylation. Multilocus sequence typing of 15 genes from 37 serotype X variant isolates and 69 isolates of eight other serotypes, 1a, 2a, 2b, 3a, 4a, 5b, X, and Y, found that all belong to a new sequence type (ST), ST91. Pulsed-field gel electrophoresis revealed 154 pulse types with 655 S. flexneri isolates analyzed and identified 57 serotype switching events. The data suggest that S. flexneri epidemics in China have been caused by a single epidemic clone, ST91, with frequent serotype switching to evade infection-induced immunity to serotypes to which the population was exposed previously. The clone has also acquired resistance to multiple antibiotics. These findings underscore the challenges to the current vaccine development and control strategies for shigellosis.
KeywordMeSH Terms
Bacterial Typing Techniques
Drug Resistance, Multiple, Bacterial
106. Singarapu  KK, Mills  JL, Xiao  R, Acton  T, Punta  M, Fischer  M, Honig  B, Rost  B, Montelione  GT, Szyperski  T,     ( 2010 )

Solution NMR structures of proteins VPA0419 from Vibrio parahaemolyticus and yiiS from Shigella flexneri provide structural coverage for protein domain family PFAM 04175.

Proteins 78 (3)
PMID : 19927321  :   DOI  :   10.1002/prot.22630     PMC  :   PMC2860719    
Abstract >>
N/A
KeywordMeSH Terms
107. Tan  K, Clancy  S, Borovilos  M, Zhou  M, Hörer  S, Moy  S, Volkart  LL, Sassoon  J, Baumann  U, Joachimiak  A,     ( 2009 )

The mannitol operon repressor MtlR belongs to a new class of transcription regulators in bacteria.

The Journal of biological chemistry 284 (52)
PMID : 19840941  :   DOI  :   10.1074/jbc.M109.062679     PMC  :   PMC2794781    
Abstract >>
Many bacteria express phosphoenolpyruvate-dependent phosphotransferase systems (PTS). The mannitol-specific PTS catalyze the uptake and phosphorylation of d-mannitol. The uptake system comprises several genes encoded in the single operon. The expression of the mannitol operon is regulated by a proposed transcriptional factor, mannitol operon repressor (MtlR) that was first studied in Escherichia coli. Here we report the first crystal structures of MtlR from Vibrio parahemeolyticus (Vp-MtlR) and its homolog YggD protein from Shigella flexneri (Sf-YggD). MtlR and YggD belong to the same protein family (Pfam05068). Although Vp-MtlR and Sf-YggD share low sequence identity (22%), their overall structures are very similar, representing a novel all alpha-helical fold, and indicate similar function. However, their lack of any known DNA-binding structural motifs and their unfavorable electrostatic properties imply that MtlR/YggD are unlikely to bind a specific DNA operator directly as proposed earlier. This structural observation is further corroborated by in vitro DNA-binding studies of E. coli MtlR (Ec-MtlR), which detected no interaction of Ec-MtlR with the well characterized mannitol operator/promoter region. Therefore, MtlR/YggD belongs to a new class of transcription factors in bacteria that may regulate gene expression indirectly as a part of a larger transcriptional complex.
KeywordMeSH Terms
108. Kiyono  M, Sone  Y, Nakamura  R, Pan-Hou  H, Sakabe  K,     ( 2009 )

The MerE protein encoded by transposon Tn21 is a broad mercury transporter in Escherichia coli.

FEBS letters 583 (7)
PMID : 19265693  :   DOI  :   10.1016/j.febslet.2009.02.039    
Abstract >>
In order to clarify the physiological role of the merE gene of transposon Tn21, a pE4 plasmid that contained the merR gene of plasmid pMR26 from Pseudomonas strain K-62, and the merE gene of Tn21 from the Shigella flexneri plasmid NR1 (R100) was constructed. Bacteria with plasmid pE4 (merR-o/p-merE) were more hypersensitive to CH(3)Hg(I) and Hg(II), and took up significantly more CH(3)Hg(I) and Hg(II), than the isogenic strain. The MerE protein encoded by pE4 was localized in the membrane cell fraction, but not in the soluble fraction. Based on these experimental results, we suggest for the first time that the merE gene is a broad mercury transporter mediating the transport of both CH(3)Hg(I) and Hg(II) across the bacterial membrane.
KeywordMeSH Terms
109. Hu  Y, Fan  CP, Fu  G, Zhu  D, Jin  Q, Wang  DC,     ( 2008 )

Crystal structure of a glutamate/aspartate binding protein complexed with a glutamate molecule: structural basis of ligand specificity at atomic resolution.

Journal of molecular biology 382 (1)
PMID : 18640128  :   DOI  :   10.1016/j.jmb.2008.06.091    
Abstract >>
The crystal structure of a periplasmic l-aspartate/l-glutamate binding protein (DEBP) from Shigella flexneri complexed with an l-glutamate molecule has been determined and refined to an atomic resolution of 1.0 A. There are two DEBP molecules in the asymmetric unit. The refined model contains 4462 non-hydrogen protein atoms, 730 water molecules, 2 bound glutamate molecules, and 2 Tris molecules from the buffer used in crystallization. The final R(cryst) and R(free) factors are 13.61% and 16.89%, respectively. The structure has root-mean-square deviations of 0.016 A from standard bond lengths and 2.35 degrees from standard bond angles. The DEBP molecule is composed of two similarly folded domains separated by the ligand binding region. Both domains contain a central five-stranded beta-sheet that is surrounded by several alpha-helices. The two domains are linked by two antiparallel beta-strands. The overall shape of DEBP is that of an ellipsoid approximately 55 A x 45 A x 40 A in size. The binding of ligand to DEBP is achieved mostly through hydrogen bonds between the glutamate and side-chain and main-chain groups of DEBP. Side chains of residues Arg24, Ser72, Arg75, Ser90, and His164 anchor the deprotonated gamma-carboxylate group of the glutamate with six hydrogen bonds. Side chains of Arg75 and Arg90 form salt bridges with the deprotonated alpha-carboxylate group, while the main-chain amide groups of Thr92 and Thr140 form hydrogen bonds with the same group. The positively charged alpha-amino group of the L-glutamate forms salt bridge interaction with the side-chain carboxylate group of Asp182 and hydrogen bond interaction with main-chain carbonyl oxygen of Ser90. In addition to these hydrogen bond and electrostatic interactions, other interactions may also play important roles. For example, the two methylene groups from the glutamate form van der Waals interactions with hydrophobic side chains of DEBP. Comparisons with several other periplasmic amino acid binding proteins indicate that DEBP residues involved in the binding of alpha-amino and alpha-carboxylate groups of the ligand and the pattern of hydrogen bond formation between these groups are very well conserved, but the binding pocket around the ligand side chain is not, leading to the specificity of DEBP. We have identified structural features of DEBP that determine its ability of binding glutamate and aspartate, two molecules with different sizes, but discriminating against very similar glutamine and asparagine molecules.
KeywordMeSH Terms
110. Zhu  Y, Li  H, Hu  L, Wang  J, Zhou  Y, Pang  Z, Liu  L, Shao  F,     ( 2008 )

Structure of a Shigella effector reveals a new class of ubiquitin ligases.

Nature structural & molecular biology 15 (12)
PMID : 18997779  :   DOI  :   10.1038/nsmb.1517    
Abstract >>
Bacterial pathogens have evolved effector proteins with ubiquitin E3 ligase activities through structural mimicking. Here we report the crystal structure of the Shigella flexneri type III effector IpaH3, a member of the leucine-rich repeat (LRR)-containing bacterial E3 family. The LRR domain is structurally similar to Yersinia pestis YopM and potentially binds to substrates. The structure of the C-terminal E3 domain differs from the typical RING- and HECT-type E3s. IpaH3 synthesizes a Lys48-linked ubiquitin chain, and the reaction requires noncovalent binding between ubiquitin and a specific E2, UbcH5. Free ubiquitin serves as an acceptor for IpaH3-catalyzed ubiquitin transfer. Cys363 within a conserved CXD motif acts as a nucleophile to catalyze ubiquitin transfer through a transthiolation reaction. The D365N mutant is devoid of E3 activities but turns into a potent ubiquitin-E2 thioesterase. Our analysis establishes a structurally and mechanistically distinct class of ubiquitin ligases found exclusively in pathogenic or symbiotic bacteria.
KeywordMeSH Terms
111. Schmitt  MP, Payne  SM,     ( 1991 )

Genetic analysis of the enterobactin gene cluster in Shigella flexneri.

Journal of bacteriology 173 (2)
PMID : 1846151  :   DOI  :   10.1128/jb.173.2.816-825.1991     PMC  :   PMC207076    
Abstract >>
The genes for transport and synthesis of the phenolate siderophore enterobactin are present on the chromosomes of both Ent+ and Ent- clinical isolates of Shigella flexneri. To determine why Ent- S. flexneri isolates fail to express a functional enterobactin system, the structure and expression of enterobactin genes were examined. Several alterations may be responsible for the inability of S. flexneri to express enterobactin. (i) The mRNA levels produced from the entC and fepB genes were not derepressed in low-iron media. (ii) DNA sequence analysis of the entC-fepB intergenic region revealed an 83-bp noncontiguous deletion in the putative fepB leader sequence. The deleted sequences are in a region which would be capable of forming extensive stem-and-loop structures. (iii) An amber codon in the 5' portion of the entC gene was also detected. (iv) An IS1 element, previously mapped to the Ent- S. flexneri enterobactin gene cluster, was found to lie within a potential transcriptional termination sequence in the entF-fepE intergenic region. (v) A mutation responsible for the inactivation of the entF gene was mapped to the entF coding region by using entF hybrid gene fusions. (vi) A comparison of outer membrane profiles from an E. coli strain harboring the cloned fepA gene from either an Ent+ or Ent- Shigella isolate revealed that the Ent- FepA protein is present in the outer membrane but at greatly reduced levels than that of the Ent+ FepA protein. This observation, along with additional studies, suggests that the Ent- FepA may be defective in translation and/or translocation.
KeywordMeSH Terms
Enterobactin
Genes, Bacterial
Multigene Family
112. von Rhein  C, Bauer  S, Simon  V, Ludwig  A,     ( 2008 )

Occurrence and characteristics of the cytolysin A gene in Shigella strains and other members of the family Enterobacteriaceae.

FEMS microbiology letters 287 (2)
PMID : 18754791  :   DOI  :   10.1111/j.1574-6968.2008.01290.x    
Abstract >>
Cytolysin A (ClyA, HlyE, SheA) is a hemolytic pore-forming toxin found in Escherichia coli and Salmonella enterica serovars Typhi and Paratyphi A. In the present study, analysis of several Shigella strains revealed that they harbor only nonfunctional clyA gene copies that have been inactivated either by the integration of insertion sequence (IS) elements (Shigella dysenteriae, Shigella boydii, and Shigella sonnei strains) or by a frameshift mutation (Shigella flexneri). Shigella dysenteriae and S. boydii strains also exhibited IS-associated deletions at the clyA locus. PCR and Southern blot analyses as well as database searches indicated that clyA-related DNA sequences are completely absent in strains belonging to various other genera of the family Enterobacteriaceae. According to these data, ClyA may play a role only for a rather small subset of the enteric bacteria.
KeywordMeSH Terms
113. Close  SM, Kado  CI,     ( 1991 )

The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity.

Journal of bacteriology 173 (17)
PMID : 1832152  :   DOI  :   10.1128/jb.173.17.5449-5456.1991     PMC  :   PMC208257    
Abstract >>
The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.
KeywordMeSH Terms
Genes, Bacterial
Plasmids
Suppression, Genetic
114. Goldman  SR, Tu  Y, Goldberg  MB,     ( 2008 )

Differential regulation by magnesium of the two MsbB paralogs of Shigella flexneri.

Journal of bacteriology 190 (10)
PMID : 18359815  :   DOI  :   10.1128/JB.00151-08     PMC  :   PMC2395013    
Abstract >>
Shigella flexneri, a gram-negative enteric pathogen, is unusual in that it contains two nonredundant paralogous genes that encode the myristoyl transferase MsbB (LpxM) that catalyzes the final step in the synthesis of the lipid A moiety of lipopolysaccharide. MsbB1 is encoded on the chromosome, and MsbB2 is encoded on the large virulence plasmid present in all pathogenic shigellae. We demonstrate that myristoyl transferase activity due to MsbB2 is detected in limited magnesium medium, but not in replete magnesium medium, whereas that due to MsbB1 is detected under both conditions. MsbB2 increases overall hexa-acylation of lipid A under limited magnesium conditions. Regulation of MsbB2 by magnesium occurs at the level of transcription and is dependent on the conserved magnesium-inducible PhoPQ two-component regulatory pathway. Direct hexanucleotide repeats within the promoter upstream of msbB2 were identified as a putative PhoP binding site, and mutations within the repeats led to diminished PhoP-dependent expression of a transcriptional fusion of lacZ to this promoter. Thus, the virulence plasmid-encoded paralog of msbB is induced under limited magnesium in a PhoPQ-dependent manner. PhoPQ regulates the response of many Enterobacteriaceae to environmental signals, which include modifications of lipid A that confer increased resistance of the organism to stressful environments and antimicrobial peptides. The findings reported here are the first example of gene duplication in which one paralog has selectively acquired the mechanism for differential regulation by PhoPQ. Our findings provide molecular insight into the mechanisms by which each of the two MsbB proteins of S. flexneri likely contributes to pathogenesis.
KeywordMeSH Terms
115. Slagowski  NL, Kramer  RW, Morrison  MF, LaBaer  J, Lesser  CF,     ( 2008 )

A functional genomic yeast screen to identify pathogenic bacterial proteins.

PLoS pathogens 4 (1)
PMID : 18208325  :   DOI  :   10.1371/journal.ppat.0040009     PMC  :   PMC2211553    
Abstract >>
Many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. Identification of these translocated proteins is essential to understanding pathogenesis. Yet, their identification remains limited. This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens. Translocated proteins have been observed to confer toxic phenotypes when expressed in the yeast Saccharomyces cerevisiae. This observation suggests that yeast growth inhibition can be used as an indicator of protein translocation in functional genomic screens. However, limited information is available regarding the behavior of non-translocated proteins in yeast. We developed a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel. We observed that expression of half of the 19 Shigella translocated proteins tested but almost none of the 20 non-translocated Shigella proteins nor approximately 1,000 Francisella tularensis proteins significantly inhibited yeast growth. Not only does this study establish that yeast growth inhibition is a sensitive and specific indicator of translocated proteins, but we also identified a new substrate of the Shigella type III secretion system (TTSS), IpaJ, previously missed by other experimental approaches. In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes. By providing positive rather than negative indication of activity our assay complements existing approaches for identification of translocated proteins. In addition, because this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to genetically manipulate or dangerous to culture.
KeywordMeSH Terms
Genes, Fungal
Genome, Fungal
116. Rohde  JR, Breitkreutz  A, Chenal  A, Sansonetti  PJ, Parsot  C,     ( 2007 )

Type III secretion effectors of the IpaH family are E3 ubiquitin ligases.

Cell host & microbe 1 (1)
PMID : 18005683  :   DOI  :   10.1016/j.chom.2007.02.002    
Abstract >>
Many bacteria pathogenic for plants or animals, including Shigella spp., which is responsible for shigellosis in humans, use a type III secretion apparatus to inject effector proteins into host cells. Effectors alter cell signaling and host responses induced upon infection; however, their precise biochemical activities have been elucidated in very few cases. Utilizing Saccharomyces cerevisiae as a surrogate host, we show that the Shigella effector IpaH9.8 interrupts pheromone response signaling by promoting the proteasome-dependent destruction of the MAPKK Ste7. In vitro, IpaH9.8 displayed ubiquitin ligase activity toward ubiquitin and Ste7. Replacement of a Cys residue that is invariant among IpaH homologs of plant and animal pathogens abolished the ubiquitin ligase activity of IpaH9.8. We also present evidence that the IpaH homolog SspH1 from Salmonella enterica can ubiquitinate ubiquitin and PKN1, a previously identified SspH1 interaction partner. This study assigns a function for IpaH family members as E3 ubiquitin ligases.
KeywordMeSH Terms
117. Hu  GZ, Chen  HY, Si  HB, Deng  LX, Wei  ZY, Yuan  L, Kuang  XH,     ( 2008 )

Phenotypic and molecular characterization of TEM-116 extended-spectrum beta-lactamase produced by a Shigella flexneri clinical isolate from chickens.

FEMS microbiology letters 279 (2)
PMID : 18093137  :   DOI  :   10.1111/j.1574-6968.2007.01017.x    
Abstract >>
Extended-spectrum beta-lactamases (ESBLs) produced by a clinical isolate of Shigella flexneri from chickens were detected with confirmatory phenotypic tests of the Clinical and Laboratory Standards Institute, and minimum inhibitory concentrations of several antibacterial drugs against the isolate were determined by the twofold dilution method. The genotype and subtype of the ESBL-producing S. flexneri isolate were identified by PCR amplifying of ESBL genes and DNA sequencing analysis. The results revealed that the isolate was able to produce ESBLs. They were resistant to third-generation cephalosporins such as ceftiofur and ceftriaxone and showed characteristics of multidrug resistance. The ESBL gene from the S. flexneri isolate was of the TEM type. Sequence analysis indicated that the TEM-type gene had 99.1% and 99.2% identity to TEM-1D ESBL and TEM-1 beta-lactamase, respectively, at the nucleotide level. The amino acid sequence inferred from the TEM-type gene revealed three substitutions compared with the TEM-1 and TEM-1D enzymes: Ser51Gly, Val82Ila and Ala182Val. When it was compared with TEM-116 (99.8% identity), there were only two mutations (A(151)G and T(403)C) in the TEM-type gene, resulting in the substitution of Ser to Gly at position 51 in the amino acid sequence. The TEM type was a TEM-116 derivative.
KeywordMeSH Terms
118. Vorontsov  II, Minasov  G, Brunzelle  JS, Shuvalova  L, Kiryukhina  O, Collart  FR, Anderson  WF,     ( 2007 )

Crystal structure of an apo form of Shigella flexneri ArsH protein with an NADPH-dependent FMN reductase activity.

Protein science : a publication of the Protein Society 16 (11)
PMID : 17962405  :   DOI  :   10.1110/ps.073029607     PMC  :   PMC2211697    
Abstract >>
The arsH gene or its homologs are a frequent part of the arsenic resistance system in bacteria and eukaryotes. Although a specific biological function of the gene product is unknown, the ArsH protein was annotated as a member of the NADPH-dependent FMN reductase family based on a conserved (T/S)XRXXSX(T/S) fingerprint motif common for FMN binding proteins. Presented here are the first crystal structure of an ArsH protein from Shigella flexneri refined at 1.7 A resolution and results of enzymatic activity assays that revealed a strong NADPH-dependent FMN reductase and low azoreductase activities. The ArsH apo protein has an alpha/beta/alpha-fold typical for FMN binding proteins. The asymmetric unit consists of four monomers, which form a tetramer. Buried surface analysis suggests that this tetramer is likely to be the relevant biological assembly. Dynamic light scattering experiments are consistent with this hypothesis and show that ArsH in solution at room temperature does exist predominantly in the tetrameric form.
KeywordMeSH Terms
119. Venkatesan  MM, Buysse  JM, Hartman  AB,     ( 1991 )

Sequence variation in two ipaH genes of Shigella flexneri 5 and homology to the LRG-like family of proteins.

Molecular microbiology 5 (10)
PMID : 1791758  :   DOI  :   10.1111/j.1365-2958.1991.tb02089.x    
Abstract >>
Oligonucleotide primers derived from the ipaH7.8 sequence have been used to determine the boundaries of DNA sequence homology among five ipaH genes on the invasion plasmid (pWR100) of Shigella flexneri 5, strain M9OT-W. The primary structure of IpaH4.5 has been established from DNA sequence analysis. The first 197 amino acids in IpaH7.8 were replaced in IpaH4.5 by a unique set of 251 amino acids, generating two related proteins with variable and conserved sequences. The amino-terminal region of IpaH4.5 displayed an internal repeat structure, also seen in IpaH7.8, characteristic of members of the leucine-rich glycoprotein (LRG) family. The DNA sequences of ipaH2.5 and ipaH1.4 indicate that these genes are truncated versions of ipaH7.8. Western blot analysis of a lambda gt11 ipaH recombinant (W7) subclone demonstrated that the antigenicity of IpaH7.8 resides outside the leucine-rich repetitive region.
KeywordMeSH Terms
Antigens, Bacterial
Genes, Bacterial
Genetic Variation
120. Tang  SS, Carlin  NI, Talukder  KA, Cam  PD, Verma  NK,     ( 2016 )

Shigella flexneri serotype 1c derived from serotype 1a by acquisition of gtrIC gene cluster via a bacteriophage.

BMC microbiology 16 (1)
PMID : 27349637  :   DOI  :   10.1186/s12866-016-0746-z     PMC  :   PMC4924310    
Abstract >>
Shigella spp. are the primary causative agents of bacillary dysentery. Since its emergence in the late 1980s, the S. flexneri serotype 1c remains poorly understood, particularly with regard to its origin and genetic evolution. This article provides a molecular insight into this novel serotype and the gtrIC gene cluster that determines its unique immune recognition. A PCR of the gtrIC cluster showed that serotype 1c isolates from different geographical origins were genetically conserved. An analysis of sequences flanking the gtrIC cluster revealed remnants of a prophage genome, in particular integrase and tRNA(Pro) genes. Meanwhile, Southern blot analyses on serotype 1c, 1a and 1b strains indicated that all the tested serotype 1c strains may have had a common origin that has since remained distinct from the closely related 1a and 1b serotypes. The identification of prophage genes upstream of the gtrIC cluster is consistent with the notion of bacteriophage-mediated integration of the gtrIC cluster into a pre-existing serotype. This is the first study to show that serotype 1c isolates from different geographical origins share an identical pattern of genetic arrangement, suggesting that serotype 1c strains may have originated from a single parental strain. Analysis of the sequence around the gtrIC cluster revealed a new site for the integration of the serotype converting phages of S. flexneri. Understanding the origin of new pathogenic serotypes and the molecular basis of serotype conversion in S. flexneri would provide information for developing cross-reactive Shigella vaccines.
KeywordMeSH Terms
Bacillary dysentery
Evolutionary origin
Glucosyltransferase
Serotype 1c
Serotype-conversion
Shigella flexneri
Serogroup
121. Yamada  M, Sasakawa  C, Okada  N, Makino  SI, Yoshikawa  M,     ( 1989 )

Molecular cloning and characterization of chromosomal virulence region kcpA of Shigella flexneri.

Molecular microbiology 3 (2)
PMID : 2668687  :   DOI  :   10.1111/j.1365-2958.1989.tb01809.x    
Abstract >>
In Shigella flexneri, in addition to several well-recognized plasmid-borne virulence loci, at least three genetic loci implicated in pathogenesis have been recognized on the chromosome. To understand more about the pathogenesis of bacillary dysentery at a molecular level, the genetically recognized but previously unidentified KcpA region (one of the chromosomal regions near purE) was cloned and sequenced. A single translatable open reading frame encoding a 12310 Dalton protein corresponding to the minicell product was found. Immunofluorescence microscopy, as well as optical and electron microscopic comparison of tissue-cultured cells and guinea-pigs' eyes infected with wild-type or kcpA mutant bacteria, revealed that the kcpA product is required by invading bacteria for spread into adjacent cells.
KeywordMeSH Terms
Cloning, Molecular
DNA, Bacterial
122. Sankaran  K, Ramachandran  V, Subrahmanyam  YV, Rajarathnam  S, Elango  S, Roy  RK,     ( 1989 )

Congo red-mediated regulation of levels of Shigella flexneri 2a membrane proteins.

Infection and immunity 57 (8)
PMID : 2663721  :   PMC  :   PMC313456    
Abstract >>
The ability of Shigella spp. to bind Congo red from agar medium is generally correlated with their virulence properties. We used a metabolically active culture of Shigella flexneri 2a to determine the effect of Congo red on its membrane protein profiles. Virulent S. flexneri grown in the presence of Congo red at 37 degrees C showed increased levels of three proteins with Mrs of 43,000, 58,000, and 63,000 (43K, 58K, and 63K proteins) in the Sarkosyl-soluble membrane fractions. The observed phenomenon was temperature dependent. At 30 or 42 degrees C the protein levels remained unaffected by the presence of Congo red. Similar regulation of the levels of the 43K, 58K, and 63K membrane proteins was also observed with Shigella dysenteriae 1 and enteroinvasive Escherichia coli, but not with enteropathogenic E. coli. The cellular uptake of Congo red seemed to be essential, but not sufficient, for regulation. All three proteins reacted with human convalescent-phase sera in immunoblots of S. flexneri 2a Sarkosyl-soluble membrane fractions. Using the 43K-specific antiserum as the primary antibody, by indirect immunofluorescence studies, we detected an increase in the level of the 43K protein in S. flexneri which had invaded epithelial cells. These observations strongly indicate that the 43K, 58K, and 63K proteins are virulence associated. We propose that the observed regulatory effect of Congo red on membrane proteins of S. flexneri is mediated through induction. Since the same regulatory effect was also observed during the invasion of epithelial cells by S. flexneri, it is suggested that Congo red mimics some host tissue factor in vitro.
KeywordMeSH Terms
123. Sasakawa  C, Adler  B, Tobe  T, Okada  N, Nagai  S, Komatsu  K, Yoshikawa  M,     ( 1989 )

Functional organization and nucleotide sequence of virulence Region-2 on the large virulence plasmid in Shigella flexneri 2a.

Molecular microbiology 3 (9)
PMID : 2552264  :   DOI  :   10.1111/j.1365-2958.1989.tb00269.x    
Abstract >>
The 7 kb virulence Region-2 of the large (virulence) plasmid in Shigella flexneri 2a encodes several proteins required for invasion of intestinal epithelial cells. Insertion and deletion mutagenesis, DNA subcloning and SDS-polyacrylamide gel electrophoresis of proteins synthesized in minicells demonstrated five genes in this region. They encode 24, 18, 62 (IpaB), 41 (IpaC) and 37 (IpaD)-kiloDalton (kD) proteins. Complementation of Tn5-induced mutations in Region-2 with the above plasmid constructs indicated that Region-2 consists of two operons and that the three Ipa proteins are essential for the virulence phenotype. The transcriptional organization determined by Northern blotting, S1 nuclease protection and the effect of Tn5 insertions on expression of the Ipa proteins revealed that Region-2 has three promoters that transcribe RNAs of 4.0, 4.5 and 7.5 kb. The 4.0 kb RNA was the transcript for the operon encoding the 24, 18 kD, IpaB and C proteins and the 4.5 kb RNA for the ipaD gene. In addition, the full-length RNA of 7.5 kb which covers Region-2 supplemented full expression of the Ipa proteins. The 7663 nucleotides of Region-2 were determined to confirm the five open reading frames encoding 23,655, 17,755, 62,168, 41,077 and 36,660 Dalton proteins, respectively, and their regulatory sequences.
KeywordMeSH Terms
124. Lett  MC, Sasakawa  C, Okada  N, Sakai  T, Makino  S, Yamada  M, Komatsu  K, Yoshikawa  M,     ( 1989 )

virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the virG protein and determination of the complete coding sequence.

Journal of bacteriology 171 (1)
PMID : 2644195  :   DOI  :   10.1128/jb.171.1.353-359.1989     PMC  :   PMC209595    
Abstract >>
On the 230-kilobase-pair (kb) virulence plasmid of Shigella flexneri 2a strain YSH6000, at least seven separate genetic determinants have been identified. One of them, an approximately 4-kb region, virG, that is required for the Sereny reaction, was extensively studied to examine the role of the virG region. The phenotype of a VirG- mutant (M94) of YSH6000 in the cytoplasm of cultured MK cells was characterized by a kinetic study of the invading shigellae. The observed phenotype of M94 in the cytoplasm indicated that the virG locus is not required for multiplication of the invading shigellae, but is essential for their spread to adjacent cells. The DNA region necessary for the VirG function was localized to a 3.6-kb DNA sequence on the 230-kb plasmid. A 130-kilodalton polypeptide was confirmed to be the virG product. External labeling of bacteria with 125I indicated that the 130-kilodalton virG protein is exposed on the bacterial surface. The nucleotide sequence of 4,472 bp, which contains the functional virG gene and its own regulatory sequence, was determined, and a large open reading frame encoding 1,102 amino acid residues was identified.
KeywordMeSH Terms
DNA-Binding Proteins
Genes, Bacterial
Plasmids
Transcription Factors
125. Adler  B, Sasakawa  C, Tobe  T, Makino  S, Komatsu  K, Yoshikawa  M,     ( 1989 )

A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence-associated antigens of Shigella flexneri.

Molecular microbiology 3 (5)
PMID : 2474742  :   DOI  :   10.1111/j.1365-2958.1989.tb00210.x    
Abstract >>
The expression of plasmid-encoded, invasion-related antigens lpa b, c and d of Shigella flexneri was found to be positively regulated at transcriptional level by a 33kD protein produced by the previously defined, virulence-associated Region 1 on the SalI fragment B of the 230 kb invasion plasmid. The gene (designated virB) was identified and its nucleotide sequence determined. No Ipa b or c was produced in the absence of an intact virB gene although lower levels of d were produced. The previously reported regulatory activity of the virF gene some 30 kb distance away was shown to act exclusively through virB. In contrast, the activation of the virG gene necessary for intercellular spread occurred directly by virF without the requirement for virB. This study thus ascribes a critical function to a previously recognized, but functionally undefined, virulence locus on the large invasion plasmid of S. flexneri. The virF gene appears to have a central role in activation of the 230 kb plasmid-encoded virulence genes.
KeywordMeSH Terms
Gene Expression Regulation
Plasmids
Virulence Factors
126. Bernardini  ML, Mounier  J, d'Hauteville  H, Coquis-Rondon  M, Sansonetti  PJ,     ( 1989 )

Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin.

Proceedings of the National Academy of Sciences of the United States of America 86 (10)
PMID : 2542950  :   DOI  :   10.1073/pnas.86.10.3867     PMC  :   PMC287242    
Abstract >>
The capacity of Shigella to spread within the cytosol of infected epithelial cells and to infect adjacent cells is critical for the development of infection foci, which lead to mucosal abscesses. Shigella is a nonmotile microorganism that appears to utilize host cell microfilaments to generate intra- as well as intercellular movements, since this movement was inhibited by cytochalasin D and involvement of F-actin was demonstrated by direct labeling of infected cells with the specific dye N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin. Such movements led to the formation of extracellular protrusions, which may explain cell to cell spread. icsA, a locus necessary for intra- and intercellular spread, was identified on the Shigella flexneri virulence plasmid pWR100. This locus was cloned and shown to express a 120-kDa outer membrane protein, which plays an important role in the interactions established between host cell microfilaments and the bacterial surface, thus leading to intracellular movement.
KeywordMeSH Terms
Plasmids
127. Parent  KN, Erb  ML, Cardone  G, Nguyen  K, Gilcrease  EB, Porcek  NB, Pogliano  J, Baker  TS, Casjens  SR,     ( 2014 )

OmpA and OmpC are critical host factors for bacteriophage Sf6 entry in Shigella.

Molecular microbiology 92 (1)
PMID : 24673644  :   DOI  :   10.1111/mmi.12536     PMC  :   PMC4034267    
Abstract >>
Despite being essential for successful infection, the molecular cues involved in host recognition and genome transfer of viruses are not completely understood. Bacterial outer membrane proteins A and C co-purify in lipid vesicles with bacteriophage Sf6, implicating both outer membrane proteins as potential host receptors. We determined that outer membrane proteins A and C mediate Sf6 infection by dramatically increasing its rate and efficiency. We performed a combination of in vivo studies with three omp null mutants of Shigella flexneri, including classic phage plaque assays and time-lapse fluorescence microscopy to monitor genome ejection at the single virion level. Cryo-electron tomography of phage 'infecting' outer membrane vesicles shows the tail needle contacting and indenting the outer membrane. Lastly, in vitro ejection studies reveal that lipopolysaccharide and outer membrane proteins are both required for Sf6 genome release. We conclude that Sf6 phage entry utilizes either outer membrane proteins A or C, with outer membrane protein A being the preferred receptor.
KeywordMeSH Terms
128. Demers  JP, Habenstein  B, Loquet  A, Kumar Vasa  S, Giller  K, Becker  S, Baker  D, Lange  A, Sgourakis  NG,     ( 2014 )

High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy.

Nature communications 5 (N/A)
PMID : 25264107  :   DOI  :   10.1038/ncomms5976     PMC  :   PMC4251803    
Abstract >>
We introduce a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state nuclear magnetic resonance (ssNMR) chemical shifts and distance constraints define the local secondary structure, protein fold and inter-subunit interactions. Finally, Rosetta structure calculations provide a general framework to integrate the different sources of structural information. Combining a 7.7-? cryo-EM density map and 996 ssNMR distance constraints, the structure of the type-III secretion system needle of Shigella flexneri is determined to a precision of 0.4 ?. The calculated structures are cross-validated using an independent data set of 691 ssNMR constraints and scanning transmission electron microscopy measurements. The hybrid model resolves the conformation of the non-conserved N terminus, which occupies a protrusion in the cryo-EM density, and reveals conserved pore residues forming a continuous pattern of electrostatic interactions, thereby suggesting a mechanism for effector protein translocation.
KeywordMeSH Terms
Cryoelectron Microscopy
Magnetic Resonance Spectroscopy
129. Keszei  AF, Tang  X, McCormick  C, Zeqiraj  E, Rohde  JR, Tyers  M, Sicheri  F,     ( 2014 )

Structure of an SspH1-PKN1 complex reveals the basis for host substrate recognition and mechanism of activation for a bacterial E3 ubiquitin ligase.

Molecular and cellular biology 34 (3)
PMID : 24248594  :   DOI  :   10.1128/MCB.01360-13     PMC  :   PMC3911519    
Abstract >>
IpaH proteins are bacterium-specific E3 enzymes that function as type three secretion system (T3SS) effectors in Salmonella, Shigella, and other Gram-negative bacteria. IpaH enzymes recruit host substrates for ubiquitination via a leucine-rich repeat (LRR) domain, which can inhibit the catalytic domain in the absence of substrate. The basis for substrate recognition and the alleviation of autoinhibition upon substrate binding is unknown. Here, we report the X-ray structure of Salmonella SspH1 in complex with human PKN1. The LRR domain of SspH1 interacts specifically with the HR1b coiled-coil subdomain of PKN1 in a manner that sterically displaces the catalytic domain from the LRR domain, thereby activating catalytic function. SspH1 catalyzes the ubiquitination and proteasome-dependent degradation of PKN1 in cells, which attenuates androgen receptor responsiveness but not NF-�eB activity. These regulatory features are conserved in other IpaH-substrate interactions. Our results explain the mechanism whereby substrate recognition and enzyme autoregulation are coupled in this class of bacterial ubiquitin ligases.
KeywordMeSH Terms
130. Wang  Y, Song  C, Duan  G, Zhu  J, Yang  H, Xi  Y, Fan  Q,     ( 2013 )

Transposition of ISEcp1 modulates blaCTX-M-55-mediated Shigella flexneri resistance to cefalothin.

International journal of antimicrobial agents 42 (6)
PMID : 24207017  :   DOI  :   10.1016/j.ijantimicag.2013.08.009    
Abstract >>
The aim of this study was to uncover the mechanisms underlying Shigella flexneri resistance to cefalothin. In this study, a resistance-related S. flexneri isolate, S. flexneri YDC, was obtained from S. flexneri mel-1998023/zz pre-incubated with cefalothin at a dose of 0.5 �� the minimum inhibitory concentration. The ISEcp1 coding element was identified upstream of bla(CTX-M-55) in S. flexneri YDC. To further determine the role of ISEcp1 in S. flexneri resistance, plasmids containing bla(CTX-M-55) recombinant with or without the ISEcp1 sequence were constructed and named as pCTX and pISECTX, respectively. It was shown that Escherichia coli DH5�\(pISECTX) was resistant to all �]-lactams tested. In contrast, E. coli DH5�\(pCTX) was sensitive to all except �]-lactams cefazolin and cefalothin. In addition, reverse transcription PCR showed that expression levels of bla(CTX-M-55) were higher in E. coli DH5�\(pISECTX). The Clinical and Laboratory Standards Institute (CLSI) assay demonstrated that extended-spectrum �]-lactamase was only positively detected in E. coli DH5�\(pISECTX) but not in E. coli DH5�\(pCTX). Taken together, these results suggest that the translocated ISEcp1 element upstream of bla(CTX-M-55) is required for overexpression of bla(CTX-M-55), leading to cephalosporin resistance.
KeywordMeSH Terms
Extended-spectrum β-lactamase
ISEcp1
bla(CTX-M-55)
beta-Lactam Resistance
DNA Transposable Elements
Recombination, Genetic
131. Azmi  IJ, Khajanchi  BK, Akter  F, Hasan  TN, Shahnaij  M, Akter  M, Banik  A, Sultana  H, Hossain  MA, Ahmed  MK, Faruque  SM, Talukder  KA,     ( 2014 )

Fluoroquinolone resistance mechanisms of Shigella flexneri isolated in Bangladesh.

PloS one 9 (7)
PMID : 25028972  :   DOI  :   10.1371/journal.pone.0102533     PMC  :   PMC4100904    
Abstract >>
To investigate the prevalence and mechanisms of fluoroquinolone resistance in Shigella species isolated in Bangladesh and to compare with similar strains isolated in China. A total of 3789 Shigella isolates collected from Clinical Microbiology Laboratory of icddr,b, during 2004-2010 were analyzed for antibiotic susceptibility. Analysis of plasmids, plasmid-mediated quinolone-resistance genes, PFGE, and sequencing of genes of the quinolone-resistance-determining regions (QRDR) were conducted in representative strains isolated in Bangladesh and compared with strains isolated in Zhengding, China. In addition, the role of efflux-pump was studied by using the efflux-pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP). Resistance to ciprofloxacin in Shigella species increased from 0% in 2004 to 44% in 2010 and S. flexneri was the predominant species. Of Shigella spp, ciprofloxacin resistant (CipR) strains were mostly found among S. flexneri (8.3%), followed by S. sonnei (1.5%). Within S. flexneri (n = 2181), 14.5% were resistance to ciprofloxacin of which serotype 2a was predominant (96%). MIC of ciprofloxacin, norfloxacin, and ofloxacin were 6-32 mg/L, 8-32 mg/L, and 8-24 mg/L, respectively in S. flexneri 2a isolates. Sequencing of QRDR genes of resistant isolates showed double mutations in gyrA gene (Ser83Leu, Asp87Asn/Gly) and single mutation in parC gene (Ser80Ile). A difference in amino acid substitution at position 87 was found between strains isolated in Bangladesh (Asp87Asn) and China (Asp87Gly) except for one. A novel mutation at position 211 (His��Tyr) in gyrA gene was detected only in the Bangladeshi strains. Susceptibility to ciprofloxacin was increased by the presence of CCCP indicating the involvement of energy dependent active efflux pumps. A single PFGE type was found in isolates from Bangladesh and China suggesting their genetic relatedness. Emergence of fluoroquinolone resistance in Shigella undermines a major challenge in current treatment strategies which needs to be followed up by using empirical therapeutic strategies.
KeywordMeSH Terms
132. Kobayashi  T, Ogawa  M, Sanada  T, Mimuro  H, Kim  M, Ashida  H, Akakura  R, Yoshida  M, Kawalec  M, Reichhart  JM, Mizushima  T, Sasakawa  C,     ( 2013 )

The Shigella OspC3 effector inhibits caspase-4, antagonizes inflammatory cell death, and promotes epithelial infection.

Cell host & microbe 13 (5)
PMID : 23684308  :   DOI  :   10.1016/j.chom.2013.04.012    
Abstract >>
Caspase-mediated inflammatory cell death acts as an intrinsic defense mechanism against infection. Bacterial pathogens deploy countermeasures against inflammatory cell death, but the mechanisms by which they do this remain largely unclear. In a screen for Shigella flexneri effectors that regulate cell death during infection, we discovered that Shigella infection induced acute inflammatory, caspase-4-dependent epithelial cell death, which is counteracted by the bacterial OspC3 effector. OspC3 interacts with the caspase-4-p19 subunit and inhibits its activation by preventing caspase-4-p19 and caspase-4-p10 heterodimerization by depositing the conserved OspC3 X1-Y-X?-D-X? motif at the putative catalytic pocket of caspase-4. Infection of guinea pigs with a Shigella ospC3-deficient mutant resulted in enhanced inflammatory cell death and associated symptoms, correlating with decreased bacterial burdens. Salmonella Typhimurium and enteropathogenic Escherichia coli infection also induced caspase-4-dependent epithelial death. These findings highlight the importance of caspase-4-dependent innate immune responses and demonstrate that Shigella delivers a caspase-4-specific inhibitor to delay epithelial cell death and promote infection.
KeywordMeSH Terms
Cell Death
Host-Pathogen Interactions
133. Sun  Q, Lan  R, Wang  J, Xia  S, Wang  Y, Wang  Y, Jin  D, Yu  B, Knirel  YA, Xu  J,     ( 2013 )

Identification and characterization of a novel Shigella flexneri serotype Yv in China.

PloS one 8 (7)
PMID : 23936172  :   DOI  :   10.1371/journal.pone.0070238     PMC  :   PMC3728103    
Abstract >>
Shigella flexneri is the major cause of bacterial shigellosis in developing countries. S. flexneri is divided into at least 19 serotypes, the majority of which are modifications of the same basic O-antigen by glucosylation and/or O-acetylation of its sugar residues by phage encoded serotype-converting genes. Recently, a plasmid encoded phosphoethanolamine (PEtN) modification of the O-antigen has been reported, which is responsible for the presence of the MASF IV-1 determinant and results in conversion of traditional serotypes X, 4a and Y to novel serotypes Xv, 4av and Yv, respectively. In this study, we characterized 19 serotype Yv strains isolated in China. A variant of the O-antigen phosphoethanolamine transferase gene opt (formerly called lpt-O) carried by a pSFxv_2-like plasmid was found in serotype Yv strains, which specifies the phosphorylation pattern on the O-antigen of this serotype. For the majority of the O-antigen units, the PEtN modification occurs on Rha(III), while for a minority, modifications occur on both Rha(II) and Rha(III). Serotype-specific gene detection and PFGE analysis suggested that these serotype Yv isolates were originated from serotypes Y, Xv and 2a by acquisition of an opt-carrying plasmid and/or inactivation of serotype-specific gene gtrII or gtrX. These data, combined with those of serotypes Xv and 4av reported earlier, demonstrate that the plasmid-encoded PEtN modification is an important serotype conversion mechanism in S. flexneri, in addition to glucosylation and O-acetylation.
KeywordMeSH Terms
134. Di Martino  ML, Fioravanti  R, Barbabella  G, Prosseda  G, Colonna  B, Casalino  M,     ( 2013 )

Molecular evolution of the nicotinic acid requirement within the Shigella/EIEC pathotype.

International journal of medical microbiology : IJMM 303 (8)
PMID : 24120364  :   DOI  :   10.1016/j.ijmm.2013.09.007    
Abstract >>
Nicotinamide adenine dinucleotide (NAD) is a crucial cofactor in several anabolic and catabolic reactions. NAD derives from quinolinic acid (QUIN) which in Escherichia coli is obtained through a pyridine salvage pathway or a de novo synthesis pathway. In the latter case, two enzymes, L-aspartate oxidase (NadB) and quinolinate synthase (NadA), are required for the synthesis of QUIN. In contrast to its E. coli ancestor, Shigella spp., the causative agent of bacillary dissentery, lacks the de novo pathway and strictly requires nicotinic acid for growth (Nic? phenotype). This phenotype depends on the silencing of the nadB and nadA genes and its pathoadaptive nature is suggested by the observation that QUIN attenuates the Shigella invasive process. Shigella shares the pathogenicity mechanism with enteronvasive E. coli (EIEC), a group of pathogenic E. coli. On the basis of this similarity EIEC and Shigella have been grouped into a single E. coli pathotype. However EIEC strains do not constitute a homogeneous group and do not possess the complete set of characters that define Shigella strains. In this work we have analysed thirteen EIEC strains belonging to different serotypes and originating from different geographic areas. We show that, in contrast to Shigella, only some EIEC strains require nicotinic acid for growth in minimal medium. Moreover, by studying the emergence of the Nic? phenotype in all serotypes of S. flexneri, as well as in S. sonnei and S. dysenteriae, we describe which molecular rearrangements occurred and which mutations are responsible for the inactivation of the nadA and nadB genes. Our data confirm that the genome of Shigella is extremely dynamic and support the hypothesis that EIEC might reflect an earlier stage of the pathoadaptation process undergone by Shigella.
KeywordMeSH Terms
Evolution
NAD biosynthesis
Pathoadaptive mutations
Pathogenic E. coli
Shigella
Evolution
NAD biosynthesis
Pathoadaptive mutations
Pathogenic E. coli
Shigella
Evolution
NAD biosynthesis
Pathoadaptive mutations
Pathogenic E. coli
Shigella
Evolution, Molecular
135. Sanada  T, Kim  M, Mimuro  H, Suzuki  M, Ogawa  M, Oyama  A, Ashida  H, Kobayashi  T, Koyama  T, Nagai  S, Shibata  Y, Gohda  J, Inoue  J, Mizushima  T, Sasakawa  C,     ( 2012 )

The Shigella flexneri effector OspI deamidates UBC13 to dampen the inflammatory response.

Nature 483 (7391)
PMID : 22407319  :   DOI  :   10.1038/nature10894    
Abstract >>
Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type �L�L�L secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol-CBM (CARD-BCL10-MALT1) complex-TRAF6-nuclear-factor-�eB signalling pathway. We determined the 2.0 ? crystal structure of OspI, which contains a putative cysteine-histidine-aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13-TRAF6 complex.
KeywordMeSH Terms
Adaptor Proteins, Signal Transducing
136. Zhao  XJ,     ( 1990 )

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r.

Molecular & general genetics : MGG 222 (2��3��)
PMID : 2274037  :   DOI  :   10.1007/bf00633842    
Abstract >>
The complete nucleotide sequences of the recA genes from Escherichia coli B/r, Shigella flexneri, Erwinia carotovora and Proteus vulgaris were determined. The DNA sequence of the coding region of the E. coli B/r gene contained a single nucleotide change compared with the E. coli K12 gene sequence whereas the S. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to the E. coli K12 RecA protein. The DNA sequences of the recA genes from E. carotovora and P. vulgaris were 80% and 74% homologous, respectively, to the E. coli K12 gene. The predicted amino acid sequences of the E. carotovora and P. vulgaris RecA proteins were 91% and 85% identical respectively, to that of E. coli K12. The RecA proteins from both P. vulgaris and E. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.
KeywordMeSH Terms
DNA, Bacterial
Serine Endopeptidases
137. Dienemann  C, Bøggild  A, Winther  KS, Gerdes  K, Brodersen  DE,     ( 2011 )

Crystal structure of the VapBC toxin-antitoxin complex from Shigella flexneri reveals a hetero-octameric DNA-binding assembly.

Journal of molecular biology 414 (5)
PMID : 22037005  :   DOI  :   10.1016/j.jmb.2011.10.024     PMC  :   PMC3384007    
Abstract >>
Toxin-antitoxin (TA) loci are common in archaea and prokaryotes and allow cells to rapidly adapt to changing environmental conditions through release of active regulators of metabolism. Many toxins are endonucleases that target cellular mRNA and tRNAs, while the antitoxins tightly wrap around the toxins to inhibit them under normal circumstances. The antitoxins also bind to operators in the promoter regions of the cognate TA operon and thereby regulate transcription. For enteric vapBC TA loci, the VapC toxins specifically cleave tRNA(fMet) and thus down-regulate protein synthesis. Here, we describe the crystal structure of the intact Shigella flexneri VapBC TA complex, determined to 2.7 ? resolution. Both in solution and in the crystal structure, four molecules of each protein combine to form a large and globular hetero-octameric assembly with SpoVT/AbrB-type DNA-binding domains at each end and a total molecular mass of about 100 kDa. The structure gives new insights into the inhibition of VapC toxins by VapB and provides the molecular basis for understanding transcriptional regulation through VapB dimerization.
KeywordMeSH Terms
Shigella flexneri
138. Bhattacharya  D, Bhattacharjee  H, Thamizhmani  R, Sayi  DS, Bharadwaj  AP, Singhania  M, Sugunan  AP, Roy  S,     ( 2011 )

Prevalence of the plasmid-mediated quinolone resistance determinants among clinical isolates of Shigella sp. in Andaman & Nicobar Islands, India.

Letters in applied microbiology 53 (2)
PMID : 21615433  :   DOI  :   10.1111/j.1472-765X.2011.03092.x    
Abstract >>
This study was carried out to find the prevalence of various plasmid-mediated quinolone-resistant (PMQR) determinants among the quinolone-resistant clinical isolates of Shigella sp. from paediatric patients in Andaman & Nicobar Islands. A total of 106 quinolone-resistant Shigella isolates obtained from paediatric patients during hospital-based surveillance from January 2003 to June 2010 were screened for the presence of various PMQR determinants. Of 106 isolates, 8 (7.5%) showed the presence of aac (6')-Ib-cr and 3 (2.8%) harboured the qnrB genes with 2 (1.9%) of these isolates showing the presence of both. All the 9 isolates had uniform mutations in gyrA (S83L) and in parC (S80I). The prevalence of fluoroquinolone-acetylating aminoglycoside acetyltransferase {aac (6')-Ib-cr} gene is higher than qnrB gene among the clinical Shigella isolates. These PMQR determinants were detected in the Shigella isolates obtained from 2008-2010, indicating that it happens in a stepwise manner following the multiple mutations in quinolone resistance-determining regions increase or extend resistance to quinolones or fluoroquinolones. The prevalence of these genes are of grave concern as it may be horizontally transferred to other human pathogenic bacteria and can lead to therapeutic failure as a consequence of antimicrobial resistance, not only for the islands but also for the entire south-east region. The results obtained should encourage further studies on the implications of the presence, distribution, association and variation of these determinants in our quest for understanding PMQR.
KeywordMeSH Terms
139.     ( 1997 )

Structures of the reduced and mercury-bound forms of MerP, the periplasmic protein from the bacterial mercury detoxification system.

Biochemistry 36 (23)
PMID : 9188683  :   DOI  :   10.1021/bi9631632    
Abstract >>
Bacteria carrying plasmids with the mer operon, which encodes the proteins responsible for the bacterial mercury detoxification system, have the ability to transport Hg(II) across the cell membrane into the cytoplasm where it is reduced to Hg(0). This is significant because metallic mercury is relatively nontoxic and volatile and thus can be passively eliminated. The structures of the reduced and mercury-bound forms of merP, the periplasmic protein, which binds Hg(II) and transfers it to the membrane transport protein merT, have been determined in aqueous solution by multidimensional NMR spectroscopy. The 72-residue merP protein has a betaalpha betabeta alphabeta fold with the two alpha helices overlaying a four-strand antiparallel beta sheet. Structural differences between the reduced and mercury-bound forms of merP are localized to the metal binding loop containing the consensus sequence GMTCXXC. The structure of the mercury-bound form of merP shows that Hg(II) is bicoordinate with the Cys side chain ligands, and this is confirmed by the chemical shift frequency of the 199Hg resonance.
KeywordMeSH Terms
Proteins
140.     ( 1997 )

Plasmid maintenance functions of the large virulence plasmid of Shigella flexneri.

Journal of bacteriology 179 (11)
PMID : 9171415  :   DOI  :   10.1128/jb.179.11.3670-3675.1997     PMC  :   PMC179163    
Abstract >>
The large virulence plasmid pMYSH6000 of Shigella flexneri contains a replicon and a plasmid maintenance stability determinant (Stb) on adjacent SalI fragments. The presence of a RepFIIA replicon on the SalI C fragment was confirmed, and the complete sequence of the adjacent SalI O fragment was determined. It shows homology to part of the transfer (tra) operon of the F plasmid. Stb stabilizes a partition-defective P1 miniplasmid in Escherichia coli. A 1.1-kb region containing a homolog of the F trbH gene was sufficient to confer stability. However, the trbH open reading frame could be interrupted without impairing stability. Deletion analysis implicated the involvement of two small open reading frames, STBORF1 and STBORF2, that fully overlap trbH in the opposite direction. These open reading frames are closely related to the vagC and vagD genes of the Salmonella dublin virulence plasmid and to open reading frame pairs in the F trbH region and in the chromosomes of Dichelobacter nodosus and Haemophilus influenzae. Stb appears to promote better-than-random distribution of plasmid copies and is a plasmid incompatibility determinant. The F homolog does not itself confer stability but exerts incompatibility against the activity of the Stb system. Stb is likely to encode either an active partition system or a postsegregational killing system. It shows little similarity to previously studied plasmid stability loci, but the genetic organization of STBORF1 and STBORF2 resembles that of postsegregational killing mechanisms.
KeywordMeSH Terms
141.     ( 1997 )

Transferable hyperproduction of TEM-1 beta-lactamase in Shigella flexneri due to a point mutation in the pribnow box.

Antimicrobial agents and chemotherapy 41 (2)
PMID : 9021210  :   PMC  :   PMC163732    
Abstract >>
TEM-1 hyperproduction in two ampicillin-sulbactam-resistant Shigella flexneri strains was studied. In both strains the blaTEM gene was encoded as a single copy on a large conjugatively transferable plasmid. A single G-->T transversion at position 1 of the -10 consensus sequence was identified to be the mechanism of TEM-1 hyperproduction.
KeywordMeSH Terms
142.     ( 1997 )

Modulation of bacterial entry into epithelial cells by association between vinculin and the Shigella IpaA invasin.

The EMBO journal 16 (10)
PMID : 9184218  :   DOI  :   10.1093/emboj/16.10.2717     PMC  :   PMC1169882    
Abstract >>
Shigella flexneri is the causative agent of bacillary dysentery in humans. Shigella invasion of epithelial cells is characterized by cytoskeletal rearrangements and formation of cellular projections engulfing the bacterium in a macropinocytic process. We show here that vinculin, a protein involved in linking actin filaments to the plasma membrane, is a direct target of Shigella during cell invasion. IpaA, a Shigella protein secreted upon cell contact, rapidly associates with vinculin during bacterial invasion. Although defective for cell entry, an ipaA mutant is still able to induce foci of actin polymerization, but differs from wild-type Shigella in its ability to recruit vinculin and alpha-actinin. Presumably, IpaA-vinculin interaction initiates the formation of focal adhesion-like structures required for efficient invasion.
KeywordMeSH Terms
Bacterial Adhesion
Pinocytosis
143.     ( 1997 )

The modified nucleoside 2-methylthio-N6-isopentenyladenosine in tRNA of Shigella flexneri is required for expression of virulence genes.

Journal of bacteriology 179 (18)
PMID : 9294434  :   DOI  :   10.1128/jb.179.18.5777-5782.1997     PMC  :   PMC179466    
Abstract >>
The virulence of the human pathogen Shigella flexneri is dependent on both chromosome- and large-virulence-plasmid-encoded genes. A kanamycin resistance cassette mutation in the miaA gene (miaA::Km Sma), which encodes the tRNA N6-isopentyladenosine (i6A37) synthetase and is involved in the first step of the synthesis of the modified nucleoside 2-methylthio-N6-isopentenyladenosine (ms2i6A), was transferred to the chromosome of S. flexneri 2a by phage P1 transduction. In the wild-type bacterium, ms2i6A37 is present in position 37 (next to and 3' of the anticodon) in a subset of tRNA species-reading codons starting with U (except tRNA(Ser) species SerI and SerV). The miaA::Km Sma mutant of S. flexneri accordingly lacked ms2i6A37 in its tRNA. In addition, the mutant strains showed reduced expression of the virulence-related genes ipaB, ipaC, ipaD, virG, and virF, accounting for sixfold-reduced contact hemolytic activity and a delayed response in the focus plaque assay. A cloned sequence resulting from PCR amplification of the wild-type Shigella chromosome and exhibiting 99% homology with the nucleotide sequence of the Escherichia coli miaA gene complemented the virulence-associated phenotypes as well as the level of the modified nucleoside ms2i6A in the tRNA of the miaA mutants. In the miaA mutant, the level of the virulence-associated protein VirF was reduced 10-fold compared with the wild type. However, the levels of virF mRNA were identical in the mutant and in the wild type. These findings suggest that a posttranscriptional mechanism influenced by the presence of the modified nucleoside ms2i6A in the tRNA is involved in the expression of the virF gene. The role of the miaA gene in the virulence of other Shigella species and in enteroinvasive E. coli was further generalized.
KeywordMeSH Terms
Alkyl and Aryl Transferases
Gene Expression Regulation, Bacterial
144.     ( 1997 )

Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin-based motility.

Molecular microbiology 25 (3)
PMID : 9302008  :   DOI  :   10.1046/j.1365-2958.1997.4681827.x    
Abstract >>
Shigella pathogenesis involves bacterial invasion of colonic epithelial cells and movement of bacteria through the cytoplasm and into adjacent cells by means of actin-based motility. The Shigella protein IcsA (VirG) is unipolar on the bacterial surface and is both necessary and sufficient for actin-based motility. IcsA is inserted into the outer membrane as a 120-kDa polypeptide that is subsequently slowly cleaved, thereby releasing the 95-kDa amino-terminal portion into the culture supernatant. IcsP, the major Shigella protease that cleaves IcsA, was identified and cloned. It has significant sequence similarity to the E. coli serine proteases, OmpP and OmpT. Disruption of icsP in serotype 2a S. flexneri leads to a marked reduction in IcsA cleavage, increased amounts of IcsA associated with the bacterium and altered distribution of IcsA on the bacterial surface. The icsP mutant displays significantly increased rates of actin-based motility, with a mean speed 27% faster than the wild-type strain; moreover, a significantly greater percentage of the icsP mutant moves in the cytoplasm. Yet, plaque formation on epithelial monolayers by the mutant was not altered detectably. These data suggest that IcsA, and not a host protein, is limiting in the rate of actin-based motility of wild-type serotype 2a S. flexneri.
KeywordMeSH Terms
145.     ( 1997 )

SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri.

Molecular microbiology 23 (5)
PMID : 9076742  :   DOI  :   10.1046/j.1365-2958.1997.2871652.x    
Abstract >>
The spreading ability of Shigella flexneri, a facultative intracellular Gram-negative bacterium, within the host-cell cytoplasm is the result of directional assembly and accumulation of actin filaments at one pole of the bacterium. IcsA/VirG, the 120 kDa outer membrane protein that is required for intracellular motility, is located at the pole of the bacterium where actin polymerization occurs. Bacteria growing in laboratory media and within infected cells release a certain proportion of the surface-exposed IcsA after proteolytic cleavage. In this study, we report the characterization of the sopA gene which is located on the virulence plasmid and encodes the protein responsible for the cleavage of IcsA. The deduced amino acid sequence of SopA exhibits 60% identity with those of the OmpT and OmpP outer membrane proteases of Escherichia coli. The construction and phenotypic characterization of a sopA mutant demonstrated that SopA is required for exclusive polar localization of IcsA on the bacterial surface and proper expression of the motility phenotype in infected cells.
KeywordMeSH Terms
Escherichia coli Proteins
Hydrolases
146.     ( 1996 )

Identification and characterization of ispA, a Shigella flexneri chromosomal gene essential for normal in vivo cell division and intracellular spreading.

Molecular microbiology 19 (3)
PMID : 8830250  :   DOI  :   10.1046/j.1365-2958.1996.405941.x    
Abstract >>
The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild-type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra- and inter-cellular spreading ability. Sequencing of Tn10-flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB. Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.
KeywordMeSH Terms
Genes, Bacterial
Membrane Proteins
147.     ( 1993 )

MxiD, an outer membrane protein necessary for the secretion of the Shigella flexneri lpa invasins.

Molecular microbiology 7 (1)
PMID : 8437520  :   DOI  :   10.1111/j.1365-2958.1993.tb01097.x    
Abstract >>
The invasive phenotype of Shigella flexneri is conferred by a 220 kb virulence plasmid, pWR100, that encodes both the lpa proteins, which are involved in the entry process, and factors which are required for the export and correct localization of the lpa proteins. We have characterized the mxiD gene, whose expression, like that of the ipa operon, is regulated by temperature. After inactivation of mxiD, the mutant strain was unable to invade HeLa cells and to provoke keratoconjunctivitis in guinea-pigs. Analysis of culture supernatants indicated that wild-type S. flexneri secretes about nine polypeptides and that secretion of several of these, including lpaA, lpaB, and lpaC, is abolished in the mxiD mutant. Examination of the membrane proteins of the wild-type and mxiD strains suggested that MxiD is an outer membrane protein. Amino acid sequence comparison revealed that MxiD is homologous to the YscC protein of Yersinia enterocolitica and to the C-terminal region of the PulD protein of Klebsiella pneumoniae. Both YscC and PulD are involved in extracellular protein secretion. These results indicate that MxiD is an essential component of the lpa secretion apparatus.
KeywordMeSH Terms
Adhesins, Bacterial
DNA-Binding Proteins
Genes, Bacterial
Transcription Factors
148.     ( 1996 )

A Shigella flexneri invasion plasmid gene, ipgH, with homology to IS629 and sequences encoding bacterial sugar phosphate transport proteins.

Gene 175 (1��2��)
PMID : 8917071  :   DOI  :   10.1016/0378-1119(96)00115-1    
Abstract >>
Sequences representing the 2684 bp flanking the ipaH4.5 gene on the invasion plasmid of Shigella flexneri 5 indicate an unusual fusion gene, designated ipgH, in which the first 27 amino acids (aa) are identical to ORF2 of IS629. The aa sequence encoded by the remainder of ipgH bears significant homology to Escherichia coli and to Salmonella typhimurium GlpT and UhpT proteins and to the S. typhimurium PgtP protein, which are involved in the uptake of high-energy sugar phosphates from an external source.
KeywordMeSH Terms
Organic Anion Transporters
149.     ( 1997 )

IpaB, a Shigella flexneri invasin, colocalizes with interleukin-1 beta-converting enzyme in the cytoplasm of macrophages.

Infection and immunity 65 (2)
PMID : 9009343  :   PMC  :   PMC176126    
Abstract >>
Shigellae are the most prevalent etiological agents of dysentery. A crucial step in shigella pathogenesis is the induction of macrophage apoptosis. The invasion plasmid antigen B (IpaB) is necessary and sufficient to induce macrophage programmed cell death. IpaB activates apoptosis by binding to interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) or a highly homologous protease. Here, we show that IpaB is disseminated throughout the cytoplasm of shigella-infected macrophages as detected by both immunofluorescence and immunoelectron microscopy. The cytoplasmic distribution of IpaB requires phagosome escape, and it is specific to IpaB, since lipopolysaccharide, used here as a bacterial marker, remains closely associated with the bacteria. In double-labeling experiments, we show that IpaB and ICE colocalize in the cytoplasm of the macrophage, suggesting that soon after secretion, IpaB binds to ICE to initiate apoptosis and to promote the cleavage of IL-1 beta.
KeywordMeSH Terms
Adhesins, Bacterial
150.     ( 1996 )

Evolutionary perspective on a composite Shigella flexneri 2a virulence plasmid-borne locus comprising three distinct genetic elements.

FEMS microbiology letters 144 (1)
PMID : 8870246  :   DOI  :   10.1111/j.1574-6968.1996.tb08502.x    
Abstract >>
Nucleotide sequence analysis of a Shigella flexneri 2a virulence plasmid-borne locus revealed that it comprised three distinct genetic elements: a stretch of colicin 1a/1b-linked sequence, a truncated IS911 element, and a third element containing two ORFs that shared a high level of similarity to a Salmonella-specific chromosomal sequence. Examination of other known IS911-like sequences showed that these sequences also were frequently associated with other accessory elements and appeared to be prone to partial deletion events. Analysis of the data led to a model of the evolution of this unusual composite locus.
KeywordMeSH Terms
151.     ( 1996 )

Identification of sigma S-dependent genes associated with the stationary-phase acid-resistance phenotype of Shigella flexneri.

Molecular microbiology 21 (5)
PMID : 8885264  :   DOI  :   10.1046/j.1365-2958.1996.00058.x    
Abstract >>
Shigella flexneri grown to stationary phase has the ability to survive for several hours at pH 2.5. This acid resistance, which may contribute to the low infective dose associated with shigellosis, is dependent upon the expression of the stationary-phase-specific sigma factor sigma S. Using random TnphoA and TnlacZ mutagenesis we isolated five acid-sensitive mutants of S. flexneri, which had lost their ability to survive at pH 2.5 for 2 h in vitro. Each transposon insertion with flanking S. flexneri DNA was cloned and sequenced. Database searches indicated that two TnlacZ mutants had an insertion within the hdeA gene, which is the first gene in the hdeAB operon. Acid resistance was restored in one of these mutants by a plasmid carrying the entire hdeAB operon. Further sequence analysis from the remaining TnlacZ and two TnphoA mutants demonstrated that they all had insertions within a previously unidentified open reading frame (ORF), which is directly downstream from the gadB gene. This putative ORF encodes a protein that has homology to a number of inner membrane amino acid antiporters. A 1.8 kb polymerase chain reaction (PCR) product containing this gene was cloned, which was able to restore acid resistance in each mutant. These fusions were induced during entry into late exponential phase and were positively regulated by RpoS. We confirmed that the expression of the acid-resistance phenotype in acidified minimal media was dependent upon the supplementation of glutamic acid and that this glutamate-dependent system was RpoS regulated. Southern hybridization revealed that both the gadC and hdeAB loci are absent in Salmonella. An rpoS deletion mutant of S. flexneri was also constructed to confirm the important role played by this gene in acid resistance. This rpoS- derivative was extremely acid sensitive. Two-dimensional gel electrophoresis of this mutant revealed that it no longer expressed 27 proteins in late log phase that were present in its isogenic parent. These data indicate that the expression of acid resistance in S. flexneri may be multifactorial and involve proteins located at different subcellular locations.
KeywordMeSH Terms
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
Genes, Bacterial
152.     ( 1996 )

The integrons In0, In2, and In5 are defective transposon derivatives.

Journal of bacteriology 178 (15)
PMID : 8755869  :   DOI  :   10.1128/jb.178.15.4429-4437.1996     PMC  :   PMC178208    
Abstract >>
The class 1 integrons In0, In2, and In5, found in different locations in pVS1, Tn21, and pSCH884, have closely related structures. All three integrons contain an insertion sequence, IS1326, that is a new member of the IS21 family. IS1326 has caused deletions of adjacent 3'-conserved segment and transposition module sequences, and all three integrons retain a complete copy of only one of four genes required for transposition of related transposons and are thus defective transposon derivatives. In2 contains an additional insertion sequence, IS1353, located within IS1326. IS1353 is a member of the IS3 family and appears to have been acquired after the integron was inserted into an ancestral mercury resistance transposon to create the ancestor of Tn21 and several other transposons that are close relatives of Tn21.
KeywordMeSH Terms
DNA Transposable Elements
153.     ( 1993 )

Eight genes in region 5 that form an operon are essential for invasion of epithelial cells by Shigella flexneri 2a.

Journal of bacteriology 175 (8)
PMID : 8385666  :   DOI  :   10.1128/jb.175.8.2334-2346.1993     PMC  :   PMC204522    
Abstract >>
The 7-kb region 5 on the large 230-kb plasmid pMYSH6000 in Shigella flexneri 2a YSH6000 is one of the virulence-associated DNA segments required for the invasion of epithelial cells (C. Sasakawa, K. Kamata, T. Sakai, S. Makino, M. Yamada, N. Okada, and M. Yoshikawa, J. Bacteriol. 170:2480-2484, 1988). To elucidate the functional organization of region 5 and to determine the virulence-associated genes encoded by region 5, we performed insertion and deletion mutagenesis, DNA subcloning, and complete nucleotide sequencing of region 5 and found that region 5 contained 11 open reading frames (ORFs) named ORF-1 through ORF-11 which could be translated into proteins with molecular masses of 15.1, 47.5, 13.2, 33.0, 33.4, 24.2, 9.4, 28.5, 39.9, 9.1, and 10.4 kDa, respectively. Complementation tests of the 14 Tn5-induced noninvasive mutants of region 5 with the above plasmid constructs have indicated that region 5 consists of an operon and that ORF-2 through ORF-9, but not ORF-1, ORF-10, and ORF-11, are essential for invasion, and 7 of 8 ORFs (ORF-2 and ORF-4 through ORF-9) and presumably the remaining ORF (ORF-3) are required for secretion of the Ipa proteins. The transcriptional organization, as determined by a promoter-proving vector, S1 nuclease protection, and primer extension RNA sequencing analysis revealed that region 5 is transcribed from a promoter located 47 bp upstream of the 5' end of ORF-2 for the 47.5-kDa protein and that the promoter activity identified was regulated by the virB gene, the transcriptional activator on the 230-kb plasmid.
KeywordMeSH Terms
Genes, Bacterial
Operon
154.     ( 1996 )

Identification and characterization of phoN-Sf, a gene on the large plasmid of Shigella flexneri 2a encoding a nonspecific phosphatase.

Journal of bacteriology 178 (15)
PMID : 8755883  :   DOI  :   10.1128/jb.178.15.4548-4554.1996     PMC  :   PMC178222    
Abstract >>
A gene encoding a nonspecific phosphatase, named PhoN-Sf, was identified on the large virulence plasmid (pMYSH6000) of Shigella flexneri 2a YSH6000. The phosphatase activity in YSH6000 was observed under high-phosphate conditions. However, it was found that low-phosphate conditions induced a slightly higher level of activity. The nucleotide sequence of the phoN-Sf region cloned from pMYSH6000 possessing the phoN-Sf gene encoded 249 amino acids with a typical signal sequence at the N terminus. The deduced amino acid sequence of the PhoN-Sf protein revealed significant homology to sequences of nonspecific acid phosphatases of other bacteria, such as Providencia stuartii (PhoN, 83.2%), Morganella morganii (PhoC, 80.6%), Salmonella typhimurium (PhoN, 47.8%), and Zymomonas mobilis (PhoC, 34.8%). The PhoN-Sf protein was purified, and its biochemical properties were characterized. The apparent molecular mass of the protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was calculated to be 27 kDa. The 20 amino acids at the N terminus corresponded to the 20 amino acid residues following the putative signal sequence of PhoN-Sf protein deduced from the nucleotide sequence. The PhoN-Sf activity had a pH optimum of 6.6, and the optimum temperature was 37 degrees C. The enzymatic activity was inhibited by diisopropyl fluorophosphate, N-bromosuccinimide, or dithiothreitol but not by EDTA. The subcellular localization of the PhoN-Sf protein in YSH6000 revealed that the protein was found predominantly in the periplasm. Examination of Shigella and enteroinvasive Escherichia coli strains for PhoN-Sf production by immunoblotting with the PhoN-specific antibody and for the presence of phoN-Sf DNA by using a phoN-Sf probe indicated that approximately one-half of the strains possessed the phoN-Sf gene on the large plasmid and expressed the PhoN-Sf protein. The Tn5 insertion mutants of YSH6000 possessing phoN-Sf::Tn5 still retained wild-type levels of invasiveness, as well as the subsequent spreading capacity in MK2 epithelial cell monolayers, thus suggesting that the PhoN-Sf activity is not involved in expression of the virulence phenotypes of Shigella strains under in vitro conditions.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
155.     ( 1995 )

Partial DNA sequence of a beta-lactamase produced by a Shigella flexneri strain.

Microbios 83 (335)
PMID : 8538491  :  
Abstract >>
A probe was constructed by radioactive labelling, and enzymatically a DNA fragment of plasmid pMAM-1, which codes for a beta-lactamase in Shigella flexneri UCSM 129, was obtained by amplification of a small part of the gene using the polymerase chain reaction technique (PCR). Since previous published work indicated that this beta-lactamase was of the TEM type, the primers used to amplify the gene were two highly conserved DNA regions in all TEM beta-lactamases. A 500 bp DNA probe was obtained which, by hybridization assays, facilitated the identification of restriction fragments of the plasmid containing the beta-lactamase gene. Two DNA fragments were sequenced by the Sanger method adapted to the PCR technique, and the sequence obtained showed a 100% homology with beta-lactamases TEM-1, TEM-2, TEM-13 and TEM-19. An intragenic restriction site, detected for Pst I, suggested that there is only one copy of the beta-lactamase gene per plasmid copy.
KeywordMeSH Terms
Genes, Bacterial
156.     ( 1993 )

purU, a source of formate for purT-dependent phosphoribosyl-N-formylglycinamide synthesis.

Journal of bacteriology 175 (21)
PMID : 8226647  :   DOI  :   10.1128/jb.175.21.7066-7073.1993     PMC  :   PMC206834    
Abstract >>
A gene designated purU has been identified and characterized. purU is adjacent to tyrT at min 27.7 on the Escherichia coli chromosome. The gene codes for a 280-amino-acid protein. The C-terminal segment of PurU from residues 84 to 280 exhibits 27% identity with 5'-phosphoribosylglycinamide (GAR) transformylase, the product of purN. Primer extension mapping and assays of lacZ in a promoter probe vector identified two promoters giving mono- and bi-cistronic purU mRNA. Neither mRNA was regulated by purines. Mutations in either of two pairs of genes are required to block synthesis of 5'-phosphoribosyl-N-formylglycinamide (FGAR) from GAR: purN purT (purT encodes an alternative formate-dependent GAR transformylase) or purN purU. On the basis of the growth of purU, purN, and purU purN mutants, it appears that PurU provides the major source of formate for the purT-dependent synthesis of FGAR.
KeywordMeSH Terms
Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor
Escherichia coli Proteins
Genes, Bacterial
Hydroxymethyl and Formyl Transferases
157.     ( 1995 )

Extracellular transport of VirG protein in Shigella.

The Journal of biological chemistry 270 (52)
PMID : 8537341  :   DOI  :   10.1074/jbc.270.52.30874    
Abstract >>
The ability of Shigella to spread within and between epithelial cells is a prerequisite for causing bacillary dysentery and requires the function encoded by the virG gene on the large plasmid. The outer membrane VirG (IcsA) protein is essential for bacterial spreading by eliciting polar deposition of filamentous actin (F-actin) in the cytoplasm of epithelial cells. Recent studies have indicated that an N-terminal 80-kDa VirG portion is exposed on the bacterial cell surface and released into the external medium, while the following 37-kDa C-terminal portion is embedded in the outer membrane, although little is known about the extracellular transport of the VirG protein. In this study, we attempted to elucidate the export pathway of VirG protein across the outer membrane and found that the C-terminal 37-kDa portion, termed VirG beta-core, serves as the self-transporter for the secretion of the preceding 80-kDa portion from the periplasmic side of the outer membrane to the external side. Indeed, foreign polypeptides such as MalE or PhoA covalently linked to the N terminus of VirG beta-core were transported to the external side of the outer membrane, and it was further shown that the folding structure of the passenger polypeptide at the periplasmic side of the outer membrane interferes with its translocation. Analysis of the secondary structure of VirG beta-core predicted that the critical structural property was a beta-barrel channel consisting of amphipathic anti-parallel transmembrane beta-strands, interspersed by hairpin turns and loops. These results thus strongly suggest that the secretion of VirG protein from Shigella is similar to the export system utilized by the IgA protease of Neisseria.
KeywordMeSH Terms
158.     ( 1995 )

MxiG, a membrane protein required for secretion of Shigella spp. Ipa invasins: involvement in entry into epithelial cells and in intercellular dissemination.

Molecular microbiology 17 (3)
PMID : 8559065  :   DOI  :   10.1111/j.1365-2958.1995.mmi_17030461.x    
Abstract >>
Entry of Shigella flexneri into epithelial cells involves secretory proteins, the Ipa proteins, and their dedicated secretion apparatus, the Mxi-Spa translocon, which is encoded by the mxi and spa operons. We have characterized the mxiG gene that is located at the proximal part of the mxi operon. Inactivation of mxiG abolished lpa secretion, which indicates that MxiG is an essential component of the Mxi-Spa translocon. Immunoblotting analysis of membrane fractions suggests that the 42 kDa MxiG protein is associated with both the inner and outer membranes. Taking advantage of the complementation of the mxiG mutant by a plasmid carrying a wild-type copy of mxiG (which restored Ipa secretion, entry into HeLa cells, and cell-to-cell spread) we mutagenized the mxiG gene carried by the complementing plasmid to replace the RGD motif of MxiG by RAD. This mutation (mxiG*), which had no effect on the stability of the protein, did not affect Ipa secretion in vitro or entry into HeLa cells, but impaired intercellular dissemination. Therefore, MxiG and possibly proteins secreted by the Mxi-Spa translocation are involved not only in entry but also in spread of Shigella between epithelial cells.
KeywordMeSH Terms
Adhesins, Bacterial
159.     ( 1994 )

Molecular cloning of the wild-type and mutant thyA gene from Shigella flexneri Y.

Microbiology and immunology 38 (4)
PMID : 7935051  :   DOI  :   10.1111/j.1348-0421.1994.tb01782.x    
Abstract >>
The thyA gene which codes for thymidylate synthase has been cloned and sequenced from the wild-type Shigella flexneri Y strain SH4 and a thyA mutant TSF21 after amplifying the gene by polymerase chain reaction (PCR). The nucleotide sequence revealed 98% homology to the E. coli K-12 thyA gene. The sequence of the wild-type thyA gene of Shigella flexneri Y was identical with that of the thyA mutant except that the residue T at position 345 was replaced by residue A in the thyA mutant. This change would cause a predicted amino acid substitution of leucine at position 44 in the polypeptide product of the wild type by glutamine in the mutant. Thus, Leu44 may be critical in enzymatic activity of the thyA gene product thymidylate synthase.
KeywordMeSH Terms
Cloning, Molecular
Genes, Bacterial
160.     ( 1994 )

Identification and characterization of a chromosomal virulence gene, vacJ, required for intercellular spreading of Shigella flexneri.

Molecular microbiology 11 (1)
PMID : 8145644  :   DOI  :   10.1111/j.1365-2958.1994.tb00287.x    
Abstract >>
Intercellular spreading of shigellae is a prerequisite for shigellosis, although the molecular mechanisms underlying the phenomenon are still largely obscure. To elucidate some of these mechanisms, we performed random Tn10 insertion mutagenesis in Shigella flexneri YSH6000T and found a chromosomal locus in the NotI-J segment responsible for bacterial spreading. The locus affected in the mutant, designated vacJ, was neither involved in the invasion of epithelial cells nor in intracellular movement, but was required for intercellular spread. The vacJ mutant was capable of forming bacterium-containing membranous protrusions within the infected cell, but had diminished ability to move from the protrusions into the cytoplasm of the adjacent epithelial cells. Cloning and sequencing of the vacJ region indicated that the vacJ gene encoded a 28.0 kDa protein possessing a signal peptide at the N-terminus, which contained the motif characteristic of lipoproteins. The analysis of the vacJ product indicated that VacJ was exposed on the bacterial surface. The vacJ gene was distributed among shigellae and enteroinvasive Escherichia coli, and the constructed vacJ mutants failed to spread intercellularly, indicating that vacJ is a chromosomal gene essential for the pathogenicity of shigellae.
KeywordMeSH Terms
161.     ( 1994 )

Inhibition of Agrobacterium tumefaciens oncogenicity by the osa gene of pSa.

Journal of bacteriology 176 (18)
PMID : 8083162  :   DOI  :   10.1128/jb.176.18.5697-5703.1994     PMC  :   PMC196773    
Abstract >>
The IncW plasmid pSa originally derived from Shigella flexneri completely inhibits the tumor-inducing ability of Agrobacterium tumefaciens when it is resident in this organism. Oncogenic inhibition is mediated through the expression of the osa gene on pSa. This gene is part of a 3.1-kb DNA segment of pSa that contains four open reading frames revealed by sequencing. Specific deletions and TnCAT insertions within this segment localized the oncogenic inhibitory activity to the last open reading frame, orf-4, designated osa (for oncogenic suppression activity). No promoter exists immediately upstream of the coding sequence of osa since TnCAT insertions or deletions into orf-3 caused the loss of oncogenic inhibition. Deletion analysis showed that the promoter of orf-1 is required for osa transcription. The first three orfs have no role in oncogenic inhibition, since osa alone placed under the control of a constitutive Pkm promoter completely inhibited A. tumefaciens oncogenicity. This inhibition of oncogenicity by osa is not limited to a specific host plant but appears to show broad host specificity. Because the osa-encoded product has close homologies to the fiwA-encoded product of the IncP plasmid RP1, osa may be involved in fertility inhibition that would prevent or reduce the formation of stable mating pairs and T-DNA transfer between A. tumefaciens and plants.
KeywordMeSH Terms
Endonucleases
Genes, Bacterial
Micrococcal Nuclease
162.     ( 1993 )

Characterization of the Shigella flexneri ipgD and ipgF genes, which are located in the proximal part of the mxi locus.

Infection and immunity 61 (5)
PMID : 8478058  :   PMC  :   PMC280755    
Abstract >>
The Shigella flexneri invasion process requires the synthesis of the Ipa proteins and their secretion by specific factors encoded by the mxi and spa genes, which are clustered upstream from the ipa operon. We report here the characterization of the ipgD, ipgE, and ipgF genes, which are located in the 5' end of the mxi locus. Analysis of IpgF-PhoA fusions endowed with high levels of alkaline phosphatase activity confirmed the functionality of a classical signal sequence detected in the sequence of IpgF. The ipgD and ipgF genes were each inactivated on the large virulence plasmid by insertion of a nonpolar cassette; each of the ipgD and ipgF mutants thus constructed showed the same invasive phenotype as the wild-type strain and was able to provoke keratoconjunctivitis in guinea pigs. It thus appears that two genes located at the ipa-proximal part of the mxi locus are not directly involved in invasion. Analysis of concentrated culture supernatants of the wild-type and ipgD strains indicated that secretion of one polypeptide, whose size was consistent with that predicted for the IpgD protein (60 kDa), was abolished in the ipgD mutant.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
163.     ( 1994 )

vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase (Tgt) of Escherichia coli K-12.

Journal of bacteriology 176 (15)
PMID : 8045893  :   DOI  :   10.1128/jb.176.15.4627-4634.1994     PMC  :   PMC196283    
Abstract >>
The genetic determinants required for invasion of epithelial cells by Shigella flexneri and for the subsequent bacterial spreading are encoded by the large virulence plasmid. Expression of the virulence genes is under the control of various genes on the large plasmid as well as on the chromosome. We previously identified one of the virulence-associated loci near phoBR in the NotI-C fragment of the chromosome of S. flexneri 2a YSH6000 and designated the locus vacC. The vacC mutant showed decreased levels of IpaC, and IpaD proteins as well as transcription of ipa, an operon essential for bacterial invasion (N. Okada, C. Sasakawa, T. Tobe, M. Yamada, S. Nagai, K. A. Talukder, K. Komatsu, S. Kanegasaki, and M. Yoshikawa, Mol. Microbiol. 5:187-195, 1991). To elucidate the molecular nature of the vacC locus, we cloned the vacC region from YSH6000 on a 1.8-kb SalI-BamHI DNA fragment. The nucleotide sequence of the 1,822-bp vacC clone was highly (> 98%) homologous to the tgt region of Escherichia coli K-12, which is located at 9.3 min on the linkage map. Complementation tests indicated that the vacC function was encoded by an open reading frame expressing a 42.5-kDa protein, which corresponded to the tgt gene of E. coli K-12, coding for tRNA-guanine transglycosylase (Tgt) (K. Reuter, R. Slany, F. Ullrich, and H. Kersten, J. Bacteriol. 173:2256-2264, 1991). The cloned tgt gene from E. coli K-12 restored the virulence phenotype to the vacC mutant of YSH6000. Characterization of the vacC mutant indicated that levels of VirG, a protein essential for bacterial spreading, and VirF, the positive regulator for the expression of the virG and ipaBCD operons, decreased significantly compared with those of the wild type. Similar phenotypic changes occurred in vacC mutants constructed by insertion of a neomycin resistance gene in shigellae and enteroinvasive E. coli strains, consistent with the hypothesis that the vacC (tgt) gene contributes to the pathogenicity of Shigella flexneri.
KeywordMeSH Terms
164.     ( 1994 )

Nucleotide sequence of the rhamnose biosynthetic operon of Shigella flexneri 2a and role of lipopolysaccharide in virulence.

Journal of bacteriology 176 (8)
PMID : 8157605  :   DOI  :   10.1128/jb.176.8.2362-2373.1994     PMC  :   PMC205360    
Abstract >>
N1308, a chromosomal Tn5 mutant of Shigella flexneri 2a, was described previously as a lipopolysaccharide (LPS) mutant with a short O side chain. N1308 formed foci, but not plaques, in LLC-MK2 cell monolayers and was negative in the Ser?ny test. In this study, the wild-type locus inactivated in N1308 was cloned and further defined by means of complementation analysis. A 4.3-kb BstEII-XhoI fragment of S. flexneri 2a YSH6200 DNA was sufficient to restore both normal LPS and virulence phenotype to the mutant. DNA sequencing of this region revealed four genes, rfbA, rfbB, rfbC, and rfbD, encoding the enzymes required for the biosynthesis of activated rhamnose. The four genes were expressed in Escherichia coli, and the expected protein products were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N1308 was shown to have normal levels of surface IpaC and IpaD, while a Western blot (immunoblot) of whole-cell lysates or outer membrane fractions indicated an elevated level of appropriately localized VirG. An in vitro invasion assay revealed that N1308 had normal primary invasive capacity and was able to multiply and move normally within the initial infected cell. However, it exhibited a significant reduction in its ability to spread from cell to cell in the monolayer. A double immunofluorescence assay revealed differences between LLC-MK2 cells infected with the wild-type YSH6000 and those infected with N1308. The wild-type bacteria elicited the formation of the characteristic F-actin tails, whereas N1308 failed to do so. However, N1308 was capable of inducing deposition of F-actin, which accumulated in a peribacterial fashion with only slight, if any, unipolar accumulation of the cytoskeletal protein.
KeywordMeSH Terms
Lipopolysaccharides
165.     ( 1994 )

Intergeneric transfer and recombination of the 6-phosphogluconate dehydrogenase gene (gnd) in enteric bacteria.

Proceedings of the National Academy of Sciences of the United States of America 91 (21)
PMID : 7937867  :   DOI  :   10.1073/pnas.91.21.10227     PMC  :   PMC44991    
Abstract >>
The gnd gene, encoding 6-phosphogluconate dehydrogenase (EC 1.1.1.44), was sequenced in 87 strains of 15 species assigned to five nominal genera of the Enterobacteriaceae, including 36 isolates of Salmonella enterica and 32 strains of Escherichia coli. In S. enterica, the effective (realized) rate of recombination of horizontally transferred gnd sequences is only moderately higher than the rates for other chromosomal housekeeping genes. In contrast, recombination at gnd has occurred with such high frequency in Escherichia coli that the indicated evolutionary relationships among strains are not congruent with those estimated by sequence analysis of other genes and by multilocus enzyme electrophoresis. E. coli and S. enterica apparently have not exchanged gnd sequences, but those of several strains of E. coli have been imported from species of Citrobacter and Klebsiella. The relatively frequent exchange of gnd within and among taxonomic groups of the Enterobacteriaceae, compared with other housekeeping genes, apparently results from its close linkage with genes that are subject to diversifying selection, including those of the rfb region determining the structure of the O antigen polysaccharide.
KeywordMeSH Terms
Biological Evolution
Gene Transfer Techniques
Genes, Bacterial
166.     ( 1994 )

Characterization of the dTDP-rhamnose biosynthetic genes encoded in the rfb locus of Shigella flexneri.

Molecular microbiology 11 (2)
PMID : 8170390  :   DOI  :   10.1111/j.1365-2958.1994.tb00308.x    
Abstract >>
The nucleotide sequence of the proximal half of the rfb region of Shigella flexneri has been determined, and the genes encoding enzymes involved in the biosynthesis of dTDP-rhamnose have been identified. These genes show strong homology to the rfb genes encoding dTDP-rhamnose biosynthesis in Salmonella enterica serovar typhimurium (strain LT2) and S. enterica serovar anatum (strain M32) (Jiang et al., 1991; Wang et al., 1992). An open reading frame upstream of rfbB was also identified which encoded a protein having strong similarity with GaIU, and has been designated galF. GalF has 92% amino acid sequence identity with an S. enterica LT2 gene, orf2X8, which is similarly situated upstream of rfbB (Jiang et al., 1991). The T7 expression system was utilized to identify proteins corresponding to those predicted from DNA sequence analysis. The similarity of the predicted proteins with proteins that are functionally identical or related, and with others of unknown function from the Yersinia enterocolitica O3 rfb region, and in the Escherichia coli K-12 rff region are also described. We have re-addressed the assignment of each gene of the dTDP-rhamnose pathway with the known enzymes of the pathway, in particular rfbC and rfbD. A reporter plasmid to detect genes encoding enzymes of the dTDP-rhamnose pathway is described. An analysis of the intergenic region between galF and rfbB has been made, and comparison with the same region from S. enterica LT2 discussed.
KeywordMeSH Terms
Genes, Bacterial
167.     ( 1993 )

Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement.

Journal of bacteriology 175 (8)
PMID : 8468279  :   DOI  :   10.1128/jb.175.8.2189-2196.1993     PMC  :   PMC204503    
Abstract >>
Shigella flexneri uses elements of the host cell cytoskeleton to move within cells and from cell to cell. IcsA, an S. flexneri protein involved in this movement, was purified and studied in vitro. IcsA bound the radiolabelled ATP analog 3'(2')-O-(4-benzoyl)benzoyl-ATP and hydrolyzed ATP. In addition, the surface localization of IcsA on both extracellular and intracellular shigellae was unipolar. Further, in HeLa cells infected with shigellae, IcsA antiserum labelled the actin tail throughout its length, thereby suggesting that IcsA interacts with elements within the tail. Localization of IcsA within the tail at a distance from the bacterium would require its secretion; we demonstrate here that in vitro IcsA is secreted into the culture supernatant in a cleaved form.
KeywordMeSH Terms
DNA-Binding Proteins
Transcription Factors
168.     ( 1994 )

Molecular characterization of intact, but cryptic, flagellin genes in the genus Shigella.

Molecular microbiology 12 (2)
PMID : 8057852  :   DOI  :   10.1111/j.1365-2958.1994.tb01016.x    
Abstract >>
Flagellin genes (fliC) were detected in two species of the genus Shigella. The fliCSF gene cloned from Shigella flexneri produced normal-type flagella in an Escherichia coli delta fliC strain while the fliCSS genes from two Shigella sonnei strains produced curly-type flagella and their expression is repressible by Salmonella FljA repressor. The fliCSF gene (1650 bp) shared high similarity with the E. coli fliCE gene not only in the 5' and 3' constant sequences but also in the upstream and downstream sequences. The fliCSS genes (1572 bp) shared high similarity with the Salmonella typhimurium fliCS gene in the operator and 3' constant sequences and also shared high similarity with the fliCE gene in the downstream sequence, suggesting that the fliCSS gene has undergone horizontal transfer and recombination. Differences in nucleotide sequences of the central variable regions among the four fliC genes, including fliCE and fliCS, suggest that they started differentiation in each lineage approximately 80 million years ago. Loss of motility in Shigella seems to be evolutionarily a recent event.
KeywordMeSH Terms
Genes, Bacterial
169. Fukuda  I, Suzuki  T, Munakata  H, Hayashi  N, Katayama  E, Yoshikawa  M, Sasakawa  C,     ( 1995 )

Cleavage of Shigella surface protein VirG occurs at a specific site, but the secretion is not essential for intracellular spreading.

Journal of bacteriology 177 (7)
PMID : 7896693  :   DOI  :   10.1128/jb.177.7.1719-1726.1995     PMC  :   PMC176798    
Abstract >>
The large plasmid-encoded outer membrane protein VirG (IcsA) of Shigella flexneri is essential for bacterial spreading by eliciting polar deposition of filamentous actin (F-actin) in the cytoplasm of epithelial cells. Recent studies have indicated that VirG is located at one pole on the surface of the bacterium and secreted into the culture supernatant and that in host cells it is localized along the length of the F-actin tail. The roles of these VirG phenotypes in bacterial spreading still remain to be elucidated. In this study, we examined the surface-exposed portion of the VirG protein by limited trypsin digestion of S. flexneri YSH6000 and determined the sites for VirG processing during secretion into the culture supernatant. Our results indicated that the 85-kDa amino-terminal portion of VirG is located on the external side of the outer membrane, while the 37-kDa carboxy-terminal portion is embedded in it. The VirG cleavage required for release of the 85-kDa protein into the culture supernatant occurred at the Arg-Arg bond at positions 758 to 759. VirG-specific cleavage was observed in Shigella species and enteroinvasive Escherichia coli, which requires an as yet unidentified protease activity governed by the virB gene on the large plasmid. To investigate whether the VirG-specific cleavage occurring in extracellular and intracellular bacteria is essential for VirG function in bacterial spreading, the Arg-Arg cleavage site was modified to an Arg-Asp or Asp-Asp bond. The virG mutants thus constructed were capable of unipolar deposition of VirG on the bacterial surface but were unable to cleave VirG under in vitro or in vivo conditions. However, these mutants were still capable of eliciting aggregation of F-actin at one pole, spreading into adjacent cells, and giving rise to a positive Sereny test. Therefore, the ability to cleave and secrete VirG in Shigella species is not a prerequisite for intracellular spreading.
KeywordMeSH Terms
170. Fasano  A, Noriega  FR, Maneval  DR, Chanasongcram  S, Russell  R, Guandalini  S, Levine  MM,     ( 1995 )

Shigella enterotoxin 1: an enterotoxin of Shigella flexneri 2a active in rabbit small intestine in vivo and in vitro.

The Journal of clinical investigation 95 (6)
PMID : 7769126  :   DOI  :   10.1172/JCI117991     PMC  :   PMC295972    
Abstract >>
Culture filtrates of Shigella flexneri 2a strain M4243 grown in iron-depleted medium, caused significant fluid accumulation in rabbit ileal loops. Also, when tested in Ussing chambers, a greater rise in potential difference and short circuit current was seen with such filtrates compared with the medium control. Analogous filtrates from two M4243 derivatives lacking the 140-MD invasiveness plasmid (either M4243avir or BS103) retained 60-65% of the wild-type enterotoxic activity. Ultrafiltration and gel exclusion size fractionation of M4243 filtrate revealed that the activity was approximately 60 kD. SDS-PAGE performed on this fraction showed 18 bands, 5 of which reacted with human convalescent sera. Genes encoding this enterotoxin, named ShET1 for Shigella enterotoxin 1, were cloned from the S. flexneri 2a chromosome, and two separate open reading frames of 534 and 186 bp were sequenced. These observations suggest that S. flexneri 2a elaborates two distinct enterotoxins: ShET1, encoded by genes located on the chromosome, and ShET2, encoded by a gene on the 140-MD invasiveness plasmid. ShET1, which is composed of two distinct subunits and is elaborated in vivo, where it elicits an immune response, may be important in the pathogenesis of diarrheal illness due to S. flexneri 2a.
KeywordMeSH Terms
171. Scholten  M, Janssen  R, Bogaarts  C, van Strien  J, Tommassen  J,     ( 1995 )

The pho regulon of Shigella flexneri.

Molecular microbiology 15 (2)
PMID : 7746146  :   DOI  :   10.1111/j.1365-2958.1995.tb02239.x    
Abstract >>
Growth of Escherichia coli K-12 in low-phosphate conditions results in the induction of the synthesis of many proteins, including the outer membrane porin PhoE, alkaline phosphatase, and the Pst system for the transport of phosphate (P1). This response is controlled by a two-component regulatory system of which PhoB and PhoR are the response-regulator and the sensor/kinase, respectively. When Shigella flexneri was starved for P1, neither PhoE nor alkaline phosphatase was produced. However, induction of the synthesis of the PstS protein was observed, indicating that S. flexneri contains a functional PhoB/PhoR regulatory system. Consistent with this notion, the introduction of the E. coli phoA gene in S. flexneri resulted in the induction of alkaline phosphatase synthesis under phosphate limitation. However, introduction of phoE on a plasmid did not lead to the expression of PhoE protein, indicating that S. flexneri PhoB does not recognize the phoE promoter region. The phoB gene was cloned and sequenced and in the deduced amino acid sequence two deviations from that of E. coli PhoB were detected. Site-directed mutagenesis revealed that one of these deviations, i.e. Leu-172, which is Arg in E. coli PhoB, is responsible for the lack of expression of the PhoE protein in S. flexneri.
KeywordMeSH Terms
Escherichia coli Proteins
Periplasmic Binding Proteins
Regulon
172. Nataro  JP, Seriwatana  J, Fasano  A, Maneval  DR, Guers  LD, Noriega  F, Dubovsky  F, Levine  MM, Morris  JG,     ( 1995 )

Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains.

Infection and immunity 63 (12)
PMID : 7591128  :   PMC  :   PMC173677    
Abstract >>
We have employed a molecular genetic approach to characterize the nature of enteroinvasive Escherichia coli (EIEC) enterotoxic activity, as previously observed in Ussing chambers (A. Fasano, B.A. Kay, R.G. Russell, D.R. Maneval, Jr., and M.M. Levine, Infect. Immun. 58:3717-3723, 1990). The screening of TnphoA mutants of EIEC yielded a single insertion mutant which had significantly reduced levels of enterotoxic activity in the Ussing chamber assay. DNA flanking the insertion was used as a probe to screen for EIEC cosmid clones which conferred secretogenic activity. Such screening resulted in the identification of two overlapping cosmid clones which elicited significant changes in mucosal short-circuit current (Isc). Subcloning and nucleotide sequence analysis of a DNA fragment from one of the cosmid clones led to the identification of a single open reading frame which conferred this enterotoxic activity. By DNA hybridization, this gene (designated sen for shigella enterotoxin) was found in 75% of EIEC strains and 83% of Shigella strains and was localized to the inv plasmid of Shigella flexneri 2457T. By PCR, a sen gene with 99.7% nucleotide identity was cloned and sequenced from 2457T. A deletion in the EIEC sen gene was constructed by allelic exchange, resulting in significantly lower rises in Isc than were elicited by the wild-type parent; however, significant enterotoxic activity remained in the sen deletion mutant. To purify the Sen protein, the gene was cloned into the multiple cloning site of the expression vector pKK223-3. Purification of the sen gene product yielded a protein with a molecular mass of 63 kDa which elicited rises in Isc in the Ussing chamber. We believe that the sen gene product may constitute all or part of a novel enterotoxin in EIEC and Shigella spp.
KeywordMeSH Terms
Escherichia coli Proteins
173.     ( 1994 )

The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD.

The EMBO journal 13 (22)
PMID : 7957095  :   PMC  :   PMC395485    
Abstract >>
Shigella species are enteropathogens that invade epithelial cells of the human colon. Entry into epithelial cells is triggered by the IpaB, IpaC and IpaD proteins which are translocated into the medium through the specific Mxi-Spa machinery. In vitro, Shigella cells secrete only a small fraction of the Ipa proteins, the majority of which remains in the cytoplasm. We show here that upon interaction with cultured epithelial cells or in the presence of fetal bovine serum, S.flexneri release pre-synthesized Ipa molecules from the cytoplasm into the environment. Evidence is presented that IpaB and IpaD are essential for both blocking secretion through the Mxi-Spa translocon in the absence of a secretion-inducing signal and controlling secretion of the Ipa proteins in the presence of a signal. Subcellular localization and analysis of the molecular interactions of the Ipa proteins indicate that IpaB and IpaD associate transiently in the bacterial envelope. We propose that IpaB and IpaD, by interacting in the secretion apparatus, modulate secretion.
KeywordMeSH Terms
Adhesins, Bacterial
174. Watarai  M, Tobe  T, Yoshikawa  M, Sasakawa  C,     ( 1995 )

Disulfide oxidoreductase activity of Shigella flexneri is required for release of Ipa proteins and invasion of epithelial cells.

Proceedings of the National Academy of Sciences of the United States of America 92 (11)
PMID : 7761426  :   DOI  :   10.1073/pnas.92.11.4927     PMC  :   PMC41820    
Abstract >>
Secretion of IpaB, IpaC, and IpaD proteins of Shigella flexneri, essential for the invasion of epithelial cells, requires a number of proteins encoded by the spa and mxi loci on the large plasmid. Introduction of dsbA::Tn5 into S.flexneri from Escherichia coli K-12 reduced invasiveness, which resulted from a decrease in the capacity to release IpaB, IpaC, and IpaD proteins into the external medium. Examination of the surface-presented Ipa proteins of the dsbA mutant, however, revealed Ipa proteins at levels similar to those on wild-type cells. Since the defective phenotype was similar to that of the spa32 mutant of S. flexneri and the Spa32 sequence possessed two Cys residues, the effect of dsbA mutation of the folding structure of Spa32 under reducing conditions and on the surface expression of Spa32 was investigated. The results indicated that Spa32 was a disulfide-containing protein whose correctly folded structure was required for its presentation on the outer membrane. Indeed, replacing either one of the two Cys residues in Spa32 with Ser by site-directed mutagenesis reduced its capacity to release Ipa proteins into the external medium and led to the accumulation of Spa32 protein in the periplasm. These results indicated that the DsbA protein performs an essential function during the invasion of mammalian cells, by facilitating transport of the Spa32 protein across the outer membrane.
KeywordMeSH Terms
175.     ( 1994 )

Extracellular association and cytoplasmic partitioning of the IpaB and IpaC invasins of S. flexneri.

Cell 79 (3)
PMID : 7954817  :   DOI  :   10.1016/0092-8674(94)90260-7    
Abstract >>
Shigella species cause bacillary dysentery in humans by invading colonic epithelial cells. IpaB and IpaC, two major invasins of these pathogens, are secreted into the extracellular milieu. We show here that IpaB and IpaC form a complex in the extracellular medium and that each binds independently to a 17 kDa polypeptide, IpgC, in the bacterial cytoplasm. The IpgC polypeptide was found to be necessary for bacterial entry into epithelial cells, to stabilize the otherwise unstable IpaB protein, and to prevent the proteolytic degradation of IpaC that occurs through its association with unprotected IpaB. We propose that IpgC, which is not secreted and thus acts as a molecular chaperone, serves as a receptor that prevents premature oligomerization of IpaB and IpaC within the cytoplasm of Shigella cells.
KeywordMeSH Terms
Adhesins, Bacterial
Cell Compartmentation
176.     ( 1994 )

Integrons found in different locations have identical 5' ends but variable 3' ends.

Journal of bacteriology 176 (20)
PMID : 7929000  :   DOI  :   10.1128/jb.176.20.6286-6294.1994     PMC  :   PMC196970    
Abstract >>
The positions of the outer boundaries of the 5'- and 3'-conserved segment sequences of integrons found at several different locations have been determined. The position of the 5' end of the 5'-conserved segment is the same for six independently located integrons, In1 (R46), In2 (Tn21), In3 (R388), In4 (Tn1696), In5 (pSCH884), and In0 (pVS1). However, the extent of the 3'-conserved segment differs in each integron. The sequences of In2 and In0 diverge first from the conserved sequence, and their divergence point corresponds to the 3'-conserved segment endpoint defined previously (H.W. Stokes and R.M. Hall, Mol. Microbiol. 3:1669-1683, 1989), which now represents the endpoint of a 359-base deletion in In0 and In2. The sequence identity in In3, In1, In4, and In5 extends beyond this point, but each sequence diverges from the conserved sequence at a different point within a short region. Insertions of IS6100 were identified adjacent to the end of the conserved region in In1 and 123 bases beyond the divergence point of In4. These 123 bases are identical to the sequence found at the mer end of the 11.2-kb insertion in Tn21 but are inverted. In5 and In0 are bounded by the same 25-base inverted repeat that bounds the 11.2-kb insert in Tn21, and this insert now corresponds to In2. However, while In0, In2, and In5 have features characteristic of transposable elements, differences in the structures of these three integrons and the absence of evidence of mobility currently preclude the identification of all of the sequences associated with a functional transposon of this type.
KeywordMeSH Terms
177. Sandlin  RC, Lampel  KA, Keasler  SP, Goldberg  MB, Stolzer  AL, Maurelli  AT,     ( 1995 )

Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane.

Infection and immunity 63 (1)
PMID : 7528731  :   PMC  :   PMC172982    
Abstract >>
Mutations in the lipopolysaccharide (LPS) of Shigella spp. result in attenuation of the bacteria in both in vitro and in vivo models of virulence, although the precise block in pathogenesis is not known. We isolated defined mutations in two genes, galU and rfe, which directly affect synthesis of the LPS of S. flexneri 2a, in order to determine more precisely the step in virulence at which LPS mutants are blocked. The galU and rfe mutants invaded HeLa cells but failed to generate the membrane protrusions (fireworks) characteristic of intracellular motility displayed by wild-type shigellae. Furthermore, the galU mutant was unable to form plaques on a confluent monolayer of eucaryotic cells and the rfe mutant generated only tiny plaques. These observations indicated that the mutants were blocked in their ability to spread from cell to cell. Western immunoblot analysis of expression of IcsA, the protein essential for intracellular motility and intercellular spread, demonstrated that both mutants synthesized IcsA, although they secreted less of the protein to the extracellular medium than did the wild-type parent. More strikingly, the LPS mutants showed aberrant surface localization of IcsA. Unlike the unipolar localization of IcsA seen in the wild-type parent, the galU mutant expressed the protein in a circumferential fashion. The rfe mutant had an intermediate phenotype in that it displayed some localization of IcsA at one pole while also showing diffuse localization around the bacterium. Given the known structures of the LPS of wild-type S. flexneri 2a, the rfe mutant, and the galU mutant, we hypothesize that the core and O-antigen components of LPS are critical elements in the correct unipolar localization of IcsA. These observations indicate a more precise role for LPS in Shigella pathogenesis.
KeywordMeSH Terms
Escherichia coli Proteins
178. Faubladier  M, Bouché  JP,     ( 1994 )

Division inhibition gene dicF of Escherichia coli reveals a widespread group of prophage sequences in bacterial genomes.

Journal of bacteriology 176 (4)
PMID : 7508908  :   DOI  :   10.1128/jb.176.4.1150-1156.1994     PMC  :   PMC205167    
Abstract >>
The genomes of various eubacteria were analyzed by Southern blot hybridization to detect sequences related to the segment of the defective lambdoid prophage Kim which encodes DicF RNA, an antisense inhibitor of cell division gene ftsZ in Escherichia coli K-12. Among the homologous sequences found, one fragment from E. coli B, similar to a piece of Rac prophage, and two fragments from Shigella flexneri were cloned and sequenced. dicF-like elements similar to transcriptional terminators were found in each sequence, but unlike dicF these had no effect on division in E. coli K-12. Like dicF, these sequences are flanked by secondary structures which form potential sites for RNase III recognition. Coding sequences located upstream from the dicF-like feature in E. coli B are related to gene sieB of bacteriophage lambda, while sequences downstream of the S. flexneri elements are similar to the immunity region of satellite bacteriophage P4. Under hybridization conditions in which only strong sequence homologies were detected in E. coli B and S. flexneri, the genomes of a large variety of microorganisms, including some gram-positive bacteria, hybridized to the dicF probe. Our results suggest that dicF and its flanking regions are markers of a widespread family of prophage-like elements of different origins.
KeywordMeSH Terms
Cytoskeletal Proteins
Genome, Bacterial
179. Tobe  T, Yoshikawa  M, Mizuno  T, Sasakawa  C,     ( 1993 )

Transcriptional control of the invasion regulatory gene virB of Shigella flexneri: activation by virF and repression by H-NS.

Journal of bacteriology 175 (19)
PMID : 7691791  :   DOI  :   10.1128/jb.175.19.6142-6149.1993     PMC  :   PMC206708    
Abstract >>
Expression of invasion genes encoded by the large 230-kb plasmid of Shigella flexneri is controlled by the virB gene, which is itself activated by another regulator, virF. Transcription of the invasion genes is temperature regulated, since they are activated in bacteria grown at 37 but not at 30 degrees C. Recently, we have shown that the thermoregulated expression of invasion genes is mediated by thermal activation of virB transcription (T. Tobe, S. Nagai, B. Adler, M. Yoshikawa, and C. Sasakawa, Mol. Microbiol. 5:887-893, 1991). It has also been shown that a mutation that inactivates H-NS, the product of virR (hns), derepresses transcription of virB. To elucidate the molecular mechanisms underlying virB activation, we determined the location of the transcription start site and found it to be 54 bp upstream of the 5' end of the virB coding sequence. Deletion analysis revealed that transcriptional activation by virF requires a DNA segment of 110 bp extending upstream of the transcription start site. By using a protein binding assay with crude extracts of S. flexneri harboring the malE'-'virF fusion gene, which was able to activate virB transcription, two protein species, one of 70 kDa (MalE'-'VirF fusion) and another of 16 kDa (H-NS), were shown to bind specifically to the virB promoter region. DNA footprinting analysis indicated that the VirF fusion and H-NS proteins bound to the upstream sequence spanning from -17 to -117 and to the sequence from -20 to +20, in which virB transcription starts, respectively. In an vitro transcription assay, the VirF fusion protein was shown to activate virB transcription while the H-NS protein blocked it. virB activation was seen only when negatively supercoiled DNA was used as a template. In in vivo studies, virB transcription was significantly decreased by adding novobiocin, a gyrase inhibitor, into the culture medium while virB transcription was increased by mutating hns. These in vitro and in vivo studies indicated that transcription of virB is activated through VirF binding to the upstream sequence of the virB promoter in a DNA-topology-dependent manner and is directly repressed by H-NS binding to the virB transcription start site.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
Genes, Regulator
Promoter Regions, Genetic
Transcription, Genetic
Virulence Factors
180. Uchiya  K, Tobe  T, Komatsu  K, Suzuki  T, Watarai  M, Fukuda  I, Yoshikawa  M, Sasakawa  C,     ( 1995 )

Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading.

Molecular microbiology 17 (2)
PMID : 7494473  :   DOI  :   10.1111/j.1365-2958.1995.mmi_17020241.x    
Abstract >>
A novel virulence gene (virA) was identified upstream of the virG gene on the large plasmid of Shigella flexneri 2a YSH6000. Characterization of virA mutants infecting MK2 epithelial cell monolayers revealed that their invasive capacity was decreased to less than one fifth of the wild-type level. Nevertheless, the bacteria were capable of expressing and secreting IpaB, IpaC and IpaD proteins. The virA mutants were also impaired in their ability to spread intercellularly, since the bacteria gave rise to a small number of foci in a focus-plaque-forming test with MK2 cells. Although virG expression was slightly decreased in the virA mutants, introduction of a cloned virG gene into a virA mutant, N1945, failed to restore spreading ability. Although, introduction of a cloned virA gene into N1945 restored invasiveness and spreading ability, the reduced virG transcription level was not affected, indicating that the reduced virG expression in virA mutants does not play a major role in defective intercellular spreading. The nucleotide sequence of the virA region revealed that the virA gene was located 528 bp upstream of the virG gene, in the opposite orientation. The deduced amino acid sequence of the VirA protein indicated a 44.7 kDa protein with no homology to known proteins. The VirA protein was secreted into the culture supernatant, a process that required the Mxi and Spa loci. The expression of virA was under the control of the virB gene, the positive regulator of the ipa, mxi and spa operons. These results indicate that virA is a new member of the invasion regulon directed by virB and that the VirA function is involved in invasion and intercellular spreading.
KeywordMeSH Terms
Virulence Factors
181. Benjelloun-Touimi  Z, Sansonetti  PJ, Parsot  C,     ( 1995 )

SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion.

Molecular microbiology 17 (1)
PMID : 7476198  :   DOI  :   10.1111/j.1365-2958.1995.mmi_17010123.x    
Abstract >>
In addition to Ipa proteins and IcsA, which are involved in entry into epithelial cells and intercellular spread, respectively, Shigella secretes a 110 kDa protein, designated SepA. We report the identification, cloning, and nucleotide sequence determination of the sepA gene, analysis of SepA secretion, and construction and characterization of a sepA mutant. The sepA gene is carried by the virulence plasmid and codes for a 150 kDa precursor. Upon secretion, which does not involve accessory proteins encoded by the virulence plasmid, the precursor is converted to a mature protein of 110 kDa by two cleavages removing an N-terminal signal sequence and a C-terminal fragment. Extensive similarities were detected between the sequence of the first 500 residues of mature SepA and the N-terminal region of IgA1 proteases from Neisseria gonorrhoeae and Haemophilus influenzae, the Tsh haemagglutinin of an avian pathogenic Escherichia coli, and the Hap protein involved in adhesion and penetration of H. influenzae. The C-terminal domain of the SepA precursor, which is not present in the secreted protein, exhibits sequence similarity with pertactin of Bordetella pertussis and the ring-forming protein of Helicobacter mustelae. Construction and phenotypic characterization of a sepA mutant indicated that SepA is required neither for entry into cultured epithelial cells nor for intercellular dissemination. However, in the rabbit ligated ileal loop model, the sepA mutant exhibited an attenuated virulence, which suggests that SepA might play a role in tissue invasion.
KeywordMeSH Terms
182. Misra  TK, Brown  NL, Fritzinger  DC, Pridmore  RD, Barnes  WM, Haberstroh  L, Silver  S,     ( 1984 )

Mercuric ion-resistance operons of plasmid R100 and transposon Tn501: the beginning of the operon including the regulatory region and the first two structural genes.

Proceedings of the National Academy of Sciences of the United States of America 81 (19)
PMID : 6091128  :   DOI  :   10.1073/pnas.81.19.5975     PMC  :   PMC391841    
Abstract >>
The mercuric ion-resistance operons of plasmid R100 (originally from Shigella) and transposon Tn501 (originally from a plasmid isolated in Pseudomonas) have been compared by DNA sequence analysis. The sequences for the first 1340 base pairs of Tn501 are given with the best alignment with the comparable 1319 base pairs of R100. The homology between the two sequences starts at base 58 after the end of the insertion sequence IS-1 of R100. The sequences include the transcriptional regulatory region, and the homology is particularly strong in regions just upstream from potential transcriptional initiation sites. The trans-acting regulatory gene merR consists of 180 base pairs in both cases and codes for a highly basic polypeptide of 60 amino acids, which is also rich in serine. The Tn501 and R100 merR genes differ in 25 of the 180 base positions, and the resulting polypeptides differ in seven amino acids. The regulatory region before the major transcription initiation site contains potential -35 and -10 sequences and dyad symmetrical sequences, which may be the merR binding sites for transcriptional regulation. The first structural gene, merT, encodes a highly hydrophobic polypeptide of 116 amino acids. The R100 and Tn501 merT genes differ in 17% of their positions, leading to 14 (12%) amino acid changes. This region had previously been shown to encode a protein governing membrane transport of mercuric ions. The second structural gene, merC, would give a 91 amino acid polypeptide with a hydrophobic amino-terminal segment. The Tn501 and R100 merC genes differ at 37 base positions, leading to 10 amino acid changes.
KeywordMeSH Terms
183. Barrineau  P, Gilbert  P, Jackson  WJ, Jones  CS, Summers  AO, Wisdom  S,     ( 1984 )

The DNA sequence of the mercury resistance operon of the IncFII plasmid NR1.

Journal of molecular and applied genetics 2 (6)
PMID : 6530603  :  
Abstract >>
The DNA sequence has been determined for a 3.8-kilobase region which encodes the mercury resistance (mer) operon of the IncFII plasmid NR1. The sequence reveals four open reading frames which could encode proteins of 12,391, 9,429, 14,965, and 58,781 daltons. On the basis of their sizes, amino acid compositions, hydropathicities, and estimated isoelectric points, the peptides encoded by these open reading frames correspond to the four previously described Hg-inducible proteins detected in minicells carrying mer+ plasmids. The NR1 mer locus is 63.4% GC overall, and the Hg(II) reductase protein sequence is 90% homologous to that of Tn501. The region encoding the merR (positive regulatory) function has three open reading frames. The smallest of these possible merR peptides (6,457 daltons) begins approximately 280 bp to the right of the adjacent IS1b and reads towards the structural genes of the mer operon. The next largest reading frame (13,139 daltons) in the merR region begins 37 bp to the left of the beginning of the smallest peptide and also reads towards the structural genes. The largest reading frame (15,907 daltons) in the merR region lies on the complementary strand and reads away from the structural genes towards IS1b. Although attempts to visualize the merR gene product were not successful, in vitro mutagenesis allows us to eliminate the largest reading frame as a merR candidate. We were also able to show that approximately 50% of the smallest detectable mer peptide (9,429 daltons) is located in the periplasm.
KeywordMeSH Terms
Drug Resistance, Microbial
Plasmids
184. Campos  M, Bayer  E, González  H, Schmeer  K, Stevanovic  S, Jouchim  H, Bocaz  G, Vásquez  O,     ( 1995 )

N-terminal amino-acid sequence of beta-lactamase from Shigella flexneri UCSF-129.

Microbios 82 (333)
PMID : 7476560  :  
Abstract >>
A beta-lactamase (EC 3.5.2.6 penicillinase, penicillin amino beta-lactam-hydrolase) was purified from Shigella flexneri USCF-129 by an efficient two-stage procedure involving chromatography in Sephadex G-75 and HPLC on a C18-reverse phase column. The homogeneity of the purified enzyme was confirmed by capillary zone electrophoresis (CZE), HPLC electrospray mass spectrometry (LC-ESMS) and amino acid sequence analyses. The highly purified enzyme was a monomeric protein with a molecular mass of 28.903 +/- 2 Da, as determined by LC-ESMS. The amino acid sequence of the first 49 N-terminal residues of this beta-lactamase revealed 100% similarity with the mature forms of the plasmid coded Escherichia coli enzymes (plasmid pBR 322 and R6K) a TEM-type beta-lactamase.
KeywordMeSH Terms
185. Morona  R, Mavris  M, Fallarino  A, Manning  PA,     ( 1994 )

Characterization of the rfc region of Shigella flexneri.

Journal of bacteriology 176 (3)
PMID : 7507920  :   DOI  :   10.1128/jb.176.3.733-747.1994     PMC  :   PMC205111    
Abstract >>
The O antigen of the Shigella flexneri lipopolysaccharide (LPS) is an important virulence determinant and immunogen. We have isolated S. flexneri mutants which produce a semi-rough LPS by using an O-antigen-specific phage, Sf6c. Western immunoblotting was used to show that the LPS produced by the semi-rough mutants contained only one O-antigen repeat unit. Thus, the mutants are deficient in production of the O-antigen polymerase and were termed rfc mutants. Complementation experiments were used to locate the rfc adjacent to the rfb genes on plasmid clones previously isolated and containing this region (D. F. Macpherson, R. Morona, D. W. Beger, K.-C. Cheah, and P. A. Manning, Mol. Microbiol 5:1491-1499, 1991). A combination of deletions and subcloning analysis located the rfc gene as spanning a 2-kb region. Insertion of a kanamycin resistance cartridge into a SalI site in this region inactivated the rfc gene. The DNA sequence of the rfc region was determined. An open reading frame spanning the SalI site was identified and encodes a protein with a predicted molecular mass of 43.7 kDa. The predicted protein is highly hydrophobic and showed little sequence homology with any other protein. Comparison of its hydropathy plot with that of other Rfc proteins from Salmonella enterica (typhimurium) and Salmonella enterica (muenchen) revealed that the profiles were similar and that the proteins have 12 or more potential membrane-spanning segments. A comparison of the S. flexneri rfc gene and protein product with other rfc and rfc-like proteins revealed that they have a similarly low percentage of G + C content and have similar codon usage, and all have a high percentage of rare codons. An attempt to identify the S. flexneri Rfc protein was unsuccessful, although proteins encoded upstream and downstream of the rfc gene could be identified. Examination of the distribution of rare or minor codons in the rfc gene revealed that it has several minor codons within the first 25 amino acids. This is in contrast to the upstream gene rfbG, which also has a high percentage of rare codons but whose gene product could be detected. The positioning of the rare codons in the rfc gene may restrict translation and suggests that minor isoaccepting tRNA species may be involved in translational regulation of rfc expression. The low percentage of G + C content of rfc genes may be a consequence of the selection pressure to maintain this form of control.
KeywordMeSH Terms
Genes, Bacterial
186. Venkatesan  MM, Buysse  JM, Kopecko  DJ,     ( 1988 )

Characterization of invasion plasmid antigen genes (ipaBCD) from Shigella flexneri.

Proceedings of the National Academy of Sciences of the United States of America 85 (23)
PMID : 3057506  :   DOI  :   10.1073/pnas.85.23.9317     PMC  :   PMC282730    
Abstract >>
The large invasion plasmid of Shigella flexneri M9OT-W was used to generate recombinant plasmids carrying the ipaA, -B, -C, and -D genes, whose products are associated with the entry of the bacteria into colonic epithelial cells. Complete DNA sequences of ipaB, -C, and -D were determined. The proteins predicted (62, 42, and 37 kDa, respectively) from the nucleotide sequences lack a signal-peptide sequence. Hydrophilic segments of the IpaB and IpaC proteins were found to overlap known epitopic domains of these membrane antigens. Analysis of total RNA demonstrated that temperature control of ipa gene expression occurs at the level of transcription. Multiple mRNA bands were detected by using ipa gene fragments as hybridization probes, and a putative transcript map for the ipa genes was constructed. Comparison of this map with the DNA sequence reveals a complex system of ipa gene regulation.
KeywordMeSH Terms
Genes, Bacterial
Plasmids
187. Cossart  P, Groisman  EA, Serre  MC, Casadaban  MJ, Gicquel-Sanzey  B,     ( 1986 )

crp genes of Shigella flexneri, Salmonella typhimurium, and Escherichia coli.

Journal of bacteriology 167 (2)
PMID : 3525518  :   DOI  :   10.1128/jb.167.2.639-646.1986     PMC  :   PMC212937    
Abstract >>
The complete nucleotide sequences of the Salmonella typhimurium LT2 and Shigella flexneri 2B crp genes were determined and compared with those of the Escherichia coli K-12 crp gene. The Shigella flexneri gene was almost like the E. coli crp gene, with only four silent base pair changes. The S. typhimurium and E. coli crp genes presented a higher degree of divergence in their nucleotide sequence with 77 changes, but the corresponding amino acid sequences presented only one amino acid difference. The nucleotide sequences of the crp genes diverged to the same extent as in the other genes, trp, ompA, metJ, and araC, which are structural or regulatory genes. An analysis of the amino acid divergence, however, revealed that the catabolite gene activator protein, the crp gene product, is the most conserved protein observed so far. Comparison of codon usage in S. typhimurium and E. coli for all genes sequenced in both organisms showed that their patterns were similar. Comparison of the regulatory regions of the S. typhimurium and E. coli crp genes showed that the most conserved sequences were those known to be essential for the expression of E. coli crp.
KeywordMeSH Terms
188. Baudry  B, Kaczorek  M, Sansonetti  PJ,     ( 1988 )

Nucleotide sequence of the invasion plasmid antigen B and C genes (ipaB and ipaC) of Shigella flexneri.

Microbial pathogenesis 4 (5)
PMID : 3071655  :  
Abstract >>
The nucleotide sequence of a 4.8 kilobase (kb) HindIII fragment from pWR100, the virulence plasmid of Shigella flexneri 5, was determined and analysed. This fragment encodes polypeptides b (62 kilodalton, kD) and c (43 kD) which have already been described as two of the four immunogenic polypeptides of Shigellae. The nucleotide sequence revealed that in addition to the ipaB and ipaC genes encoding polypeptides b and c, a third complete open reading frame was found within the fragment. The gene, named ippI, encoded a 17 kD polypeptide. The deduced amino acids sequence of polypeptides b and c showed no signal peptide but presence of highly hydrophobic domains compatible with a transmembraneous location. The surprising A and T richness of the three genes as compared with the Escherichia coli and Shigella genomes, resulted in a biased codon usage, and raises the question of the origin of the sequences.
KeywordMeSH Terms
189. Chinault  AC, Blakesley  VA, Roessler  E, Willis  DG, Smith  CA, Cook  RG, Fenwick  RG,     ( 1986 )

Characterization of transferable plasmids from Shigella flexneri 2a that confer resistance to trimethoprim, streptomycin, and sulfonamides.

Plasmid 15 (2)
PMID : 3517903  :  
Abstract >>
A set of plasmids conferring resistance to several antibiotics, including the combination of trimethoprim and sulfamethoxazole, has been isolated from Escherichia coli following conjugative cotransfer from a clinical isolate of Shigella flexneri 2a. One of the plasmids, pCN1, was shown by subcloning and DNA sequencing to carry a gene encoding a trimethoprim-insensitive dihydrofolate reductase identical to that found in E. coli transposon 7. This plasmid was also shown to confer resistance to both streptomycin and spectinomycin by production of an adenylyltransferase that inactivated the drugs and the gene encoding this enzyme has also been sequenced. A second plasmid from the set, pCN2, was shown to inactivate streptomycin by a phosphotransferase mechanism and also to confer resistance to sulfonamides. The third plasmid from the set could not be correlated with a drug-resistance phenotype, but does appear to play a crucial role in plasmid mobilization.
KeywordMeSH Terms
Genes, Bacterial
R Factors
190. Brown  NL, Misra  TK, Winnie  JN, Schmidt  A, Seiff  M, Silver  S,     ( 1986 )

The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system.

Molecular & general genetics : MGG 202 (1)
PMID : 3007931  :   DOI  :   10.1007/bf00330531    
Abstract >>
The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence, and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes.
KeywordMeSH Terms
DNA Transposable Elements
Genes
Genes, Bacterial
Operon
R Factors
191. Tait  RC, Rempel  H, Rodriguez  RL, Kado  CI,     ( 1985 )

The aminoglycoside-resistance operon of the plasmid pSa: nucleotide sequence of the streptomycin-spectinomycin resistance gene.

Gene 36 (1��2��)
PMID : 2998941  :   DOI  :   10.1016/0378-1119(85)90073-3    
Abstract >>
The nucleotide sequence of the probable C terminus of the kanamycin-resistance gene (KmR) and the probable complete sequence of the streptomycin-spectinomycin-resistance gene (SpR) of the IncW plasmid pSa have been determined. The two genes appear to be oriented in the same direction and separated by a spacer region of 53 bp, with transcription proceeding from the KmR gene into the SpR gene. An RNA transcript encompassing the C terminus of the KmR gene, the 53-base spacer, and the N terminus of the SpR gene has the potential to form a stem-loop structure with a free energy value of -68 kcal/mol. The SpR gene of pSa has extensive sequence homology with the aadA gene of the plasmid R538-1. Comparison of the proposed amino acid sequence of the KmR protein of pSa with those of two aminoglycoside phosphotransferases revealed a region of potential homology with those proteins.
KeywordMeSH Terms
Genes, Bacterial
Operon
R Factors
192. Sakai  T, Sasakawa  C, Makino  S, Yoshikawa  M,     ( 1986 )

DNA sequence and product analysis of the virF locus responsible for congo red binding and cell invasion in Shigella flexneri 2a.

Infection and immunity 54 (2)
PMID : 3021627  :   PMC  :   PMC260174    
Abstract >>
The DNA sequence of virF, a locus associated with virulence and the ability to bind Congo red in Shigella flexneri 2a that is located on a 140-megadalton (230-kilobase) plasmid, was determined and analyzed. It was rich in A and T. The direction of transcription of virF was determined by using a chloramphenicol resistance cartridge. An open reading frame readable in this direction was found. Three proteins, 30, 27, and 21 kilodaltons, all corresponding to those predicted from the above sequence, were produced in minicells containing the virF locus. The three proteins were expressed only weakly in minicells with the 230-kilobase plasmid.
KeywordMeSH Terms
Plasmids
193. Misra  TK, Brown  NL, Haberstroh  L, Schmidt  A, Goddette  D, Silver  S,     ( 1985 )

Mercuric reductase structural genes from plasmid R100 and transposon Tn501: functional domains of the enzyme.

Gene 34 (2��3��)
PMID : 2989109  :   DOI  :   10.1016/0378-1119(85)90134-9    
Abstract >>
The nucleotide sequence for the 2240 bp of plasmid R100 following the merC gene of the mercuric resistance operon has been determined and compared with the homologous sequence of transposon Tn501. The sequences following merC and preceding the next structural gene merA are unrelated between R100 and Tn501 and differ in length, with 72 bp in Tn501 and 509 bp in R100. The R100 sequence has a potential open reading frame (ORF) for a 140 amino acid polypeptide with a reasonable translational start signal preceding it. The merA genes contain 1686 (Tn501) and 1695 (R100) bp respectively. When optimally aligned, the merA sequences differ in 18% of their positions. These differences were clustered in specific regions. In addition, there was one nucleotide triplet in the Tn501 sequence which has no counterpart in the R100 sequence and one dodecyl-nucleotide sequence in the R100 sequence without counterpart in Tn501. Thus the predicted merA polypeptide of Tn501 contains 561 amino acids and the R100 counterpart contains 564 amino acids. Comparison of the R100 mercuric reductase sequences with that for human glutathione reductase [Krauth-Siegel et al.: Eur. J. Biochem. 121 (1982) 259-267], for which there is a 2 A resolution electron density map [Thieme et al.: J. Mol. Biol. 152 (1981) 763-782] shows a strong homology, with 26% identical amino acids and many conservative substitutions. This homology allows the conclusion that the active site of these enzymes and the contact positions for flavin adenine dinucleotide (FAD) and NADPH are highly conserved, while the amino- and carboxyl-terminal sequences differ.
KeywordMeSH Terms
194.     ( 2013 )

Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ.

Nature 496 (7443)
PMID : 23535599  :   DOI  :   10.1038/nature12004     PMC  :   PMC3722872    
Abstract >>
Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome. The ability of the myristoyl group to facilitate dynamic protein-protein and protein-membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification.
KeywordMeSH Terms
Protein Processing, Post-Translational
Proteolysis
195.     ( 2013 )

Genetic diversity of Shigella spp. and their integron content.

Foodborne pathogens and disease 10 (3)
PMID : 23489046  :   DOI  :   10.1089/fpd.2012.1250    
Abstract >>
The aim of this study was to investigate the occurrence and resistance gene content of class 1 and 2 integrons among Shigella spp. and to study the genetic diversity of isolates using the pulsed-field gel electrophoresis (PFGE) method. A total of 32 Shigella spp. were identified from 700 stool samples of patients with diarrhea from two provinces in Iran. S. sonnei (70.8%) and S. flexneri (62.5%) were the most frequent serogroups in Tehran and Razavi Khorasan provinces, respectively. Class 2 integrons were more frequent among Shigella spp. in comparison with class 1 integrons. Three different resistance gene arrays were identified among class 1 integrons. Dihydrofolate reductase (dfrA) gene cassette was detected in 78.9% of total integrons (class 1 and 2). PFGE analysis revealed clonal dissemination (62.5%) of a single clone with identical class 2 resistance gene content in Tehran province. Comparison of our Shigella pulsotypes with those published from other countries showed similar pulsotypes in India and Korea, with identical resistance profiles, which suggests dissemination of this (these) clone(s) in Asian countries. Class 2 integrons were found to be predominant among our Shigella spp. This reflects the need to monitor the acquisition and dissemination of different resistant gene cassettes among integrons. Comparison of PFGE pattern through standard procedures promoted the molecular epidemiological surveys and identification of clonal isolates in Iran and other Asian countries.
KeywordMeSH Terms
Genetic Variation
Integrons
196. Yang  W, Wei  B, Yan  R,     ( 2018 )

Amoxapine Demonstrates Incomplete Inhibition of �]-Glucuronidase Activity from Human Gut Microbiota.

SLAS discovery : advancing life sciences R & D 23 (1)
PMID : 28809607  :   DOI  :   10.1177/2472555217725264    
Abstract >>
Amoxapine has been demonstrated to be a potent inhibitor of Escherichia coli �]-glucuronidase. This study aims to explore the factors causing unsatisfactory efficacy of amoxapine in alleviating CPT-11-induced gastrointestinal toxicity in mice and to predict the outcomes in humans. Amoxapine (100 ?M) exhibited poor and varied inhibition on �]-glucuronidase activity in gut microbiota from 10 healthy individuals and their pool (pool, 11.9%; individuals, 3.6%-54.4%) with IC50 >100 ?M and potent inhibition toward E. coli �]-glucuronidase (IC50 = 0.34 ?M). p-Nitrophenol formation from p-nitrophenyl-�]-D-glucuronide by pooled and individual gut microbiota fitted classical Michaelis-Menten kinetics, showing similar affinity (Km = 113-189 ?M) but varied catalytic capability (Vmax = 53-556 nmol/h/mg). Interestingly, amoxapine showed distinct inhibitory effects (8.7%-100%) toward �]-glucuronidases of 13 bacterial isolates (including four Enterococcus, three Streptococcus, two Escherichia, and two Staphylococcus strains; gus genes belonging to OTU1, 2 or 21) regardless of their genetic similarity or bacterial origin. In addition, amoxapine inhibited the growth of pooled and individual gut microbiota at a high concentration (6.3%-30.8%, 200 ?M). Taken together, these findings partly explain the unsatisfactory efficacy of amoxapine in alleviating CPT-11-induced toxicity and predict a poor outcome of �]-glucuronidase inhibition in humans, highlighting the necessity of using a human gut microbiota community for drug screening.
KeywordMeSH Terms
CPT-11–induced toxicity
amoxapine
bacterial isolates
human gut microbiota
β-glucuronidase inhibition
CPT-11–induced toxicity
amoxapine
bacterial isolates
human gut microbiota
β-glucuronidase inhibition
CPT-11–induced toxicity
amoxapine
bacterial isolates
human gut microbiota
β-glucuronidase inhibition
CPT-11–induced toxicity
amoxapine
bacterial isolates
human gut microbiota
β-glucuronidase inhibition
CPT-11–induced toxicity
amoxapine
bacterial isolates
human gut microbiota
β-glucuronidase inhibition
Gastrointestinal Microbiome
197. Yaghoubi  S, Ranjbar  R, Soltan Dallal  MM, Shirazi  MH, Sharifi-Yazdi  MK,     ( N/A )

Frequency of Mutations in Quinolone Resistance-Determining Regions and Plasmid-Mediated Quinolone Resistance in Shigella Isolates Recovered from Pediatric Patients in Tehran, Iran: An Overlooked Problem.

Microbial drug resistance (Larchmont, N.Y.) 24 (6)
PMID : 29148915  :   DOI  :   10.1089/mdr.2017.0155    
Abstract >>
Fluoroquinolone (FQ) resistance in clinical isolates of Shigella species has been increasing reported in recent years. This study was carried out to find the mutations within the quinolone resistance-determining regions (QRDRs) and the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants among the clinical isolates of Shigella sp. in Tehran, Iran. A total of 50 Shigella isolates were collected from five teaching therapeutic centers in Tehran, Iran and analyzed for antibiotic susceptibility over a period of 20 months from July 2015 to January 2017. The PCR and direct nucleotide sequencing were used for genetic alterations in the QRDRs. The PMQR genes were detected using PCR. The results revealed four types of mutations in the QRDR of gyrA: 20 (40%) had a S83L mutation, 1 (2%) had a S83A mutation, 2 (4%) had a D87G mutation, and 1 (2%) isolate had a D87Y mutation. Mutations were also found at codon N57D, D200N, and E210K in three isolates. Seven hospitalized children had qnrS determinants, and one isolates had the mutation S83A, while two isolates had double mutations at S83L and/or D87G (Ser83Leu and Asp-87Gly). The PMQR gene-positive isolates had the single replacement of serine with leucine. In hospitalized children, two isolates had two types of PMQR determinants (qnrS and qnrA) and (qnrS and qnrB) at once. The results of this study indicate that the emergence of strains with mutations in the QRDR regions and the capture of PMQR determinants in strains may lead to failure in therapy with FQ and the widespread emergence of strains with high-level FQ resistance.
KeywordMeSH Terms
DNA gyrase
qnr gene
shigellosis
topoisomerase IV
DNA gyrase
qnr gene
shigellosis
topoisomerase IV
198.     ( 2013 )

Structural basis for the recognition of Ubc13 by the Shigella flexneri effector OspI.

Journal of molecular biology 425 (15)
PMID : 23542009  :   DOI  :   10.1016/j.jmb.2013.02.037    
Abstract >>
Ubc13 is a ubiquitin-conjugating enzyme that plays a key role in the nuclear factor-�eB signal transduction pathway in human diseases. The Shigella flexneri effector OspI affects inflammatory responses by catalyzing the deamidation of a specific glutamine residue at position 100 in Ubc13 during infection. This modification prevents the activation of the TNF (tumor necrosis factor) receptor-associated factor 6, leading to modulation of the diacylglycerol-CBM (CARD-Bcl10-Malt1) complex-TNF receptor-associated factor 6-nuclear factor-�eB signaling pathway. To elucidate the structural basis of OspI function, we determined the crystal structures of the catalytically inert OspI C62A mutant and its complex with Ubc13 at resolutions of 3.0 and 2.96?, respectively. The structure of the OspI-Ubc13 complex revealed that the interacting surfaces between OspI and Ubc13 are a hydrophobic surface and a complementary charged surface. Furthermore, we predict that the complementary charged surface of OspI plays a key role in substrate specificity determination.
KeywordMeSH Terms
GST
PDB
Protein Data Bank
Shigella flexneri
TNF receptor-associated factor 6
TRAF6
Ubc13
crystal structure
deamidase
glutathione S-transferase
ubiquitination
199.     ( 1998 )

A role for H-NS in the regulation of the virF gene of Shigella and enteroinvasive Escherichia coli.

Research in microbiology 149 (1)
PMID : 9766205  :  
Abstract >>
We have investigated the role of H-NS, one of the major components of the bacterial nucleoid, in the expression of the virF gene present on the large virulence plasmid of Shigella and enteroinvasive Escherichia coli in response to different environmental conditions. VirF is an AraC-like protein which activates at least two promoters, virB and virG, both repressed by H-NS. Band shift experiments reveal that the affinity of H-NS for the virF and virB promoters is comparable, while the affinity for the virG promoter is higher. Polyacrylamide gel electrophoresis of three DNA fragments containing the virF, the virB and the VirG promoters demonstrates, in agreement with computer predictions, that they have an intrinsically curved structure, confirming the preference of H-NS for bent DNA. In vivo transcriptional analysis of virF mRNA shows that H-NS negatively controls the expression of virF at 30 degrees C. The expression of a virF-lacZ translational fusion in E.coli wild type and in an hns-defective derivative grown at 30 degrees or 37 degrees C and at pH 6.0 or 7.0 indicates that, in the absence of H-NS, virF expression becomes insensitive to temperature and to limited pH changes. Our results strongly suggest that H-NS controls virF expression by binding to the virF promoter and by repressing its expression at low temperature and at low pH.
KeywordMeSH Terms
Virulence Factors
200.     ( 1998 )

Identification of two Shigella flexneri chromosomal loci involved in intercellular spreading.

Infection and immunity 66 (10)
PMID : 9746567  :   PMC  :   PMC108578    
Abstract >>
The ability of Shigella flexneri to multiply within colonic epithelial cells and spread to adjacent cells is essential for production of dysentery. Two S. flexneri chromosomal loci that are required for these processes were identified by screening a pool of TnphoA insertion mutants. These mutants were able to invade cultured epithelial cells but could not form wild-type plaques. Analysis of the nucleotide sequence indicated that the sites of TnphoA insertion were within two different regions that are almost identical to Escherichia coli K-12 chromosomal sequences of unknown functions. One region is located at 70 min on the E. coli chromosome, upstream of murZ, while the other is at 28 min, downstream of tonB. The mutant with the insertion at 70 min was named vpsC because it showed an altered pattern of virulence protein secretion. The vpsC mutant formed pinpoint-sized plaques, was defective in recovery from infected tissue culture cells, and was sensitive to lysis by the detergent sodium dodecyl sulfate. Recombinant plasmids carrying the S. flexneri vpsA, -B, and -C genes complemented all of the phenotypes of the vpsC mutant. A mutation in vpsA resulted in the same phenotype as the vpsC mutation, suggesting that these two genes are part of a virulence operon in S. flexneri. The mutant with the insertion at 28 min was interrupted in the same open reading frame as S. flexneri ispA. This ispA mutant could not form plaques and was defective in bacterial septation inside tissue culture cells.
KeywordMeSH Terms
Genes, Bacterial
Membrane Proteins
201.     ( 1998 )

NMR solution structure of the oxidized form of MerP, a mercuric ion binding protein involved in bacterial mercuric ion resistance.

Biochemistry 37 (26)
PMID : 9649312  :   DOI  :   10.1021/bi9803628    
Abstract >>
Mercuric ions are toxic to living organisms because of their strong affinity for cysteine residues in proteins. Some bacteria have developed a resistance mechanism whereby Hg2+ is transported into the cytoplasm and reduced to Hg0. One of the proteins involved in the transport of mercuric ion is the periplasmic binding protein MerP, which can exist both as oxidized (disulfide) and as reduced (dithiol) forms. Only the reduced form with Cys-17 and Cys-14 residues as free thiols is a potent receptor for mercuric ion. In this work the solution structure of the oxidized form of MerP has been determined by multidimensional NMR spectroscopy and compared to the NMR structures of the previously published structures of the reduced and mercury-bound forms of MerP. The mercury-bound and oxidized forms have similar tertiary structures, whereas in the reduced form there is a large rearrangement of the mercuric ion binding loop and the nearby loop comprising residues 38-41. The structural arrangement of the latter loop seems to be important for the stabilization of the surface location of the cysteine-containing loop. In the reduced form at low pH the cysteine-containing loop adopts a conformation similar to what is observed in the oxidized and mercury-bound forms. The oxidized form also differs with respect to the other two forms in the relative positions of some of the alpha-helices and beta-strands. Structural differences between the oxidized and reduced forms may help explain why the reduced form is stable in the periplasm, which is considered to be an oxidizing environment.
KeywordMeSH Terms
Proteins
202.     ( 1998 )

The vacB gene required for virulence in Shigella flexneri and Escherichia coli encodes the exoribonuclease RNase R.

The Journal of biological chemistry 273 (23)
PMID : 9603904  :   DOI  :   10.1074/jbc.273.23.14077    
Abstract >>
vacB, a gene previously shown to be required for expression of virulence in Shigella and enteroinvasive Escherichia coli, has been found to encode the 3'-5' exoribonuclease, RNase R. Thus, cloning of E. coli vacB led to overexpression of RNase R activity, and partial deletion or interruption of the cloned gene abolished this overexpression. Interruption of the chromosomal copy of vacB eliminated endogenous RNase R activity; however, the absence of RNase R by itself had no effect on cell growth. In contrast, cells lacking both RNase R and polynucleotide phosphorylase were found to be inviable. These data indicate that RNase R participates in an essential cell function in addition to its role in virulence. The identification of the vacB gene product as RNase R should aid in understanding how the virulence phenotype in enterobacteria is expressed and regulated. On the basis of this information we propose that vacB be renamed rnr.
KeywordMeSH Terms
Escherichia coli Proteins
Exoribonucleases
203.     ( 1998 )

Novel combination of mutations in the DNA gyrase and topoisomerase IV genes in laboratory-grown fluoroquinolone-resistant Shigella flexneri mutants.

Antimicrobial agents and chemotherapy 42 (11)
PMID : 9867794  :   PMC  :   PMC105997    
Abstract >>
N/A
KeywordMeSH Terms
Mutation
204.     ( 1998 )

SepA, the 110 kDa protein secreted by Shigella flexneri: two-domain structure and proteolytic activity.

Microbiology (Reading, England) 144 (Pt 7) (N/A)
PMID : 9695914  :   DOI  :   10.1099/00221287-144-7-1815    
Abstract >>
Shigellosis is characterized by a strong inflammatory response which is induced by bacteria invading the colonic mucosa. Characterization of a sepA mutant indicated that SepA, the major protein secreted by Shigella flexneri growing in laboratory media, might be involved in invasion and destruction of the host intestinal epithelium. The sequence of the first 500 residues of mature SepA (110 kDa) is homologous to that of the N-terminal region of IgA1 proteases. To investigate the potential proteolytic activity of SepA, the activity of the purified protein on a wide range of synthetic peptides was tested. SepA hydrolysed several of these substrates and the activity was inhibited by PMSF. Several peptides which were hydrolysed by SepA have been described as specific substrates for cathepsin G, a serine protease produced by polymorphonuclear leukocytes that was proposed to play a role in inflammation. However, unlike cathepsin G, SepA degraded neither fibronectin nor angiotensin I and had no effect on aggregation of human platelets. In addition, analysis of SepA hydrolysis by proteinase K suggested that the protein is composed of two domains of about 450 residues separated by a hinge region of 100 residues. The 47 kDa N-terminal domain was stable and endowed with proteolytic activity.
KeywordMeSH Terms
205.     ( 1997 )

Detection and speciation of bacteria through PCR using universal major cold-shock protein primer oligomers.

Journal of industrial microbiology & biotechnology 19 (4)
PMID : 9439003  :  
Abstract >>
The detection of bacteria using PCR is a well-established diagnostic technique. However, conventional PCR requires the use of DNA primer oligomers that are specific to the target organism and, as a consequence, a sample can only be tested for the presence of that specific target. A significant advantage would be to probe a sample for the presence of any bacteria, followed by identification. To achieve this it is necessary to identify a DNA sequence common to all bacteria. Here we demonstrate that such a sequence may be that encoding the major cold-shock proteins. Using two universal PCR primer oligomers from conserved regions of these gene homologues, we have amplified a 200 base-pair DNA sequence from more than 30 diverse Gram-positive and Gram-negative bacteria, including representatives from the genera Aeromonas, Bacillus, Citrobacter, Enterobacter, Enterococcus, Escherichia, Klebsiella, Lactobacillus, Lactococcus, Listeria, Pediococcus, Photobacterium, Proteus, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia. Sequence analysis of the amplified products confirmed a high level of DNA homology. Significantly, however, there are sufficient nucleotide variations to allow the unique allocation of each amplified sequence to its parental bacterium.
KeywordMeSH Terms
DNA Primers
206.     ( 1997 )

A mercuric ion uptake role for the integral inner membrane protein, MerC, involved in bacterial mercuric ion resistance.

The Journal of biological chemistry 272 (47)
PMID : 9368013  :   DOI  :   10.1074/jbc.272.47.29518    
Abstract >>
Bacterial detoxification of mercuric ion depends on the presence of one or more integral membrane proteins (MerT and/or MerC) whose postulated function is in transport of Hg2+ from a periplasmic Hg2+-binding protein (MerP) to cytoplasmic mercuric reductase. In this study, MerC from the Tn21-encoded mer operon was overexpressed and studied in vesicles and in purified form to clarify the role played by this protein in mercuric ion resistance. MerC-containing vesicles were found to take up mercuric ion independently of MerP. Since uptake correlated with the level of MerC expression was unaffected by osmotic pressure, and was only partially decreased in the presence of 0.05% Triton X-100, the observed uptake appears to represent mainly binding to MerC. Binding was inhibited by thiol-specific reagents, consistent with an essential role for cysteine residues. The essential thiol groups were inaccessible to hydrophilic thiol reagents, whereas hydrophobic reagents completely abolished Hg2+ binding. These observations are consistent with the predicted topology of the protein, wherein all 4 cysteine residues are either in the cytoplasm or the bilayer. A role for MerC in Hg2+ transport is thus also likely. Based on these results, a modified model for bacterial Hg2+ transport is proposed.
KeywordMeSH Terms
Cation Transport Proteins
Proteins
207.     ( 1998 )

Neural Wiskott-Aldrich syndrome protein is implicated in the actin-based motility of Shigella flexneri.

The EMBO journal 17 (10)
PMID : 9582270  :   DOI  :   10.1093/emboj/17.10.2767     PMC  :   PMC1170617    
Abstract >>
Shigella, the causative agent of bacillary dysentery, is capable of directing its own movement in the cytoplasm of infected epithelial cells. The bacterial surface protein VirG recruits host components mediating actin polymerization, which is thought to serve as the propulsive force. Here, we show that neural Wiskott-Aldrich syndrome protein (N-WASP), which is a critical target for filopodium formation downstream of Cdc42, is required for assembly of the actin tail generated by intracellular S.flexneri. N-WASP accumulates at the front of the actin tail and is capable of interacting with VirG in vitro and in vivo, a phenomenon that is not observed in intracellular Listeria monocytogenes. The verprolin-homology region in N-WASP was required for binding to the glycine-rich repeats domain of VirG, an essential domain for recruitment of F-actin on intracellular S.flexneri. Overexpression of a dominant-negative N-WASP mutant greatly inhibited formation of the actin tail by intracellular S.flexneri. Furthermore, depletion of N-WASP from Xenopus egg extracts shut off Shigella actin tail assembly, and this was restored upon addition of N-WASP protein, suggesting that N-WASP is a critical host factor for the assembly of the actin tail by intracellular Shigella.
KeywordMeSH Terms
Saccharomyces cerevisiae Proteins
208.     ( 1997 )

Identification and molecular characterization of a 27 kDa Shigella flexneri invasion plasmid antigen, IpaJ.

Microbial pathogenesis 23 (6)
PMID : 9441862  :   DOI  :   10.1006/mpat.1997.0164    
Abstract >>
Shigella species and enteroinvasive Escherichia coli contain a core set of virulence genes whose coordinated expression results in the invasion of host colonic epithelial cells and the dysenteric syndrome. A number of virulence determinants are carried by the 230 kb invasion plasmid found in all virulent strains of Shigellae. Many of these invasion plasmid genes encode immunogens that are recognized by convalescent serum, including proteins that mediate the invasion (IpaB, IpaC, IpaD) and cell spreading (VirG or IcsA and IcsB) phenotypes. In this report, we describe the molecular characterization of a novel invasion plasmid antigen from Shigella flexneri, designated IpaJ. The ipaJ gene encodes a 780 bp open reading frame (ORF), separated from the ipaR (virB) stop codon by 944 bp. The predicted amino acid sequence for IpaJ revealed a consensus signal peptide for protein export. TnphoA mutagenesis of the ipaJ ORF confirmed the presence of export signal sequences in IpaJ. Unlike ipaBCDA genes, transcription analysis of ipaJ indicated that the gene is not expressed in a temperature-dependent fashion. The IpaJ protein was expressed and purified as a His6-tagged fusion protein that reacted with convalescent sera in Western blot analyses, confirming its identification as a Shigella immunogen. Construction and phenotypic characterization of ipaJ mutants in two serotypes of S. flexneri showed that the mutants were not compromised in their ability to invade cultured epithelial cells or to form plaques on BHK cell monolayers. In addition, the ipaJ mutants were Sereny positive indicating a capacity for intercellular dissemination; however, in the limited number of guinea-pigs tested, the keratoconjunctivitis reaction appeared attenuated.
KeywordMeSH Terms
209.     ( 1997 )

Use of a novel approach, termed island probing, identifies the Shigella flexneri she pathogenicity island which encodes a homolog of the immunoglobulin A protease-like family of proteins.

Infection and immunity 65 (11)
PMID : 9353040  :   PMC  :   PMC175661    
Abstract >>
The she gene of Shigella flexneri 2a, which also harbors the internal enterotoxin genes set1A and set1B (F. R. Noriega, GenBank accession no. U35656, 1995) encodes a homolog of the virulence-related immunoglobulin A (IgA) protease-like family of secreted proteins, Tsh, EspC, SepA, and Hap, from an avian pathogenic Escherichia coli, an enteropathogenic E. coli, S. flexneri 5, and Haemophilus influenzae, respectively. To investigate the possibility that this locus was carried on a larger deletable element, the S. flexneri 2a YSH6000T she gene was insertionally disrupted by allelic exchange using a Tn10-derived tetAR(B) cassette. Then, to detect loss of the she locus, the tetracycline-resistant derivative was plated onto fusaric acid medium to select for tetracycline-sensitive revertants, which were observed to arise at a frequency of 10(-5) to 10(-6). PCR and pulsed-field gel electrophoresis analysis confirmed loss of the she::tetAR(B) locus in six independent tetracycline-sensitive isolates. Sample sequencing over a 25-kb region flanking she identified four insertion sequence-like elements, the group II intron-like sequence Sf.IntA, and the 3' end of a second IgA protease-like homolog, sigA, lying 3.6 kb downstream and in an orientation inverted with respect to she. The deletion was mapped to chromosomal NotI fragment A and determined to have a size of 51 kb. Hybridization with flanking probes confirmed that at least 17.7 kb of the 51-kb deletable element was unique to the seven she+ strains investigated, supporting the conclusion that she lay within a large pathogenicity island. The method described in this study, termed island probing, provides a useful tool to further the study of pathogenicity islands in general. Importantly, this approach could also be of value in constructing safer live attenuated bacterial vaccines.
KeywordMeSH Terms
Genes, Bacterial
210.     ( 1998 )

Induction of type III secretion in Shigella flexneri is associated with differential control of transcription of genes encoding secreted proteins.

The EMBO journal 17 (10)
PMID : 9582283  :   DOI  :   10.1093/emboj/17.10.2894     PMC  :   PMC1170630    
Abstract >>
Shigella, the etiological agent of human bacillary dysentery, invades the colonic epithelium where it induces an intense inflammatory response. Entry of Shigella into epithelial cells involves a type III secretion machinery, encoded by the mxi and spa operons, and the IpaA-D secreted proteins. In this study, we have identified secreted proteins of 46 and 60 kDa as the products of virA and ipaH9.8, respectively, the latter being a member of the ipaH multigene family. Inactivation of virA did not affect entry into epithelial cells. Using lacZ transcriptional fusions, we found that transcription of virA and four ipaH genes, but not that of the ipaBCDA and mxi operons, was markedly increased during growth in the presence of Congo red and in an ipaD mutant, two conditions in which secretion through the Mxi-Spa machinery is enhanced. Transcription of the virA and ipaH genes was also transiently activated upon entry into epithelial cells. These results suggest that transcription of the virA and ipaH genes is regulated by the type III secretion machinery and that a regulatory cascade differentially controls transcription of genes encoding secreted proteins, some of which, like virA, are not required for entry.
KeywordMeSH Terms
Antigens, Bacterial
Genes, Bacterial
Transcription, Genetic
Virulence Factors

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