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1. Ko  KS, Hong  SK, Lee  HK, Park  MY, Kook  YH,     ( 2003 )

Molecular evolution of the dotA gene in Legionella pneumophila.

Journal of bacteriology 185 (21)
PMID : 14563861  :   DOI  :   10.1128/jb.185.21.6269-6277.2003     PMC  :   PMC219400    
Abstract >>
The molecular evolution of dotA, which is related to the virulence of Legionella pneumophila, was investigated by comparing the sequences of 15 reference strains (serogroups 1 to 15). It was found that dotA has a complex mosaic structure. The whole dotA gene of Legionella pneumophila subsp. pneumophila serogroups 2, 6, and 12 has been transferred from Legionella pneumophila subsp. fraseri. A discrepancy was found between the trees inferred from the nucleotide and deduced amino acid sequences of dotA, which suggests that multiple hits, resulting in synonymous substitutions, have occurred. Gene phylogenies inferred from three different segments (the 5'-end region, the central, large periplasmic domain, and the 3'-end region) showed impressively dissimilar topologies. This was concordant with the sequence polymorphisms, indicating that each region has experienced an independent evolutionary history, and was evident even within the same domain of each strain. For example, the PP2 domain was found to have a heterogeneous structure, which led us hypothesize that the dotA gene of L. pneumophila may have originated from two or more different sources. Comparisons of synonymous and nonsynonymous substitutions demonstrated that the PP2 domain has been under strong selective pressure with respect to amino acid change. Split decomposition analysis also supported the intragenic recombination of dotA. Multiple recombinational exchange within the dotA gene, encoding an integral cytoplasmic membrane protein that is secreted, probably provided increased fitness in certain environmental niches, such as within a particular biofilm community or species of amoebae.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
2. Bernander  S, Jacobson  K, Helbig  JH, Lück  PC, Lundholm  M,     ( 2003 )

A hospital-associated outbreak of Legionnaires' disease caused by Legionella pneumophila serogroup 1 is characterized by stable genetic fingerprinting but variable monoclonal antibody patterns.

Journal of clinical microbiology 41 (6)
PMID : 12791873  :   DOI  :   10.1128/jcm.41.6.2503-2508.2003     PMC  :   PMC156525    
Abstract >>
An outbreak of 18 pneumonia cases caused by Legionella pneumophila serogroup 1 occurred at a Swedish university hospital 1996 to 1999. Eight clinical isolates obtained by culture from the respiratory tract were compared to 20 environmental isolates from the hospital and to 21 epidemiologically unrelated isolates in Sweden, mostly from patients, by using pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism analysis (AFLP), and monoclonal antibody (MAb) typing. All patients and most environmental isolates from the outbreak hospital belonged to the same genotypic cluster in both PFGE and AFLP. This genotype was distinctly different from other strains, including a cluster from a second hospital in a different part of the country. The MAb subtype of the outbreak clone was Knoxville except for three isolates that were Oxford. A variation in the MAb reactivity pattern was also found in a second genotypic cluster. These changes in the MAb reactivity pattern were due to the absence or presence of the lag-1 gene coding for an O-acetyltransferase that is responsible for expression of the lipopolysaccharide epitope recognized by MAb 3/1 of the Dresden Panel. In all MAb 3/1-positive strains, the lag-1 gene was present on a genetic element that was bordered by a direct repeat that showed a high degree of sequence homology. Due to this homology, the lag-1 gene region seemed to be an unstable element in the chromosome. MAb patterns are thus a valuable adjunct to genotyping methods in defining subgroups inside a genotypic cluster of L. pneumophila sg 1.
KeywordMeSH Terms
Disease Outbreaks
Hospitals, University
3. Ko  KS, Lee  HK, Park  MY, Kook  YH,     ( 2003 )

Mosaic structure of pathogenicity islands in Legionella pneumophila.

Journal of molecular evolution 57 (1)
PMID : 12962307  :   DOI  :   10.1007/s00239-002-2452-8    
Abstract >>
A gene complex, dot/icm, located in two independent chromosomal loci of L. pneumophila, the causative agent of Legionnaires' disease, is related to virulence. To investigate the evolutionary pattern of these pathogenicity islands of L. pneumophila, portions of four genes in the dot/icm complex, namely, dotA, dotB, icmB, and icmT, were amplified, sequenced, and phylogenetically analyzed, in addition to rpoB, which encodes an RNA polymerase beta-subunit. The nucleotide sequences and phylogenetic analyses of these five genes of 96 L. pneumophila strains revealed that several subgroups of L. pneumophila proliferated clonally. However, incongruent gene tree topologies and the results of statistical testing (Templeton Willcoxon signed-ranked and incongruence length differences tests) indicated that the evolutionary histories of these genes within the pathogenicity islands are not uniform, and that they constitute a mosaic structure. In addition, the nonuniform grouping of some reference strains suggests that intraspecific recombination might be still occurring in nature or in the laboratory.
KeywordMeSH Terms
Evolution, Molecular
Genes, Bacterial
4. Edelstein  PH, Hu  B, Higa  F, Edelstein  MA,     ( 2003 )

lvgA, a novel Legionella pneumophila virulence factor.

Infection and immunity 71 (5)
PMID : 12704109  :   DOI  :   10.1128/iai.71.5.2394-2403.2003     PMC  :   PMC153278    
Abstract >>
Several novel Legionella pneumophila virulence genes were previously discovered by use of signature-tagged mutagenesis (P. H. Edelstein, M. A. Edelstein, F. Higa, and S. Falkow, Proc. Natl. Acad. Sci. 96:8190-8195, 1999). One of these mutants appeared to be defective in multiplication in guinea pig lungs and spleens, yet it multiplies normally in guinea pig alveolar macrophages. Here we report further characterization of the mutated gene and its protein and the virulence role of the gene. The complete sequence of the gene, now called lvgA, is 627 bp long, and its protein product is approximately 27 kDa in size. lvgA was present in all 50 strains of L. pneumophila tested. No significant nucleic acid or protein homology was found in the GenBank database for the gene, nor were any distinctive motifs discovered in a search of other databases. The expression of both DotA and IcmX in the lvgA mutant was normal. Subcellular fractionation studies localized LvgA to the outer membrane fraction, and protease digestion studies suggested that at least some of the protein is surface expressed. No change in bacterial lipopolysaccharide composition or reactivity to serogroup-specific antisera was detected in the mutant. Growth competition studies with alveolar macrophages showed that the mutant was outcompeted by its parent 3-fold in 24 h and 24-fold in 48 h, in contrast to what was observed with the null phenotype in parallel testing with alveolar macrophages or with the A549 alveolar epithelial cell line. This macrophage defect of the mutant bacterium was due to slower growth, as the mutant invaded alveolar macrophages normally. Electron microscopy showed that the mutant bacterium resided in a ribosome-studded phagosome in alveolar macrophages, with no distinction from its parent. The lvgA mutant was outcompeted by its parent about sixfold in guinea pig lungs and spleens; prolonged observation of infected animals showed no late-onset virulence of the mutant. Transcomplementation of the mutant restored the parental phenotype in guinea pigs. The lvgA mutant was twofold more susceptible to killing by human beta-defensin 2 but not to killing by other cationic peptides, serum complement, or polymorphonuclear neutrophils. lvgA is a novel virulence gene that is responsible for pleiotropic functions involving both extracellular and intracellular bacterial resistance mechanisms.
KeywordMeSH Terms
5. Conover  GM, Derré  I, Vogel  JP, Isberg  RR,     ( 2003 )

The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity.

Molecular microbiology 48 (2)
PMID : 12675793  :   DOI  :   10.1046/j.1365-2958.2003.03400.x    
Abstract >>
Legionella pneumophila establishes a replication vacuole within phagocytes that requires the bacterial Dot/Icm apparatus for its formation. This apparatus is predicted to translocate effectors into host cells. We hypothesized that some translocated proteins also function to maintain the integrity of the Dot/Icm translocator. Mutations that destroy this function are predicted to result in a Dot/Icm complex that poisons the bacterium, resulting in reduced viability. To identify such mutants, strains were isolated (called lid-) that showed reduced viability on bacteriological medium in the presence of an intact Dot/Icm apparatus, but which had high viability in the absence of the translocator. Several such mutants were analysed in detail to identify candidate strains that may have lost the ability to synthesize a translocated substrate of Dot/Icm. Two such strains had mutations in the lidA gene. The LidA protein exhibits properties expected for a translocated substrate of Dot/Icm that is important for maintenance of bacterial cell integrity: it associates with the phagosomal surface, promotes replication vacuole formation, and is important for both efficient intracellular growth and high viability on bacteriological media after introduction of a plasmid that allows high level expression of the dotA gene.
KeywordMeSH Terms
6. Gal-Mor  O, Segal  G,     ( 2003 )

Identification of CpxR as a positive regulator of icm and dot virulence genes of Legionella pneumophila.

Journal of bacteriology 185 (16)
PMID : 12897011  :   DOI  :   10.1128/jb.185.16.4908-4919.2003     PMC  :   PMC166489    
Abstract >>
To date, 24 Legionella pneumophila genes (icm and dot genes) have been shown to be required for intercellular growth and host cell killing. A previous report indicated that the regulation of these genes is complicated and probably involves several regulatory proteins. In this study, a genetic screen performed in Escherichia coli identified the CpxR response regulator as an activator of the L. pneumophila icmR gene. Construction of an L. pneumophila cpxR insertion mutant showed that the expression of icmR is regulated by CpxR. In addition, a conserved CpxR binding site (GTAAA) was identified in the icmR regulatory region and L. pneumophila His-tagged CpxR protein was shown to bind to the icmR regulatory region using a mobility shift assay. Besides its dramatic effect on the icmR level of expression, the CpxR regulator was also found to affect the expression of the icmV-dotA and icmW-icmX operons, but to a lesser extent. The role of CpxA, the cognate sensor kinase of CpxR, was also examined and its effect on the icmR level of expression was found to be less pronounced than the effect of CpxR. The RpoE sigma factor, which was shown to coregulate genes together with CpxR, was examined as well, but it did not influence icm and dot gene expression. In addition, when the cpxR mutant strain, in which the expression of the icmR gene was dramatically reduced, and the cpxA and rpoE mutant strains were examined for their ability to grow inside Acanthamoeba castellanii and HL-60-derived human macrophages, no intracellular growth defect was observed. This study presents the first evidence for a direct regulator (CpxR) of an icm-dot virulence gene (icmR). The CpxR regulator together with other regulatory factors probably concerts with the expression of icm and dot genes to result in successful infection.
KeywordMeSH Terms
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
7. Lynch  D, Fieser  N, Glöggler  K, Forsbach-Birk  V, Marre  R,     ( 2003 )

The response regulator LetA regulates the stationary-phase stress response in Legionella pneumophila and is required for efficient infection of Acanthamoeba castellanii.

FEMS microbiology letters 219 (2)
PMID : 12620627  :   DOI  :   10.1016/S0378-1097(03)00050-8    
Abstract >>
In order to identify a potential regulator of virulence gene expression in Legionella pneumophila, the L. pneumophila homologue of the response regulator GacA, LetA, was identified and cloned, facilitating the generation of a L. pneumophila letA insertion mutant. The L. pneumophila letA insertion mutant was more sensitive to oxidative and acid stress than the wild-type. The letA mutant exhibited reduced infectivity and was defective for intracellular growth within Acanthamoeba castellanii. Transcription of the rpoS and dotA genes was reduced in the letA mutant. Our data indicate that the response regulator LetA functions as a regulator of the stationary-phase stress response in L. pneumophila and is required for efficient replication within A. castellanii.
KeywordMeSH Terms
8. Flieger  A, Neumeister  B, Cianciotto  NP,     ( 2002 )

Characterization of the gene encoding the major secreted lysophospholipase A of Legionella pneumophila and its role in detoxification of lysophosphatidylcholine.

Infection and immunity 70 (11)
PMID : 12379686  :   DOI  :   10.1128/iai.70.11.6094-6106.2002     PMC  :   PMC130422    
Abstract >>
We previously showed that Legionella pneumophila secretes, via its type II secretion system, phospholipase A activities that are distinguished by their specificity for certain phospholipids. In this study, we identified and characterized plaA, a gene encoding a phospholipase A that cleaves fatty acids from lysophospholipids. The plaA gene encoded a 309-amino-acid protein (PlaA) which had homology to a group of lipolytic enzymes containing the catalytic signature GDSL. In Escherichia coli, the cloned gene conferred trypsin-resistant hydrolysis of lysophosphatidylcholine and lysophosphatidylglycerol. An L. pneumophila plaA mutant was generated by allelic exchange. Although the mutant grew normally in standard buffered yeast extract broth, its culture supernatants lost greater than 80% of their ability to release fatty acids from lysophosphatidylcholine and lysophosphatidylglycerol, implying that PlaA is the major secreted lysophospholipase A of L. pneumophila. The mutant's reduced lipolytic activity was confirmed by growth on egg yolk agar and thin layer chromatography and was complemented by reintroduction of an intact copy of plaA. Overexpression of plaA completely protected L. pneumophila from the toxic effects of lysophosphatidylcholine, suggesting a role for PlaA in bacterial detoxification of lysophospholipids. The plaA mutant grew like the wild type in U937 cell macrophages and Hartmannella vermiformis amoebae, indicating that PlaA is not essential for intracellular infection of L. pneumophila. In the course of characterizing plaA, we discovered that wild-type legionellae secrete a phospholipid cholesterol acyltransferase activity, highlighting the spectrum of lipolytic enzymes produced by L. pneumophila.
KeywordMeSH Terms
Genes, Bacterial
9. Robey  M, Cianciotto  NP,     ( 2002 )

Legionella pneumophila feoAB promotes ferrous iron uptake and intracellular infection.

Infection and immunity 70 (10)
PMID : 12228295  :   DOI  :   10.1128/iai.70.10.5659-5669.2002     PMC  :   PMC128349    
Abstract >>
In order to determine the role of ferrous iron transport in Legionella pathogenesis, we identified and mutated the feoB gene in virulent Legionella pneumophila strain 130b. As it is in Escherichia coli, the L. pneumophila feoB gene was contained within a putative feoAB operon. L. pneumophila feoB insertion mutants exhibited decreased ferrous but not ferric iron uptake compared to the wild type. Growth on standard buffered charcoal yeast extract agar or buffered yeast extract broth was unaffected by the loss of L. pneumophila FeoB. However, the L. pneumophila feoB mutant had a reduced ability to grow on buffered charcoal yeast extract agar with a reduced amount of its usual iron supplementation, a phenotype that could be complemented by the addition of feoB in trans. In unsupplemented buffered yeast extract broth, the feoB mutant also had a growth defect, which was further exacerbated by the addition of the ferrous iron chelator, 2,2'-dipyridyl. The feoB mutant was also 2.5 logs more resistant to streptonigrin than wild-type 130b, confirming its decreased ability to acquire iron during extracellular growth. Decreased replication of the feoB mutant was noted within iron-depleted Hartmannella vermiformis amoebae and human U937 cell macrophages. The reduced intracellular infectivity of the feoB mutant was complemented by the introduction of a plasmid containing feoAB. The L. pneumophila feoB gene conferred a modest growth advantage for the wild type over the mutant in a competition assay within the lungs of A/J mice. Taken together, these results indicate that L. pneumophila FeoB is a ferrous iron transporter that is important for extracellular and intracellular growth, especially in iron-limited environments. These data represent the first evidence for the importance of ferrous iron transport for intracellular replication by a human pathogen.
KeywordMeSH Terms
10. Nagai  H, Kagan  JC, Zhu  X, Kahn  RA, Roy  CR,     ( 2002 )

A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes.

Science (New York, N.Y.) 295 (5555)
PMID : 11809974  :   DOI  :   10.1126/science.1067025    
Abstract >>
The intracellular pathogen Legionella pneumophila subverts vesicle traffic in eukaryotic host cells to create a vacuole that supports replication. The dot/icm genes encode a protein secretion apparatus that L. pneumophila require for biogenesis of this vacuole. Here we show that L. pneumophila produce a protein called RalF that functions as an exchange factor for the ADP ribosylation factor (ARF) family of guanosine triphosphatases (GTPases). The RalF protein is required for the localization of ARF on phagosomes containing L. pneumophila. Translocation of RalF protein through the phagosomal membrane is a dot/icm-dependent process. Thus, RalF is a substrate of the Dot/Icm secretion apparatus.
KeywordMeSH Terms
11. Rankin  S, Li  Z, Isberg  RR,     ( 2002 )

Macrophage-induced genes of Legionella pneumophila: protection from reactive intermediates and solute imbalance during intracellular growth.

Infection and immunity 70 (7)
PMID : 12065505  :   DOI  :   10.1128/iai.70.7.3637-3648.2002     PMC  :   PMC128052    
Abstract >>
A promoter-probe strategy was devised to identify genes specifically expressed by Legionella pneumophila during growth within the macrophage. Random fragments from the L. pneumophila chromosome were inserted upstream of a promoterless phage T4 td gene, and fragments that led to complementation of thymine auxotrophy during intracellular growth of the bacterium were identified. Two different selection strategies were employed to eliminate promoters that were also active during extracellular growth of the bacterium. Some of these genes were identified independently by using both of the selection strategies. The factors identified include orthologs of efflux-mediated resistance determinants and transporters, a transporter involved in protection from osmotic stress, a stress response GTP-binding protein, a response regulator, a sensor kinase, and two systems that increase the reducing potential of the bacterium, one of which encodes the L. pneumophila ortholog of ahpC. Five of the clones analyzed here were fusions to promoters that were closely linked to genes encoding three-component chemiosmotic efflux pumps that export heavy metals or toxic organic compounds. Analysis of ahpC gene expression indicates that levels increased at least sevenfold during intracellular growth of the bacterium. Inactivation of several of the genes at their chromosomal loci had no effect on the intracellular growth rate of L. pneumophila in cultured macrophages. This suggests that a number of genes with increased expression during intracellular growth may be part of redundant systems that allow survival and growth under the conditions encountered within host cells.
KeywordMeSH Terms
Gene Expression
Genes, Bacterial
12. Aragon  V, Rossier  O, Cianciotto  NP,     ( 2002 )

Legionella pneumophila genes that encode lipase and phospholipase C activities.

Microbiology (Reading, England) 148 (Pt 7)
PMID : 12101309  :   DOI  :   10.1099/00221287-148-7-2223    
Abstract >>
Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic protozoans and human macrophages. The type II protein secretion system of the Gram-negative Legionella organism promotes intracellular infection. A lipase activity and a p-nitrophenylphosphorylcholine (pNPPC) hydrolytic activity are two of the factors that are diminished in L. pneumophila type II secretion mutants. The Legionella lipase activity was found to include free fatty acid release from di- and triacylglycerol substrates, in addition to the previously reported cleavage of monoacylglycerol. In a number of other bacterial systems, the release of p-nitrophenol from pNPPC is due to a phospholipase C. In an attempt to identify exoproteins that potentiate intracellular infection, three genes were identified and mutated in L. pneumophila strain 130b that were predicted to encode either a secreted lipase or a phospholipase C. The first two genes, which were designated lipA and lipB, encoded proteins containing the lipase consensus sequence [LIV]-X-[LIVFY]-[LIVMST]-G-[HYWV]-S-X-G-[GSTAC]. Mutations in lipA in particular reduced supernatant activity against mono- and triacylglycerols. However, loss of lipA and/or lipB did not impair the ability of L. pneumophila to infect Hartmannella amoebae or U937 cell macrophages. The third L. pneumophila gene, which was denoted plcA, encoded a protein that was highly homologous with a phospholipase C from Pseudomonas fluorescens. Inactivation of plcA diminished secreted pNPPC hydrolase activity but did not influence Legionella infection of host cells. Taken together, these data indicate that L. pneumophila has multiple lipases and possibly several phospholipase C enzymes but that LipA, LipB and PlcA are not among those exoproteins required for optimal intracellular infection.
KeywordMeSH Terms
13. Fettes  PS, Forsbach-Birk  V, Lynch  D, Marre  R,     ( 2001 )

Overexpresssion of a Legionella pneumophila homologue of the E. coli regulator csrA affects cell size, flagellation, and pigmentation.

International journal of medical microbiology : IJMM 291 (5)
PMID : 11727819  :   DOI  :   10.1078/1438-4221-00141    
Abstract >>
Legionella pneumophila is an inhabitant of the aquatic environment and the causative agent of a bacterial pneumonia. We identified the presence of an L. pneumophila homologue of csrA of E. coli and rsmA of Erwinia carotovora, genes which regulate gene expression by destabilising mRNA and which have been shown to relate to environmental fitness and pathogenicity. The Legionella csrA was able to complement a csrA-negative mutant of E. coli. Overproduction of csrA in L. pneumophila lead to a reduction of flagellation and pigmentation and an increase in bacterial cell size. csrA overproduction was associated with a reduction of fliA and flaA transcripts. This suggests that similar to E. coli and Erwinia, L. pneumophila csrA is a regulator of gene expression and may contribute to the capability of the pathogen to rapidly adapt to changing environments.
KeywordMeSH Terms
Escherichia coli Proteins
14. Ko  KS, Lee  HK, Park  MY, Park  MS, Lee  KH, Woo  SY, Yun  YJ, Kook  YH,     ( 2002 )

Population genetic structure of Legionella pneumophila inferred from RNA polymerase gene (rpoB) and DotA gene (dotA) sequences.

Journal of bacteriology 184 (8)
PMID : 11914343  :   DOI  :   10.1128/jb.184.8.2123-2130.2002     PMC  :   PMC134959    
Abstract >>
The population structure of Legionella pneumophila was studied by using partial RNA polymerase gene (rpoB) and DotA gene (dotA) sequences. Trees inferred from rpoB sequences showed that two subspecies of L. pneumophila, Legionella pneumophila subsp. pneumophila and Legionella pneumophila subsp. fraseri, were clearly separated genetically. In both rpoB and dotA trees, 79 Korean isolates used in this study constituted six clonal populations, four of which (designated subgroups P-I to P-IV) were identified in L. pneumophila subsp. pneumophila and two of which (designated subgroups F-I and F-II) were identified in L. pneumophila subsp. fraseri. Although the relationships among subgroups were not identical, such subgrouping was congruent between the rpoB and dotA trees. Type strains of several serogroups did not belong to any subgroup, presumably because isolates similar to these strains were not present among our local sample of the population. There was evidence that horizontal gene transfer or recombination had occurred within L. pneumophila. Contrary to the phylogeny from rpoB and the taxonomic context, subgroups P-III and P-IV of L. pneumophila subsp. pneumophila proved to be closely related to those of L. pneumophila subsp. fraseri or showed a distinct clustering in the dotA tree. It can be inferred that dotA of subgroups P-III and P-IV has been transferred horizontally from other subspecies. The diverse distribution of serogroup 1 strains through the gene trees suggests that surface antigen-coding genes that determine serogroup can be exchanged. Thus, it can be inferred that genetic recombination has been important in the evolution of L. pneumophila.
KeywordMeSH Terms
Bacterial Proteins
15. Bumbaugh  AC, McGraw  EA, Page  KL, Selander  RK, Whittam  TS,     ( 2002 )

Sequence polymorphism of dotA and mip alleles mediating invasion and intracellular replication of Legionella pneumophila.

Current microbiology 44 (5)
PMID : 11927981  :   DOI  :   10.1007/s00284-001-0024-6    
Abstract >>
Legionella pneumophila inhabit a variety of natural and man-made aquatic environments, where they live primarily as intracellular parasites of protozoans. Given the proper exposure, however, they can cause opportunistic pneumonic infections in humans. The products of two L. pneumophila genes, dotA and mip, are part of the mechanism mediating the initial invasion of eukaryotic cells, and subsequent intracellular survival and multiplication. In this study, DNA polymorphism of the dotA and mip genes was assessed for 17 clinical and environmental isolates by nucleotide sequencing to determine the level of sequence variation, rates of molecular evolution, and history of gene divergence. The mip gene is highly conserved, whereas dotA is extremely variable, with an average level of nucleotide diversity four times greater than that of mip. Gene trees for each locus support a division of the L. pneumophila isolates into two clonal lineages. There are several disagreements between the gene trees suggesting that although L. pneumophila has a clonal population structure, genetic exchange has contributed to genotypic variation among strains in nature.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
Peptidylprolyl Isomerase
Polymorphism, Genetic
16. Viswanathan  VK, Kurtz  S, Pedersen  LL, Abu-Kwaik  Y, Krcmarik  K, Mody  S, Cianciotto  NP,     ( 2002 )

The cytochrome c maturation locus of Legionella pneumophila promotes iron assimilation and intracellular infection and contains a strain-specific insertion sequence element.

Infection and immunity 70 (4)
PMID : 11895946  :   DOI  :   10.1128/iai.70.4.1842-1852.2002     PMC  :   PMC127876    
Abstract >>
Previously, we obtained a Legionella pneumophila mutant, NU208, that is hypersensitive to iron chelators when grown on standard Legionella media. Here, we demonstrate that NU208 is also impaired for growth in media that simply lack their iron supplement. The mutant was not, however, impaired for the production of legiobactin, the only known L. pneumophila siderophore. Importantly, NU208 was also highly defective for intracellular growth in human U937 cell macrophages and Hartmannella and Acanthamoeba amoebae. The growth defect within macrophages was exacerbated by treatment of the host cells with an iron chelator. Sequence analysis demonstrated that the transposon disruption in NU208 lies within an open reading frame that is highly similar to the cytochrome c maturation gene, ccmC. CcmC is generally recognized for its role in the heme export step of cytochrome biogenesis. Indeed, NU208 lacked cytochrome c. Phenotypic analysis of two additional, independently derived ccmC mutants confirmed that the growth defect in low-iron medium and impaired infectivity were associated with the transposon insertion and not an entirely spontaneous second-site mutation. trans-complementation analysis of NU208 confirmed that L. pneumophila ccmC is required for cytochrome c production, growth under low-iron growth conditions, and at least some forms of intracellular infection. Although ccm genes have recently been implicated in iron assimilation, our data indicate, for the first time, that a ccm gene can be required for bacterial growth in an intracellular niche. Complete sequence analysis of the ccm locus from strain 130b identified the genes ccmA-H. Interestingly, however, we also observed that a 1.8-kb insertion sequence element was positioned between ccmB and ccmC. Southern hybridizations indicated that the open reading frame within this element (ISLp 1) was present in multiple copies in some strains of L. pneumophila but was absent from others. These findings represent the first evidence for a transposable element in Legionella and the first identification of an L. pneumophila strain-specific gene.
KeywordMeSH Terms
Bacterial Proteins
DNA Transposable Elements
17. Samrakandi  MM, Cirillo  SL, Ridenour  DA, Bermudez  LE, Cirillo  JD,     ( 2002 )

Genetic and phenotypic differences between Legionella pneumophila strains.

Journal of clinical microbiology 40 (4)
PMID : 11923356  :   DOI  :   10.1128/jcm.40.4.1352-1362.2002     PMC  :   PMC140379    
Abstract >>
Legionnaires' disease is a potentially lethal pneumonia that is primarily due to infection by the species Legionella pneumophila, although more than 40 other species are known. Certain L. pneumophila subgroups, particularly serogroup 1, are associated with the majority of the epidemics. The genetic bases for these differences in virulence have not been determined. Three strains, AA100, JR32, and Lp01, have been used in many molecular pathogenesis studies of L. pneumophila. We found genetic differences between these strains by PCR and Southern analyses that may be related to their ability to cause disease. We also examined the distribution of these genetic loci in clinical and environmental isolates of Legionella and found a correlation between the presence of two of these loci, rtxA and lvh, and the ability to cause disease in humans. Examination of the interactions of these strains with host cells suggested that they differ in important phenotypic characteristics including adherence, entry, and intracellular replication. Furthermore, in the mouse model of infection they display differing levels of replication in lungs. These studies emphasize the importance of further investigation into the genetic makeup of these strains, which is likely to lead to the identification of additional factors involved in Legionella pathogenesis.
KeywordMeSH Terms
18. Steinert  M, Flügel  M, Schuppler  M, Helbig  JH, Supriyono  A, Proksch  P, Lück  PC,     ( 2001 )

The Lly protein is essential for p-hydroxyphenylpyruvate dioxygenase activity in Legionella pneumophila.

FEMS microbiology letters 203 (1)
PMID : 11557138  :   DOI  :   10.1111/j.1574-6968.2001.tb10818.x    
Abstract >>
The lly locus confers fluorescence, haemolysis, brown pigmentation and an increased resistance to light in Legionella pneumophila. In this study, we correlated the pigment production of two lly-positive L. pneumophila isolates and a recombinant lly-positive Escherichia coli strain with the presence of homogentisic acid (HGA) in the culture supernatant. The detection of HGA by high performance liquid chromatography and the data analysis of the deduced amino acid sequence of the lly gene indicate that the lly locus codes for a p-hydroxyphenylpyruvate dioxygenase (HPPD). This enzyme catalyses the transformation of p-hydroxyphenylpyruvate into HGA, which subsequently oxidises and polymerises into a melanin-like pigment. One open reading frame (ORF 1) in the lly region exhibited homologies with genes of Synechocystis sp., Petroselium crispum and Streptomyces mycarofaciens that code for methyltransferases. By screening a genomic library of L. pneumophila (serogroup 1) strain Corby with a monoclonal antibody against the legiolysin (lly), we identified two recombinant E. coli clones that did not produce the brown pigment and showed no haemolysis and fluorescence. DNA sequencing revealed that both clones contained 874 nucleotides of the N-terminal part of the lly gene. The recombinant strains expressed truncated legiolysin proteins of 39.5 and 35.7 kDa and did not produce HGA. Considering the highly conserved structure of legiolysin-like HPPD genes from other organisms, we suggest that the C-terminus of the legiolysin may be essential for the enzymatic activity that conferred pigmentation via HGA polymerisation, haemolysis and fluorescence.
KeywordMeSH Terms
19. Higa  F, Edelstein  PH,     ( 2001 )

Potential virulence role of the Legionella pneumophila ptsP ortholog.

Infection and immunity 69 (8)
PMID : 11447151  :   DOI  :   10.1128/IAI.69.8.4782-4789.2001     PMC  :   PMC98565    
Abstract >>
We previously identified the Legionella pneumophila ptsP (phosphoenolpyruvate phosphotransferase) ortholog gene as a putative virulence factor in a study of signature-tagged mutagenesis using a guinea pig pneumonia model. In this study, we further defined the phenotypic properties of L. pneumophila ptsP and its complete sequence. The L. pneumophila ptsP was 2,295 bases in length. Its deduced amino acid sequence had high similarity with ptsP orthologs of Pseudomonas aeruginosa, Azotobacter vinelandii, and Escherichia coli, with nearly identical lengths. Here we show that while the mutant grew well in laboratory media, it was defective in both lung and spleen multiplication in guinea pigs. It grew slowly in guinea pig alveolar macrophages despite good uptake into the cells. Furthermore, there was minimal growth in a human alveolar epithelial cell line (A549). Transcomplementation of the L. pneumophila ptsP mutant almost completely rescued its growth in alveolar macrophages, in A549 cells, and in guinea pig lung and spleen. The L. pneumophila ptsP mutant was capable of evasion of phagosome-lysosome fusion and resided in ribosome-studded phagosomes. Pore formation activity of the mutant was normal. The L. pneumophila ptsP mutant expressed DotA and IcmX in apparently normal amounts, suggesting that the ptsP mutation did not affect dotA and icmX regulation. In addition, the mutant was resistant to serum and neutrophil killing. Taken together, these findings show that L. pneumophila ptsP is required for full in vivo virulence of L. pneumophila, most probably by affecting intracellular growth.
KeywordMeSH Terms
20. Robey  M, O'Connell  W, Cianciotto  NP,     ( 2001 )

Identification of Legionella pneumophila rcp, a pagP-like gene that confers resistance to cationic antimicrobial peptides and promotes intracellular infection.

Infection and immunity 69 (7)
PMID : 11401964  :   DOI  :   10.1128/IAI.69.7.4276-4286.2001     PMC  :   PMC98497    
Abstract >>
In the course of characterizing a locus involved in heme utilization, we identified a Legionella pneumophila gene predicted to encode a protein with homology to the product of the Salmonella enterica serovar Typhimurium pagP gene. In Salmonella, pagP increases resistance to the bactericidal effects of cationic antimicrobial peptides (CAMPs). Mutants with insertions in the L. pneumophila pagP-like gene were generated and showed decreased resistance to different structural classes of CAMPs compared to the wild type; hence, this gene was designated rcp for resistance to cationic antimicrobial peptides. Furthermore, Legionella CAMP resistance was induced by growth in low-magnesium medium. To determine whether rcp had any role in intracellular survival, mutants were tested in the two most relevant host cells for Legionnaires' disease, i.e., amoebae and macrophages. These mutants exhibited a 1,000-fold-decreased recovery during a Hartmannella vermiformis coculture. Complementation of the infectivity defect could be achieved by introduction of a plasmid containing the intact rcp gene. Mutations in rcp consistently reduced both the numbers of bacteria recovered during intracellular infection and their cytopathic capacity for U937 macrophages. The rcp mutant was also more defective for lung colonization of A/J mice. Growth of rcp mutants in buffered yeast extract broth was identical to that of the wild type, indicating that the observed differences in numbers of bacteria recovered from host cells were not due to a generalized growth defect. However, in low-Mg(2+) medium, the rcp mutant was impaired in stationary-phase survival. This is the first demonstration of a pagP-like gene, involved in resistance to CAMPs, being required for intracellular infection and virulence.
KeywordMeSH Terms
DNA-Binding Proteins
Genes, Bacterial
21. Lüneberg  E, Mayer  B, Daryab  N, Kooistra  O, Zähringer  U, Rohde  M, Swanson  J, Frosch  M,     ( 2001 )

Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila.

Molecular microbiology 39 (5)
PMID : 11251842  :   DOI  :   10.1111/j.1365-2958.2001.02314.x    
Abstract >>
We recently described the phase-variable expression of a virulence-associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild-type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high-copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non-functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high-copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA-independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.
KeywordMeSH Terms
22. Rossier  O, Cianciotto  NP,     ( 2001 )

Type II protein secretion is a subset of the PilD-dependent processes that facilitate intracellular infection by Legionella pneumophila.

Infection and immunity 69 (4)
PMID : 11254562  :   DOI  :   10.1128/IAI.69.4.2092-2098.2001     PMC  :   PMC98134    
Abstract >>
Previously, we had demonstrated that a Legionella pneumophila prepilin peptidase (pilD) mutant does not produce type IV pili and shows reduced secretion of enzymatic activities. Moreover, it displays a distinct colony morphology and a dramatic reduction in intracellular growth within amoebae and macrophages, two phenotypes that are not exhibited by a pilin (pilE(L)) mutant. To determine whether these pilD-dependent defects were linked to type II secretion, we have constructed two new mutants of L. pneumophila strain 130b. Mutations were introduced into either lspDE, which encodes the type II outer membrane secretin and ATPase, or lspFGHIJK, which encodes the pseudopilins. Unlike the wild-type and pilE(L) strains, both lspDE and lspG mutants showed reduced secretion of six pilD-dependent enzymatic activities; i.e., protease, acid phosphatase, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A. However, they exhibited a colony morphology different from that of the pilD mutant, suggesting that their surfaces are distinct. The pilD, lspDE, and lspG mutants were similarly and greatly impaired for growth within Hartmannella vermiformis, indicating that the intracellular defect of the peptidase mutant in amoebae is explained by the loss of type II secretion. When assessed for infection of U937 macrophages, both lsp mutants exhibited a 10-fold reduction in intracellular multiplication and a diminished cytopathic effect. Interestingly, the pilD mutant was clearly 100-fold more defective than the type II secretion mutants in U937 cells. These results suggest the existence of a novel pilD-dependent mechanism for promoting L. pneumophila intracellular infection of human cells.
KeywordMeSH Terms
Endopeptidases
23. Polesky  AH, Ross  JT, Falkow  S, Tompkins  LS,     ( 2001 )

Identification of Legionella pneumophila genes important for infection of amoebas by signature-tagged mutagenesis.

Infection and immunity 69 (2)
PMID : 11159993  :   DOI  :   10.1128/IAI.69.2.977-987.2001     PMC  :   PMC97977    
Abstract >>
Legionella pneumophila is a facultative intracellular gram-negative rod that causes pneumonia in humans. Free-living amoebas are thought to serve as a reservoir for Legionella infections. Signature-tagged mutagenesis was employed to identify Legionella pneumophila genes necessary for survival in the amoeba Acanthamoeba castellanii. Six mutant strains were defective in assays of invasion and intracellular growth. Four mutants also exhibited invasion and replication defects in Hartmannella vermiformis, an amoeba linked to hospital outbreaks of Legionella pneumonia. The six mutants also were tested in macrophages derived from peripheral blood mononuclear cells. Two mutants had intracellular replication defects, and two different strains entered cells less efficiently. Two transposon insertions were in known L. pneumophila genes, lspK and aroB. The other four were in novel genes. One gene has similarity to a cytochrome c-type biogenesis protein of Pseudomonas fluorescens. Another has similarity to a transcriptional activator regulating flagellar biosynthesis in Vibrio cholera. The third is similar to traA of Rhizobium sp. strain NGR234, which is involved in conjugal transfer of DNA. The fourth has no homology. By using survival in amoeba as a selection, we have isolated mutant strains with a range of phenotypes; and we have potentially identified new L. pneumophila virulence genes.
KeywordMeSH Terms
Genes, Bacterial
24. Aragon  V, Kurtz  S, Cianciotto  NP,     ( 2001 )

Legionella pneumophila major acid phosphatase and its role in intracellular infection.

Infection and immunity 69 (1)
PMID : 11119504  :   DOI  :   10.1128/IAI.69.1.177-185.2001     PMC  :   PMC97870    
Abstract >>
Legionella pneumophila is an intracellular pathogen of protozoa and alveolar macrophages. This bacterium contains a gene (pilD) that is involved in both type IV pilus biogenesis and type II protein secretion. We previously demonstrated that the PilD prepilin peptidase is crucial for intracellular infection by L. pneumophila and that the secreted pilD-dependent proteins include a metalloprotease, an acid phosphatase, an esterase/lipase, a phospholipase A, and a p-nitrophenyl phosphorylcholine hydrolase. Since mutants lacking type IV pili, the protease, or the phosphorylcholine hydrolase are not defective for intracellular infection, we sought to determine the significance of the secreted acid phosphatase activity. Three mutants defective in acid phosphatase activity were isolated from a population of mini-Tn10-mutagenized L. pneumophila. Supernatants as well as cell lysates from these mutants contained minimal acid phosphatase activity while possessing normal levels of other pilD-dependent exoproteins. Genetic studies indicated that the gene affected by the transposon insertions encoded a novel bacterial histidine acid phosphatase, which we designated Map for major acid phosphatase. Subsequent inhibitor studies indicated that Map, like its eukaryotic homologs, is a tartrate-sensitive acid phosphatase. The map mutants grew within macrophage-like U937 cells and Hartmannella amoebae to the same degree as did wild-type legionellae, indicating that this acid phosphatase is not essential for L. pneumophila intracellular infection. However, in the course of characterizing our new mutants, we gained evidence for a second pilD-dependent acid phosphatase activity that, unlike Map, is tartrate resistant.
KeywordMeSH Terms
Endopeptidases
25. Harb  OS, Abu Kwaik  Y,     ( 2000 )

Essential role for the Legionella pneumophila rep helicase homologue in intracellular infection of mammalian cells.

Infection and immunity 68 (12)
PMID : 11083821  :   DOI  :   10.1128/iai.68.12.6970-6978.2000     PMC  :   PMC97806    
Abstract >>
We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (mil) loci. In this study we characterized the mil mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar to rep helicase genes of other bacteria. Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the L. pneumophila phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the L. pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in the rep helicase homologue mutant, rendering it defective in intracellular replication.
KeywordMeSH Terms
DNA Helicases
26. Bandyopadhyay  P, Steinman  HM,     ( 2000 )

Catalase-peroxidases of Legionella pneumophila: cloning of the katA gene and studies of KatA function.

Journal of bacteriology 182 (23)
PMID : 11073912  :   DOI  :   10.1128/jb.182.23.6679-6686.2000     PMC  :   PMC111410    
Abstract >>
Legionella pneumophila, the causative organism of Legionnaires' pneumonia, contains two enzymes with catalatic and peroxidatic activity, KatA and KatB. To address the issue of redundant, overlapping, or discrete in vivo functions of highly homologous catalase-peroxidases, the gene for katA was cloned and its function was studied in L. pneumophila and Escherichia coli and compared with prior studies of katB in this laboratory. katA is induced during exponential growth and is the predominant peroxidase in stationary phase. When katA is inactivated, L. pneumophila is more sensitive to exogenous hydrogen peroxide and less virulent in the THP-1 macrophage cell line, similar to katB. Catalatic-peroxidatic activity with different peroxidatic cosubstrates is comparable for KatA and KatB, but KatA is five times more active towards dianisidine. In contrast with these examples of redundant or overlapping function, stationary-phase survival is decreased by 100- to 10,000-fold when katA is inactivated, while no change from wild type is seen for the katB null. The principal clue for understanding this discrete in vivo function was the demonstration that KatA is periplasmic and KatB is cytosolic. This stationary-phase phenotype suggests that targets sensitive to hydrogen peroxide are present outside the cytosol in stationary phase or that the peroxidatic activity of KatA is critical for stationary-phase redox reactions in the periplasm, perhaps disulfide bond formation. Since starvation-induced stationary phase is a prerequisite to acquisition of virulence by L. pneumophila, further studies on the function and regulation of katA in stationary phase may give insights on the mechanisms of infectivity of this pathogen.
KeywordMeSH Terms
27. Lüneberg  E, Zetzmann  N, Alber  D, Knirel  YA, Kooistra  O, Zähringer  U, Frosch  M,     ( 2000 )

Cloning and functional characterization of a 30 kb gene locus required for lipopolysaccharide biosynthesis in Legionella pneumophila.

International journal of medical microbiology : IJMM 290 (1)
PMID : 11043980  :   DOI  :   10.1016/S1438-4221(00)80104-6    
Abstract >>
The spontaneous Legionella pneumophila lipopolysaccharide (LPS) mutant 137, which did not bind the LPS-specific mAb 2625, was complemented with a genomic library from the parental wild-type strain. Transformants were screened for reconstitution of the wild-type LPS phenotype, able to bind mAb 2625. By this strategy, a 32,661 bp region comprising 30 open reading frames (Orfs) was identified. Orfs with significant homologies to genes encoding enzymes required for LPS or capsule biosynthesis of Gram-negative bacteria were located on the gene locus. The mutation of strain 137 could be assigned to a deletion of a cytosine residue in Orf 8. The protein encoded by Orf 8 exhibited homology to bacterial methyl-transferases. The L. pneumophila LPS gene locus included genes with deduced products likely to be involved in LPS core oligosaccharide biosynthesis (rmlA-D, rhamnosyl-transferases, acetyl-transferase) as well as LPS O-chain biosynthesis and translocation (mnaA, neuB, neuA, wecA, wzt, wzm). The neuA (Orf 25) and neuB (Orf 24) gene products were functionally characterized by complementation of the capsule negative E. coli K1 mutants EV5 and EV24, respectively. By introduction of the L. pneumophila neuA gene into E. coli EV5 and the neuB gene into EV24, expression of the K1 polysialic acid capsule could be restored. We, therefore, conclude that the biosynthesis pathway of legionaminic acid, the structural unit of the L. pneumophila Sg1 O-antigen, might be similar to the biosynthesis of sialic acid. Southern blot analysis indicated the entire gene locus to be present in L. pneumophila serogroup (Sg)1 strains, whereas only parts of the DNA stretch hybridized to DNA from Sg2 to Sg14 strains.
KeywordMeSH Terms
Genes, Bacterial
28. Heuner  K, Dietrich  C, Steinert  M, Göbel  UB, Hacker  J,     ( 2000 )

Cloning and characterization of a Legionella pneumophila-specific gene encoding a member of the LysR family of transcriptional regulators.

Molecular & general genetics : MGG 264 (1��2��)
PMID : 11016850  :   DOI  :   10.1007/s004380000310    
Abstract >>
Flagellin gene regulation in Legionella pneumophila is modulated by various environmental factors. The expression of the virulent phenotype seems to be linked genetically to flagellum expression. To better understand the mechanisms of flagellin gene expression in L. pneumophila (Lp), we screened a pool of plasmids from a L. pneumophila Corby genomic library for the ability to prevent or reduce luciferase activity in the Escherichia coli strain YK410, which harbours a Lp-pflaA-luxAB fusion. We cloned a DNA fragment encoding the N-terminal part of a protein with significant similarity to members of the LysR family of transcriptional regulators (LTTRs). The entire gene, cloned by inverse PCR, was named flaR. It encodes a protein of 302 amino acids, and computer-assisted analysis of the amino acid sequence revealed a helix-turn-helix motif located near the N-terminus of the protein. The FlaR protein exhibits 21-31% identity to various LTTRs. Furthermore, gel retardation experiments indicate that the FlaR protein is able to bind to its own promoter region and, to a lesser extent, to the flaA promoter of L. pneumophila. The flaR promoter region contains putative LysR binding motifs and two putative Fur boxes. Taken together, these results indicate that FlaR is a DNA-binding protein which belongs to the LTTR family. Southern analysis with a L. pneumophila Corby-specific flaR probe revealed homologous genes in various L. pneumophila strains, but not in the 12 nonpneumophila strains tested so far.
KeywordMeSH Terms
29. Lum  J, Cirillo  SL,     ( 2000 )

Identification of novel loci involved in entry by Legionella pneumophila.

Microbiology (Reading, England) 146 (Pt 6) (N/A)
PMID : 10846213  :   DOI  :   10.1099/00221287-146-6-1345    
Abstract >>
Legionella pneumophila is primarily an intracellular pathogen during infection; thus, the mechanisms of entry into host cells are likely to be important for pathogenesis. Several L. pneumophila mutants that display an enhanced-entry (Enh) phenotype were isolated by selecting for bacteria that enter host cells at a higher frequency than wild-type. In the course of characterizing the genetic basis of one of these mutants, C3, a strategy was developed for the isolation of laboratory-media-repressed virulence determinants from L. pneumophila. Screens for dominant mutations using a genomic DNA library from C3 resulted in the isolation of three cosmids that confer an Enh phenotype to wild-type L. pneumophila. Transposon mutagenesis of these cosmids allowed identification of three loci that affect entry. Analysis of the putative proteins encoded by these loci, designated rtxA and enhC, demonstrated similarity to repeats in the structural toxin protein and the secreted Sel-1 protein from Caenorhabditis elegans, respectively. L. pneumophila rtxA and enhC mutants display significantly reduced entry into host cells, compared to wild-type bacteria. The phenotype that the cosmids containing these loci confer is most likely due to elevated expression resulting from their presence on multicopy vectors. The use of increased gene copy number to overexpress genes that are normally repressed under laboratory growth conditions is generally applicable to the isolation of virulence determinants from L. pneumophila and other bacterial pathogens.
KeywordMeSH Terms
30. Nielsen  K, Hindersson  P, Hoiby  N, Bangsborg  JM,     ( 2000 )

Sequencing of the rpoB gene in Legionella pneumophila and characterization of mutations associated with rifampin resistance in the Legionellaceae.

Antimicrobial agents and chemotherapy 44 (10)
PMID : 10991843  :   DOI  :   10.1128/aac.44.10.2679-2683.2000     PMC  :   PMC90134    
Abstract >>
Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the rpoB gene are known to cause rifampin resistance in Escherichia coli and Mycobacterium tuberculosis, and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family Legionellaceae. Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the Legionella pneumophila rpoB gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the rpoB gene (4,104 bp) and an ORF of 384 bp representing part of the rpoC gene was obtained. A 316-bp DNA fragment in the center of the L. pneumophila rpoB gene, corresponding to a previously described site for mutations leading to rifampin resistance in M. tuberculosis, was sequenced from 18 rifampin-resistant Legionella isolates representing four species (L. bozemanii, L. longbeachae, L. micdadei, and L. pneumophila), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family Legionellaceae.
KeywordMeSH Terms
31. Fettes  PS, Susa  M, Hacker  J, Marre  R,     ( 2000 )

Characterization of the Legionella pneumophila gene ligA.

International journal of medical microbiology : IJMM 290 (3)
PMID : 10959726  :   DOI  :   10.1016/S1438-4221(00)80121-6    
Abstract >>
Legionella pneumophila is a bacterial pathogen that resides and multiplies in macrophages as well as in its natural aquatic hosts, the protozoa. Different bacterial factors contribute to pathogenicity and accompanying eukaryotic intracellular events. Sequencing of mip flanking regions revealed a gene of 2610 bp, ligA, that has no significant similarity to any of the genes identified previously. Epidemiological studies indicate that this gene is present in Legionella pneumophila, the species most often associated with cases of the Legionnaires' disease, but not in Legionella species other than L. pneumophila. The isogenic ligA deletion mutant was resistant to NaCl, and showed decreased cytotoxicity to human monocytes and decreased hemolytic activity to red blood cells. However, the most prominent effect of the L. pneumophila ligA mutant strain LEPF1 was the nearly completely reduced replication within the natural host Acanthamoeba castellanii. Since this gene is L. pneumophila specific and regulates numerous bacterial properties we designated this gene ligA for Legionella pneumophila infectivity gene A.
KeywordMeSH Terms
Peptidylprolyl Isomerase
32. Edelstein  PH, Pope  CD, Viswanathan  VK,     ( 2000 )

The Legionella pneumophila iraAB locus is required for iron assimilation, intracellular infection, and virulence.

Infection and immunity 68 (3)
PMID : 10678909  :   DOI  :   10.1128/iai.68.3.1069-1079.2000     PMC  :   PMC97250    
Abstract >>
Legionella pneumophila, a facultative intracellular parasite of human alveolar macrophages and protozoa, causes Legionnaires' disease. Using mini-Tn10 mutagenesis, we previously isolated a L. pneumophila mutant that was hypersensitive to iron chelators. This mutant, NU216, and its allelic equivalent, NU216R, were also defective for intracellular infection, particularly in iron-deficient host cells. To determine whether NU216R was attenuated for virulence, we assessed its ability to cause disease in guinea pigs following intratracheal inoculation. NU216R-infected animals yielded 1,000-fold fewer bacteria from their lungs and spleen compared to wild-type-130b-infected animals that had received a 50-fold-lower dose. Moreover, NU216R-infected animals subsequently cleared the bacteria from these sites. While infection with 130b resulted in high fever, weight loss, and ruffled fur, inoculation with NU216R did not elicit any signs of disease. DNA sequence analysis revealed that the transposon insertion in NU216R lies in the first open reading frame of a two-gene operon. This open reading frame (iraA) encodes a 272-amino-acid protein that shows sequence similarity to methyltransferases. The second open reading frame (iraB) encodes a 501-amino-acid protein that is highly similar to di- and tripeptide transporters from both prokaryotes and eukaryotes. Southern hybridization analyses determined that the iraAB locus was largely limited to strains of L. pneumophila, the most pathogenic of the Legionella species. A newly derived mutant containing a targeted disruption of iraB showed reduced ability to grow under iron-depleted extracellular conditions, but it did not have an infectivity defect in the macrophage-like U937 cells. These data suggest that iraA is critical for virulence of L. pneumophila while iraB is involved in a novel method of iron acquisition which may utilize iron-loaded peptides.
KeywordMeSH Terms
Chromosome Mapping
Open Reading Frames
33. Russo  JJ, Segal  G,     ( 1999 )

Relationships between a new type IV secretion system and the icm/dot virulence system of Legionella pneumophila.

Molecular microbiology 34 (4)
PMID : 10564519  :   DOI  :   10.1046/j.1365-2958.1999.01642.x    
Abstract >>
We describe here a Legionella pneumophila type IV secretion system that is distinct from the previously described icm/dot system. This type IV secretion system contains 11 genes (lvh) homologous to genes of other type IV secretion systems, arranged in a similar manner. The lvh genes were found to be located on a DNA island with a GC content higher than the L. pneumophila chromosome. In contrast to the icm/dot system that was shown to be required for intracellular growth in HL-60-derived human macrophages and Acanthamoeba castellanii, the lvh system was found to be dispensable for intracellular growth in these two hosts. The lvh system was found to be partially required for RSF1010 conjugation, a process that was previously shown to be completely dependent on several icm/dot genes. However, results obtained from analysis of double mutants in the icm/dot genes and the lvh genes revealed that lvh genes can substitute for some components of the icm/dot system for RSF1010 conjugation, but not for intracellular growth. These results indicate that components of the icm/dot system and components of the lvh type IV secretion system are able to interact with one another.
KeywordMeSH Terms
Virulence Factors
34. Harb  OS,     ( 2000 )

Characterization of a macrophage-specific infectivity locus (milA) of Legionella pneumophila.

Infection and immunity 68 (1)
PMID : 10603410  :   PMC  :   PMC97143    
Abstract >>
Legionella pneumophila has been shown to possess multiple genetic loci that play roles in its ability to survive within host cells. The mil (macrophage-specific infectivity loci) mutants of L. pneumophila exhibit a spectrum of defects in intracellular survival in and cytopathogenicity to macrophages and alveolar epithelial cells. This study characterizes one of the mil mutants (GB111). Intracellular growth of GB111 in macrophages was approximately 100- to 1,000-fold less than that of AA100, the parental strain, at 24 and 48 h postinfection. This defect in turn corresponded to a defect in cytopathogenicity. Sequence analysis of the affected GB111 open reading frame (ORF) revealed it to encode a putative transport protein, and the ORF was designated milA. The phenotypic defect of the milA mutant was complemented with a PCR fragment containing only milA, indicating that the defect in GB111 was due to the disruption of milA. Intracellular trafficking of the mutant was examined by laser scanning confocal microscopy. The data showed that 50% of the GB111 phagosomes colocalized with the late endosomal/lysosomal marker LAMP-2 (2 and 4 h postinfection), while less than 10% of the AA100 phagosomes colocalized with this marker. On the other hand, over 80% of the GB111 phagosomes were similar to the AA100 phagosome in that they were devoid of LAMP-1 and cathepsin D, and they were colocalized with the endoplasmic reticulum (ER) marker BiP. However, the number of GB111 phagosomes that colocalized with BiP decreased to 50% 6 h postinfection compared to that of AA100, which remained constant (80% colocalization). Thus, compared to AA100, the milA mutation caused a defect in intracellular replication, which was associated with colocalization of the phagosome with LAMP-2 and BiP, while colocalization with LAMP-1 and cathepsin D was not affected.
KeywordMeSH Terms
Genes, Bacterial
Peptidylprolyl Isomerase
35. Shuman  HA,     ( 1999 )

The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii.

Journal of bacteriology 181 (16)
PMID : 10438758  :   PMC  :   PMC93975    
Abstract >>
To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of the Escherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoS mutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in the rpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host, Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS in L. pneumophila is very different from what has previously been reported for E. coli rpoS.
KeywordMeSH Terms
36. Mintz  CS, Knirel  YA, Helbig  JH, Zähringer  U,     ( 1999 )

Molecular cloning and characterization of a locus responsible for O acetylation of the O polysaccharide of Legionella pneumophila serogroup 1 lipopolysaccharide.

Journal of bacteriology 181 (13)
PMID : 10383989  :   PMC  :   PMC93911    
Abstract >>
Complementation experiments, Tn5 mutagenesis, and DNA sequencing were used to identify a locus (lag-1) that participates in acetylation of Legionella pneumophila serogroup 1 lipopolysaccharide. Nuclear magnetic resonance analyses of lipopolysaccharides from mutant and complemented strains suggest that lag-1 is responsible for O acetylation of serogroup 1 O polysaccharide.
KeywordMeSH Terms
Genes, Bacterial
37. Shuman  HA,     ( 1999 )

Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease.

Infection and immunity 67 (7)
PMID : 10377156  :   PMC  :   PMC116561    
Abstract >>
We report the identification of a set of Legionella pneumophila genes that encode products with homology to proteins of the type II general secretion pathway of gram-negative bacteria. A strain containing a deletion-substitution mutation of two of these genes was unable to secrete the Msp protease. This strain was unable to multiply within the free-living amoeba Acanthamoeba castellanii yet was able to kill HL-60-derived macrophages. Because Msp is not required for growth in amoebae, other proteins which are important for growth in amoebae are likely secreted by this pathway.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
38. Edelstein  MA, Higa  F, Falkow  S,     ( 1999 )

Discovery of virulence genes of Legionella pneumophila by using signature tagged mutagenesis in a guinea pig pneumonia model.

Proceedings of the National Academy of Sciences of the United States of America 96 (14)
PMID : 10393970  :   DOI  :   10.1073/pnas.96.14.8190     PMC  :   PMC22210    
Abstract >>
Legionella pneumophila is the cause of Legionnaires' disease, which is a form of potentially fatal pneumonia. To identify genes required for virulence of the bacterium, a library of 1,386 L. pneumophila signature tagged transposon mutants was studied for guinea pig virulence. The mutants were screened in pools of 96 each in a guinea pig model of L. pneumophila pneumonia. Sixteen unique mutant clones were determined to have attenuated virulence after being screened twice in the animal model. All 16 mutants failed to multiply in both lungs and spleens. Four of the sixteen had no apparent defect for intracellular multiplication in macrophages. Partial DNA sequences of the interrupted genes adjacent to the transposon insertions showed that six of them had mutations in five known L. pneumophila virulence genes: dotB, dotF/icmG, dotO/icmB, icmX, and proA. Three of the sequenced clones contained mutations in genes without known homology to other published bacterial genes, and seven clones appeared to be homologous to five different known bacterial genes but are still being characterized. With this methodology, we demonstrate the existence of L. pneumophila genes responsible for non-macrophage-related virulence. The discovery of L. pneumophila virulence genes indicates the utility of the signature tagged mutagenesis technique for pulmonary pathogens.
KeywordMeSH Terms
Genes, Bacterial
39. Ratzow  S, Gaia  V, Helbig  JH, Fry  NK, Lück  PC,     ( 2007 )

Addition of neuA, the gene encoding N-acylneuraminate cytidylyl transferase, increases the discriminatory ability of the consensus sequence-based scheme for typing Legionella pneumophila serogroup 1 strains.

Journal of clinical microbiology 45 (6)
PMID : 17409215  :   DOI  :   10.1128/JCM.00261-07     PMC  :   PMC1933043    
Abstract >>
The standard sequence-based method for the typing of Legionella pneumophila serogroup 1 strains was extended by using the gspA and neuA alleles. The use of neuA as a seventh allele for typing significantly increased the index of discrimination calculated for a panel of unrelated strains (from 0.932 to 0.963) and subdivided some known large common complexes (e.g., 1,4,3,1,1,1). This modification to the standard method is proposed as the method of choice in the epidemiological investigation of L. pneumophila infections.
KeywordMeSH Terms
Bacterial Typing Techniques
Consensus Sequence
40. Engleberg  NC, Howe  DC, Rogers  JE, Arroyo  J, Eisenstein  BI,     ( 1991 )

Characterization of a Legionella pneumophila gene encoding a lipoprotein antigen.

Molecular microbiology 5 (8)
PMID : 1766377  :   DOI  :   10.1111/j.1365-2958.1991.tb00824.x    
Abstract >>
A prominent 19 kDa surface antigen of Legionella pneumophila, cloned in Escherichia coli, was found to be intimately associated with peptidoglycan. The DNA region encoding this antigen was mapped on an 11.9 kb plasmid by means of deletion analysis and transposon mutagenesis. PhoA+ gene fusions, gene-rated by TnphoA insertions into this region, confirmed the presence of a gene encoding a secreted protein. PhoA+ transposon insertions were also associated with loss of the 19 kDa antigen in immunoassays using a monoclonal antibody (mAb1E9) and the replacement of the 19 kDa antigen with larger fusion proteins in immunoblots using Legionella immune serum. A 1540bp PstI fragment carrying the gene was sequenced, and the open reading frame encoding the antigen was identified. The gene encodes a polypeptide 176 amino acid residues long and 18913Da in size. The presence of a signal sequence of 22 amino acids with a consensus sequence for cleavage by signal peptidase II indicates that the antigen is a lipoprotein, and striking similarity with peptidoglycan-associated lipoproteins (PALs) from E. coli (51% amino acid homology) and Haemophilus influenzae (55% homology) is noted. We conclude that the 19kDa antigen of L. pneumophila is the structural equivalent of the PAL found in other Gram-negative species and suggest that its post-translational acylation may explain its potency as an immunogen.
KeywordMeSH Terms
Bacterial Outer Membrane Proteins
Proteoglycans
41. Gilmour  MW, Bernard  K, Tracz  DM, Olson  AB, Corbett  CR, Burdz  T, Ng  B, Wiebe  D, Broukhanski  G, Boleszczuk  P, Tang  P, Jamieson  F, Van Domselaar  G, Plummer  FA, Berry  JD,     ( 2007 )

Molecular typing of a Legionella pneumophila outbreak in Ontario, Canada.

Journal of medical microbiology 56 (Pt 3)
PMID : 17314363  :   DOI  :   10.1099/jmm.0.46738-0     PMC  :   PMC2884934    
Abstract >>
An outbreak of Legionnaires' disease at a long-term care facility in Ontario, Canada from September to October 2005 resulted in the death of 23 residents and the illness of 112 other people. In response, molecular methods were developed to detect Legionella pneumophila in clinical lung samples and to subtype isolates from clinical and environmental samples. The targeted genetic loci included Legionella-specific virulence determinants (mip, icmO, sidA and lidA) and core bacterial determinants (ftsZ, trpS and dnaX). An established amplified fragment length polymorphism typing method provided the first indication of genetic relatedness between strains recovered from clinical samples and strains cultured from environmental samples taken from the outbreak site. These associations were verified using the European Working Group for Legionella Infections sequence-based typing protocol targeting the flaA, pilE, asd, mip, mompS and proA loci. These molecular typing methods confirmed the outbreak source as a contaminated air conditioning cooling tower.
KeywordMeSH Terms
Bacterial Typing Techniques
Disease Outbreaks
42. Hoffman  PS, Ripley  M, Weeratna  R,     ( 1992 )

Cloning and nucleotide sequence of a gene (ompS) encoding the major outer membrane protein of Legionella pneumophila.

Journal of bacteriology 174 (3)
PMID : 1732223  :   DOI  :   10.1128/jb.174.3.914-920.1992     PMC  :   PMC206170    
Abstract >>
The major outer membrane protein of Legionella pneumophila is composed of 28- and 31-kDa subunits cross-linked by interchain disulfide bonds. The oligomer is covalently anchored to the underlying peptidoglycan via the 31-kDa subunit. We have cloned the structural gene ompS encoding both proteins. Oligonucleotide probes synthesized from the codons of the N-terminal amino acid sequence of purified 28- and 31-kDa subunits were used to identify cloned sequences. A 2.9-kb HindIII fragment cloned into pBluescript (clone H151) contained the ompS gene. Nucleotide sequence analysis revealed an open reading frame of 891 bp encoding a polypeptide of 297 amino acids. A leader sequence of 21 amino acids was identified, and the mature protein contained 276 amino acids. The deduced amino acid sequence of OmpS matched the experimentally determined amino acid sequence (32 amino acids), with the exception of two cysteine residues. The deduced amino acid sequence was rich in glycine and aromatic amino acids and contained four cysteine residues, two in the amino terminus and two in the carboxy region. Primer extension analysis (total RNA from L. pneumophila) identified the transcription start at 96 to 98 bp upstream of the translation start, but no Escherichia coli-like promoter sequences were evident. While an mRNA transcript from clone H151 was detected, no cross-reactive protein was detected by immunoblotting with either monoclonal or polyclonal antibody. Attempts to subclone the gene in the absence of the putative promoter region (i.e., under the control of the lac promoter) proved unsuccessful, possibly because of overproduction lethality in E. coli. The ompS DNA sequence was highly conserved among the serogroups of L. pneumophila, and related species also exhibited homology in Southern blot analysis at a moderately high stringency. Evidence is presented to suggest that this gene may be environmentally regulated in L. pneumophila.
KeywordMeSH Terms
Bacterial Proteins
Porins
43. Wong  S, Pabbaraju  K, Burk  VF, Broukhanski  GC, Fox  J, Louie  T, Mah  MW, Bernard  K, Tilley  PA,     ( 2006 )

Use of sequence-based typing for investigation of a case of nosocomial legionellosis.

Journal of medical microbiology 55 (Pt 12)
PMID : 17108275  :   DOI  :   10.1099/jmm.0.46754-0    
Abstract >>
A fatal case of nosocomial legionellosis in a low prevalence region (Calgary, Alberta, Canada) prompted investigation into the source of infection. Hospital water systems contaminated with Legionella pneumophila have been shown to pose a risk to compromised patients. Typing of an L. pneumophila serogroup 1 strain isolated from the patient using sequence-based typing (SBT) and amplified fragment length polymorphism (AFLP) analysis linked it to a persistent and widespread strain isolated from the hospital water system establishing a nosocomial mode of acquisition. Different SBT and AFLP patterns were determined for non-epidemiologically linked cases and isolates from different hospitals.
KeywordMeSH Terms
44. Murata  T, Delprato  A, Ingmundson  A, Toomre  DK, Lambright  DG, Roy  CR,     ( 2006 )

The Legionella pneumophila effector protein DrrA is a Rab1 guanine nucleotide-exchange factor.

Nature cell biology 8 (9)
PMID : 16906144  :   DOI  :   10.1038/ncb1463    
Abstract >>
The intracellular pathogen Legionella pneumophila avoids fusion with lysosomes and subverts membrane transport from the endoplasmic reticulum to create an organelle that supports bacterial replication. Transport of endoplasmic reticulum-derived vesicles to the Legionella-containing vacuole (LCV) requires bacterial proteins that are translocated into host cells by a type IV secretion apparatus called Dot/Icm. Recent observations have revealed recruitment of the host GTPase Rab1 to the LCV by a process requiring the Dot/Icm system. Here, a visual screen was used to identify L. pneumophila mutants with defects in Rab1 recruitment. One of the factors identified in this screen was DrrA, a new Dot/Icm substrate protein translocated into host cells. We show that DrrA is a potent and highly specific Rab1 guanine nucleotide-exchange factor (GEF). DrrA can disrupt Rab1-mediated secretory transport to the Golgi apparatus by competing with endogenous exchange factors to recruit and activate Rab1 on plasma membrane-derived organelles. These data establish that intracellular pathogens have the capacity to directly modulate the activation state of a specific member of the Rab family of GTPases and thus further our understanding of the mechanisms used by bacterial pathogens to manipulate host vesicular transport.
KeywordMeSH Terms
45. Newton  HJ, Sansom  FM, Bennett-Wood  V, Hartland  EL,     ( 2006 )

Identification of Legionella pneumophila-specific genes by genomic subtractive hybridization with Legionella micdadei and identification of lpnE, a gene required for efficient host cell entry.

Infection and immunity 74 (1��3��)
PMID : 16495539  :   DOI  :   10.1128/IAI.74.3.1683-1691.2006     PMC  :   PMC1418643    
Abstract >>
Legionella pneumophila is a ubiquitous environmental organism and a facultative intracellular pathogen of humans. To identify genes that may contribute to the virulence of L. pneumophila, we performed genomic subtractive hybridization between L. pneumophila serogroup 1 strain 02/41 and L. micdadei strain 02/42. A total of 144 L. pneumophila-specific clones were sequenced, revealing 151 genes that were absent in L. micdadei strain 02/42. Low-stringency Southern hybridization was used to determine the distribution of 41 sequences, representing 40 open reading frames (ORFs) with a range of putative functions among L. pneumophila isolates of various serogroups as well as strains of Legionella longbeachae, L. micdadei, Legionella gormanii, and Legionella jordanis. Twelve predicted ORFs were L. pneumophila specific, including the gene encoding the dot/icm effector, lepB, as well as several genes predicted to play a role in lipopolysaccharide biosynthesis and cell wall synthesis and several sequences with similarity to virulence-associated determinants. A further nine predicted ORFs were in all L. pneumophila serotypes tested and an isolate of L. gormanii. These included icmD, the 5' end of a pilMNOPQ locus, and two genes known to be upregulated during growth within macrophages, cadA2 and ceaA. Disruption of an L. pneumophila-specific gene (lpg2222 locus tag) encoding a putative protein with eight tetratricopeptide repeats resulted in reduced entry into the macrophage-like cell line, THP-1, and the type II alveolar epithelial cell line, A549. The gene was subsequently renamed lpnE, for "L. pneumophila entry." In summary, this investigation has revealed important genetic differences between L. pneumophila and other Legionella species that may contribute to the phenotypic and clinical differences observed within this genus.
KeywordMeSH Terms
Genome, Bacterial
46. Allard  KA, Viswanathan  VK, Cianciotto  NP,     ( 2006 )

lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin.

Journal of bacteriology 188 (4)
PMID : 16452417  :   DOI  :   10.1128/JB.188.4.1351-1363.2006     PMC  :   PMC1367248    
Abstract >>
Under iron stress, Legionella pneumophila secretes legiobactin, a nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. Here, we have optimized conditions for legiobactin expression, shown its biological activity, and identified two genes, lbtA and lbtB, which are involved in legiobactin production. lbtA appears to be iron repressed and encodes a protein that has significant homology with siderophore synthetases, and FrgA, a previously described iron-regulated protein of L. pneumophila. lbtB encodes a protein homologous with members of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtA or lbtB were defective for legiobactin, producing 40 to 70% less CAS reactivity in deferrated chemically defined medium (CDM). In bioassays, mutant CDM culture supernatants, unlike those of the wild type, did not support growth of iron-limited wild-type bacteria in 2',2'-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant on BCYE agar without added iron. The lbtA mutant was modestly defective for growth in deferrated CDM containing the iron chelator citrate, indicating that legiobactin is required in conditions of severe iron limitation. Complementation of the lbt mutants restored both siderophore expression, as measured by the CAS assay and bioassays, and bacterial growth in deferrated, citrate-containing media. The lbtA mutant replicated as the wild type did in macrophages, amoebae, and the lungs of mice. However, L. pneumophila expresses lbtA in the macrophage, suggesting that legiobactin, though not required, may play a dispensable role in intracellular growth. The discovery of lbtAB represents the first identification of genes required for L. pneumophila siderophore expression.
KeywordMeSH Terms
Genes, Bacterial
47. Chang  B, Kura  F, Amemura-Maekawa  J, Koizumi  N, Watanabe  H,     ( 2005 )

Identification of a novel adhesion molecule involved in the virulence of Legionella pneumophila.

Infection and immunity 73 (7)
PMID : 15972519  :   DOI  :   10.1128/IAI.73.7.4272-4280.2005     PMC  :   PMC1168565    
Abstract >>
Legionella pneumophila is an intracellular bacterium, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we sought genes that are involved in virulence by screening a genomic DNA library of an L. pneumophila strain, 80-045, with convalescent-phase sera of Legionnaires' disease patients. Three antigens that reacted exclusively with the convalescent-phase sera were isolated. One of them, which shared homology with an integrin analogue of Saccharomyces cerevisiae, was named L. pneumophila adhesion molecule homologous with integrin analogue of S. cerevisiae (LaiA). The laiA gene product was involved in L. pneumophila adhesion to and invasion of the human lung alveolar epithelial cell line A549 during in vitro coculture. However, its presence did not affect multiplication of L. pneumophila within a U937 human macrophage cell line. Furthermore, after intranasal infection of A/J mice, the laiA mutant was eliminated from lungs and caused reduced mortality compared to the wild isolate. Thus, we conclude that the laiA gene encodes a virulence factor that is involved in transmission of L. pneumophila 80-045 and may play a role in Legionnaires' disease in humans.
KeywordMeSH Terms
48. Ko  KS, Miyamoto  H, Lee  HK, Park  MY, Fukuda  K, Park  BJ, Kook  YH,     ( 2006 )

Genetic diversity of Legionella pneumophila inferred from rpoB and dotA sequences.

Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 12 (3)
PMID : 16451413  :   DOI  :   10.1111/j.1469-0691.2005.01338.x    
Abstract >>
This study characterised the population structure of Legionella pneumophila by comparing the rpoB (300-bp) and dotA (360-bp) sequences of 267 isolates (18 reference strains, 149 Korean isolates and 100 Japanese isolates). In addition to the six clonal subgroups established previously, four subgroups, P-V to P-VIII, were identified. Subgroupings based on rpoB and dotA sequences were found to correlate with the source of the isolates, and this data may be useful for future epidemiological studies. Fourteen (five Korean and nine Japanese) isolates showed incongruent subgroupings in the rpoB and dotA trees, suggesting that genetic exchange among subgroups, and even among subspecies, may occur frequently in nature.
KeywordMeSH Terms
Genes, Bacterial
49. Amemura-Maekawa  J, Kura  F, Chang  B, Watanabe  H,     ( 2005 )

Legionella pneumophila serogroup 1 isolates from cooling towers in Japan form a distinct genetic cluster.

Microbiology and immunology 49 (12)
PMID : 16365527  :   DOI  :   10.1111/j.1348-0421.2005.tb03699.x    
Abstract >>
Thirty-one epidemiologically unrelated Legionella pneumophila serogroup 1 isolates (10 from cooling towers, 10 from public spas and/or hot spring baths, and 11 from patients) were analyzed by pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) using 6 loci, flaA, pilE, asd, mip, mompS, and proA. The results of PFGE and SBT analysis indicated that all 10 isolates from cooling towers clustered into a unique type, which was distinct from strains of other environmental sources.
KeywordMeSH Terms
Environmental Microbiology
50. Banerji  S, Bewersdorff  M, Hermes  B, Cianciotto  NP, Flieger  A,     ( 2005 )

Characterization of the major secreted zinc metalloprotease- dependent glycerophospholipid:cholesterol acyltransferase, PlaC, of Legionella pneumophila.

Infection and immunity 73 (5)
PMID : 15845496  :   DOI  :   10.1128/IAI.73.5.2899-2909.2005     PMC  :   PMC1087370    
Abstract >>
Legionella pneumophila, an intracellular pathogen causing a severe pneumonia, possesses distinct lipolytic activities which have not been completely assigned to specific enzymes so far. We cloned and characterized a gene, plaC, encoding a protein with high homology to PlaA, the major secreted lysophospholipase A of L. pneumophila and to other hydrolytic enzymes belonging to the GDSL family. Here we show that L. pneumophila plaC mutants possessed reduced phospholipase A and lysophospholipase A activities and lacked glycerophospholipid:cholesterol acyltransferase activity in their culture supernatants. The mutants' reduced phospholipase A and acyltransferase activities were complemented by reintroduction of an intact copy of plaC. Additionally, plaC conferred increased lysophospholipase A and glycerophospholipid:cholesterol acytransferase activities to recombinant Escherichia coli. Furthermore, PlaC was shown to be another candidate exported by the L. pneumophila type II secretion system and was activated by a factor present in the bacterial culture supernatant dependent on the zinc metalloprotease. Finally, the role of plaC in intracellular infection of Acanthamoeba castellanii and U937 macrophages with L. pneumophila was assessed, and plaC was found to be dispensable. Thus, L. pneumophila possesses another secreted lipolytic enzyme, a protein with acyltransferase, phospholipase A, and lysophospholipase A activities. This enzyme is distinguished from the previously characterized phospholipases A and lysophospholipases A by its capacity not only to cleave fatty acids from lipids but to transfer them to cholesterol. Cholesterol is an important compound of eukaryotic membranes, and an acyltransferase might be a tool for host cell modification to fit the needs of the bacterium.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
51. Buscher  BA, Conover  GM, Miller  JL, Vogel  SA, Meyers  SN, Isberg  RR, Vogel  JP,     ( 2005 )

The DotL protein, a member of the TraG-coupling protein family, is essential for Viability of Legionella pneumophila strain Lp02.

Journal of bacteriology 187 (9)
PMID : 15838018  :   DOI  :   10.1128/JB.187.9.2927-2938.2005     PMC  :   PMC1082803    
Abstract >>
Legionella pneumophila is able to survive inside phagocytic cells by an internalization route that bypasses fusion of the nascent phagosome with the endocytic pathway to allow formation of a replicative phagosome. The dot/icm genes, a major virulence system of L. pneumophila, encode a type IVB secretion system that is required for intracellular growth. One Dot protein, DotL, has sequence similarity to type IV secretion system coupling proteins (T4CPs). In other systems, coupling proteins are not required for viability of the organism. Here we report the first example of a strain, L. pneumophila Lp02, in which a putative T4CP is essential for viability of the organism on bacteriological media. This result is particularly surprising since the majority of the dot/icm genes in Lp02 are dispensable for growth outside of a host cell, a condition that does not require a functional Dot/Icm secretion complex. We were able to isolate suppressors of the Delta dotL lethality and found that many contained mutations in other components of the Dot/Icm secretion system. A systematic analysis of dot/icm deletion mutants revealed that the majority of them (20 of 26) suppressed the lethality phenotype, indicating a partially assembled secretion system may be the source of Delta dotL toxicity in the wild-type strain. These results are consistent with a model in which the DotL protein plays a role in regulating the activity of the L. pneumophila type IV secretion apparatus.
KeywordMeSH Terms
52. Edelstein  PH, Hu  B, Shinzato  T, Edelstein  MA, Xu  W, Bessman  MJ,     ( 2005 )

Legionella pneumophila NudA Is a Nudix hydrolase and virulence factor.

Infection and immunity 73 (10)
PMID : 16177332  :   DOI  :   10.1128/IAI.73.10.6567-6576.2005     PMC  :   PMC1230914    
Abstract >>
We studied the identity and function of the 528-bp gene immediately upstream of Legionella pneumophila F2310 ptsP (enzyme I(Ntr)). This gene, nudA, encoded for a Nudix hydrolase based on the inferred protein sequence. NudA had hydrolytic activity typical of other Nudix hydrolases, such as Escherichia coli YgdP, in that Ap(n)A's, in particular diadenosine pentaphosphate (Ap(5)A), were the preferred substrates. NudA hydrolyzed Ap(5)A to ATP plus ADP. Both ptsP and nudA were cotranscribed. Bacterial two-hybrid analysis showed no PtsP-NudA interactions. Gene nudA was present in 19 of 20 different L. pneumophila strains tested and in 5 of 10 different Legionella spp. other than L. pneumophila. An in-frame nudA mutation was made in L. pneumophila F2310 to determine the phenotype. The nudA mutant was an auxotroph that grew slowly in liquid and on solid media and had a smaller colony size than its parent. In addition, the mutant was more salt resistant than its parent and grew very poorly at 25 degrees C; all of these characteristics, as well as auxotrophy and slow-growth rate, were reversed by transcomplementation with nudA. The nudA mutant was outcompeted by about fourfold by the parent in competition studies in macrophages; transcomplementation almost completely restored this defect. Competition studies in guinea pigs with L. pneumophila pneumonia showed that the nudA mutant was outcompeted by its parent in both lung and spleen. NudA is of major importance for resisting stress in L. pneumophila and is a virulence factor.
KeywordMeSH Terms
53. Shohdy  N, Efe  JA, Emr  SD, Shuman  HA,     ( 2005 )

Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking.

Proceedings of the National Academy of Sciences of the United States of America 102 (13)
PMID : 15781869  :   DOI  :   10.1073/pnas.0501315102     PMC  :   PMC555709    
Abstract >>
Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.
KeywordMeSH Terms
54. Miyake  M, Watanabe  T, Koike  H, Molmeret  M, Imai  Y, Abu Kwaik  Y,     ( 2005 )

Characterization of Legionella pneumophila pmiA, a gene essential for infectivity of protozoa and macrophages.

Infection and immunity 73 (10)
PMID : 16177298  :   DOI  :   10.1128/IAI.73.10.6272-6282.2005     PMC  :   PMC1230894    
Abstract >>
The ability of Legionella pneumophila to cause pneumonia is dependent on intracellular replication within alveolar macrophages. The Icm/Dot secretion apparatus is essential for the ability of L. pneumophila to evade endocytic fusion, to remodel the phagosome by the endoplasmic reticulum (ER), and to replicate intracellularly. Protozoan and macrophage infectivity (pmi) mutants of L. pneumophila, which include 11 dot/icm mutants, exhibit defects in intracellular growth and replication within both protozoa and macrophages. In this study we characterized one of the pmi loci, pmiA. In contrast to the parental strain, the pmiA mutant is defective in cytopathogenicity for protozoa and macrophages. This is a novel mutant that exhibits a partial defect in survival within U937 human macrophage-like cells but exhibits a severe growth defect within Acanthamoeba polyphaga, which results in elimination from this host. The intracellular defects of this mutant are complemented by the wild-type pmiA gene on a plasmid. In contrast to phagosomes harboring the wild-type strain, which exclude endosomal-lysosomal markers, the pmiA mutant-containing phagosomes acquire the late endosomal-lysosomal markers LAMP-1 and LAMP-2. In contrast to the parental strain-containing phagosomes that are remodeled by the ER, there was a decrease in the number of ER-remodeled phagosomes harboring the pmiA mutant. Among several Legionella species examined, the pmiA gene is specific for L. pneumophila. The predicted amino acid sequence of the PmiA protein suggests that it is a transmembrane protein with three membrane-spanning regions. PmiA is similar to several hypothetical proteins produced by bacteria with a type IV secretion apparatus. Importantly, the defect in pmiA abolishes the pore-forming activity, which has been attributed to the Icm/Dot type IV secretion system. However, the mutant is sensitive to NaCl, and this sensitivity is abrogated in the icm/dot mutants. These results suggest that PmiA is a novel virulence factor that is involved in intracellular survival and replication of L. pneumophila in macrophages and protozoan cells.
KeywordMeSH Terms
55. Rossier  O, Cianciotto  NP,     ( 2005 )

The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection.

Infection and immunity 73 (4)
PMID : 15784543  :   DOI  :   10.1128/IAI.73.4.2020-2032.2005     PMC  :   PMC1087389    
Abstract >>
Our previous mutational analysis of Legionella pneumophila demonstrated a role for type II protein (Lsp) secretion and iron acquisition in intracellular infection and virulence. In gram-negative bacteria, the twin-arginine translocation (Tat) pathway is involved in secretion of proteins, including components of respiratory complexes, across the inner membrane to the periplasm. To assess the significance of Tat for L. pneumophila, tatB mutants were characterized. The mutants exhibited normal growth in standard media but grew slowly under low-iron conditions. They were also impaired in the Nadi assay, indicating that the function of cytochrome c oxidase is influenced by tatB. Consistent with this observation, a subunit of the cytochrome c reductase was shown to be a Tat substrate. Supernatants of the tatB mutants showed a 30% reduction in phospholipase C activity while maintaining normal levels of other Lsp secreted activities. When tested for infection of U937 macrophages, the tatB mutants showed a 10-fold reduction in growth. Double mutants lacking tatB and Lsp secretion were even more defective, suggesting tatB has an intracellular role that is independent of Lsp. tatB mutants were also impaired 20-fold in Hartmannella vermiformis amoebae cultured in the presence of an iron chelator. All mutant phenotypes were complemented by reintroduction of an intact tatB. Thus, L. pneumophila tatB plays a role in the formation of a respiratory complex, growth under low-iron conditions, the secretion of a phospholipase C activity, and intracellular infection.
KeywordMeSH Terms
56. Ninio  S, Zuckman-Cholon  DM, Cambronne  ED, Roy  CR,     ( 2005 )

The Legionella IcmS-IcmW protein complex is important for Dot/Icm-mediated protein translocation.

Molecular microbiology 55 (3)
PMID : 15661013  :   DOI  :   10.1111/j.1365-2958.2004.04435.x    
Abstract >>
The intracellular pathogen Legionella pneumophila can infect and replicate within macrophages of a human host. To establish infection, Legionella require the Dot/Icm secretion system to inject protein substrates directly into the host cell cytoplasm. The mechanism by which substrate proteins are engaged and translocated by the Dot/Icm system is not well understood. Here we show that two cytosolic components of the Dot/Icm secretion machinery, the proteins IcmS and IcmW, play an important role in substrate translocation. Biochemical analysis indicates that IcmS and IcmW form a stable protein complex. In Legionella, the IcmW protein is rapidly degraded in the absence of the IcmS protein. Substrate proteins translocated into mammalian host cells by the Dot/Icm system were identified using the IcmW protein as bait in a yeast two-hybrid screen. It was determined that the IcmS-IcmW complex interacts with these substrates and plays an important role in translocation of these proteins into mammalian cells. These data are consistent with the IcmS-IcmW complex being involved in the recognition and Dot/Icm-dependent translocation of substrate proteins during Legionella infection of host cells.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
57. Aurell  H, Farge  P, Meugnier  H, Gouy  M, Forey  F, Lina  G, Vandenesch  F, Etienne  J, Jarraud  S,     ( 2005 )

Clinical and environmental isolates of Legionella pneumophila serogroup 1 cannot be distinguished by sequence analysis of two surface protein genes and three housekeeping genes.

Applied and environmental microbiology 71 (1)
PMID : 15640199  :   DOI  :   10.1128/AEM.71.1.282-289.2005     PMC  :   PMC544207    
Abstract >>
We used gene sequencing to determine whether clinical (sporadic, epidemic, and endemic) and environmental isolates of Legionella pneumophila serogroup (sg) 1 belong to specific lineages. A total of 178 clinical and environmental L. pneumophila sg 1 isolates, defined by pulsed-field gel electrophoresis and epidemiological data as sporadic, epidemic, or endemic, were analyzed for polymorphisms in five gene fragments. The fragments belonged to three housekeeping genes (coding for aconitase [acn], aspartate-beta-semialdehyde dehydrogenase [asd], and RNA polymerase beta subunit [rpoB]) and two surface protein genes (coding for the macrophage infectivity potentiator [mip] and the major outer membrane protein [mompS]). The phylogenetic tree inferred from sequence polymorphisms of the five genes identified two large clusters, one consisting of 133 poorly differentiated strains and containing two smaller clusters (10 and 2 strains) unrelated to each other and the other consisting of 42 strains. Clinical and environmental isolates could not be distinguished on this basis, and no link between genetic background and epidemiological type was found, suggesting that other factors are responsible for differences in pathogenicity.
KeywordMeSH Terms
Sequence Analysis, DNA
Water Microbiology
58. VanRheenen  SM, Duménil  G, Isberg  RR,     ( 2004 )

IcmF and DotU are required for optimal effector translocation and trafficking of the Legionella pneumophila vacuole.

Infection and immunity 72 (10)
PMID : 15385501  :   DOI  :   10.1128/IAI.72.10.5972-5982.2004     PMC  :   PMC517542    
Abstract >>
The gram-negative bacterium Legionella pneumophila causes a severe form of pneumonia called Legionnaires' disease, characterized by bacterial replication within alveolar macrophages. Prior to intracellular replication, the vacuole harboring the bacterium must first escape trafficking to the host lysosome, a process that is dependent on the Dot/Icm type IV secretion system. To identify genes required for intracellular growth, bacterial mutants were isolated that were delayed in escape from the macrophage but which retain a minimally functional Dot/Icm machinery. The mutations were found in eight distinct genes, including three genes known to be required for optimal intracellular growth. Two of these genes, icmF and dotU, are located at one end of a cluster of genes that encode the type IV secretion system, yet both icmF and dotU lack orthologs in other type IV translocons. DotU protein is degraded in the early postexponential phase in wild-type L. pneumophila and at all growth phases in an icmF mutant. IcmF contains an extracytoplasmic domain(s) based on accessibility to a membrane-impermeant amine-reactive reagent. In the absence of either gene, L. pneumophila targets inappropriately to LAMP-1-positive compartments during macrophage infection, is defective in the formation of replicative vacuoles, and is impaired in the translocation of the effector protein SidC. Therefore, although IcmF and DotU do not appear to be part of the core type IV secretion system, these proteins are necessary for an efficiently functioning secretion apparatus.
KeywordMeSH Terms
59. Amor  JC, Swails  J, Zhu  X, Roy  CR, Nagai  H, Ingmundson  A, Cheng  X, Kahn  RA,     ( 2005 )

The structure of RalF, an ADP-ribosylation factor guanine nucleotide exchange factor from Legionella pneumophila, reveals the presence of a cap over the active site.

The Journal of biological chemistry 280 (2)
PMID : 15520000  :   DOI  :   10.1074/jbc.M410820200    
Abstract >>
The Legionella pneumophila protein RalF is secreted into host cytosol via the Dot/Icm type IV transporter where it acts to recruit ADP-ribosylation factor (Arf) to pathogen-containing phagosomes in the establishment of a replicative organelle. The presence in RalF of the Sec7 domain, present in all Arf guanine nucleotide exchange factors, has suggested that recruitment of Arf is an early step in pathogenesis. We have determined the crystal structure of RalF and of the isolated Sec7 domain and found that RalF is made up of two domains. The Sec7 domain is homologous to mammalian Sec7 domains. The C-terminal domain forms a cap over the active site in the Sec7 domain and contains a conserved folding motif, previously observed in adaptor subunits of vesicle coat complexes. The importance of the capping domain and of the glutamate in the "glutamic finger," conserved in all Sec7 domains, to RalF functions was examined using three different assays. These data highlight the functional importance of domains other than Sec7 in Arf guanine nucleotide exchange factors to biological activities and suggest novel mechanisms of regulation of those activities.
KeywordMeSH Terms
60. Roth  SB, Jalava  J, Ruuskanen  O, Ruohola  A, Nikkari  S,     ( 2004 )

Use of an oligonucleotide array for laboratory diagnosis of bacteria responsible for acute upper respiratory infections.

Journal of clinical microbiology 42 (9)
PMID : 15365022  :   DOI  :   10.1128/JCM.42.9.4268-4274.2004     PMC  :   PMC516371    
Abstract >>
We developed a diagnostic array of oligonucleotide probes targeting species-specific variable regions of the genes encoding topoisomerases GyrB and ParE of respiratory bacterial pathogens. Suitable broad-range primer sequences were designed based on alignment of gyrB/parE sequences from nine different bacterial species. These species included Corynebacterium diphtheriae, Fusobacterium necrophorum, Haemophilus influenzae, Legionella pneumophila, Moraxella catarrhalis, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. Specific probe sequences were selected by comparative analysis against the European Bioinformatics Database, as well as gyrB/parE sequences generated for this study. To verify specificity, at least six initial oligonucleotide probe sequences per bacterial species were tested by hybridization on a solid glass support using culture collection strains as templates. Finally, three oligonucleotide probes per bacterial species were utilized to examine 65 middle ear fluid and 29 throat swab samples. The sensitivities of the developed assay compared to classic culture from middle ear fluid samples for H. influenzae, M. catarrhalis, and S. pneumoniae were 96 (93 for culture), 73 (93 for culture), and 100% (78% for culture), respectively. No cross-reactivity with bacterial species belonging to the normal oral flora was observed when the 29 throat swab samples were studied. The sensitivity of the assay to detect S. pyogenes from these samples was 93% (80% for culture). These results provide a proof of concept for the diagnostic use of microarray technology based on broad-range topoisomerase gene amplification, followed by hybridization and specific detection of bacterial species.
KeywordMeSH Terms
61. Zusman  T, Feldman  M, Halperin  E, Segal  G,     ( 2004 )

Characterization of the icmH and icmF genes required for Legionella pneumophila intracellular growth, genes that are present in many bacteria associated with eukaryotic cells.

Infection and immunity 72 (6)
PMID : 15155646  :   DOI  :   10.1128/IAI.72.6.3398-3409.2004     PMC  :   PMC415720    
Abstract >>
Legionella pneumophila, the causative agent of Legionnaires' disease, replicates intracellularly within a specialized phagosome of mammalian and protozoan host cells, and the Icm/Dot type IV secretion system has been shown to be essential for this process. Unlike all the other known Icm/Dot proteins, the IcmF protein, which was described before, and the IcmH protein, which is characterized here, have homologous proteins in many bacteria (such as Yersinia pestis, Salmonella enterica, Rhizobium leguminosarum, and Vibrio cholerae), all of which associate with eukaryotic cells. Here, we have characterized the L. pneumophila icmH and icmF genes and found that both genes are present in 16 different Legionella species examined. The icmH and icmF genes were found to be absolutely required for intracellular multiplication in Acanthamoeba castellanii and partially required for intracellular growth in HL-60-derived human macrophages, for immediate cytotoxicity, and for salt sensitivity. Mutagenesis of the predicted ATP/GTP binding site of IcmF revealed that the site is partially required for intracellular growth in A. castellanii. Analysis of the regulatory region of the icmH and icmF genes, which were found to be cotranscribed, revealed that it contains at least two regulatory elements. In addition, an icmH::lacZ fusion was shown to be activated during stationary phase in a LetA- and RelA-dependent manner. Our results indicate that although the icmH and icmF genes probably have a different evolutionary origin than the rest of the icm/dot genes, they are part of the icm/dot system and are required for L. pneumophila pathogenesis.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
62. Bachman  MA, Swanson  MS,     ( 2004 )

The LetE protein enhances expression of multiple LetA/LetS-dependent transmission traits by Legionella pneumophila.

Infection and immunity 72 (6)
PMID : 15155631  :   DOI  :   10.1128/IAI.72.6.3284-3293.2004     PMC  :   PMC415668    
Abstract >>
Legionella pneumophila colonizes freshwater amoebae and can also replicate within alveolar macrophages. When their nutrient supply is exhausted, replicating bacteria become cytotoxic, motile, and infectious, which is thought to promote transmission to a new amoeba. The differentiation of L. pneumophila is coordinated by the sigma factors RpoS and FliA and the two-component regulator LetA/LetS and is enhanced by the letE locus. Here we demonstrate that letE promotes motility by increasing expression of the flagellin gene flaA but has little impact on the transcription of fliA, the flagellar sigma factor gene. In addition to promoting motility, letE induces the characteristic shape, pigment, and heat resistance of stationary-phase L. pneumophila. To gain insight into how letE promotes the expression of the transmission phenotype, we designed molecular genetic experiments to discriminate between the following three models: letE mutations are polar on milX; letE encodes a small novel protein; or, by analogy to csrB, letE encodes a regulatory RNA that sequesters CsrA to relieve repression. We report that letE encodes an activator protein, as it does not complement an Escherichia coli csrB mutant, it directs the synthesis of an approximately 12-kDa polypeptide, and a letE nonsense mutation eliminates function. A monocistronic letE RNA is abundant during the exponential phase, and its decay during the stationary phase requires RpoS and LetA/LetS. We also discuss how the LetE protein may interact with LetA/LetS and CsrA to enhance L. pneumophila differentiation to a transmissible form.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
63. Lammertyn  E, Van Mellaert  L, Meyen  E, Lebeau  I, De Buck  E, Anné  J, Geukens  N,     ( 2004 )

Molecular and functional characterization of type I signal peptidase from Legionella pneumophila.

Microbiology (Reading, England) 150 (Pt 5)
PMID : 15133109  :   DOI  :   10.1099/mic.0.26973-0    
Abstract >>
Legionella pneumophila is a facultative intracellular Gram-negative rod-shaped bacterium that has become an important cause of both community-acquired and nosocomial pneumonia. Numerous studies concerning the unravelling of the virulence mechanism of this important pathogen have been initiated. As evidence is now accumulating for the involvement of protein secretion systems in bacterial virulence in general, the type I signal peptidase (LepB) of L. pneumophila was of particular interest. This endopeptidase plays an essential role in the processing of preproteins carrying a typical amino-terminal signal peptide, upon translocation across the cytoplasmic membrane. This paper reports the cloning and the transcriptional analysis of the L. pneumophila lepB gene encoding the type I signal peptidase (SPase). Reverse transcription PCR experiments showed clear lepB expression when L. pneumophila was grown both in culture medium, and also intracellularly in Acanthamoeba castellanii, a natural eukaryotic host of L. pneumophila. In addition, LepB was shown to be encoded by a polycistronic mRNA transcript together with two other proteins, i.e. a LepA homologue and a ribonuclease III homologue. SPase activity of the LepB protein was demonstrated by in vivo complementation analysis in a temperature-sensitive Escherichia coli lepB mutant. Protein sequence and predicted membrane topology were compared to those of leader peptidases of other Gram-negative human pathogens. Most strikingly, a strictly conserved methionine residue in the substrate binding pocket was replaced by a leucine residue, which might influence substrate recognition. Finally it was shown by in vivo experiments that L. pneumophila LepB is a target for (5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate, a specific inhibitor of type I SPases.
KeywordMeSH Terms
Cloning, Molecular
Transcription, Genetic
64. Flieger  A, Rydzewski  K, Banerji  S, Broich  M, Heuner  K,     ( 2004 )

Cloning and characterization of the gene encoding the major cell-associated phospholipase A of Legionella pneumophila, plaB, exhibiting hemolytic activity.

Infection and immunity 72 (5)
PMID : 15102773  :   DOI  :   10.1128/iai.72.5.2648-2658.2004     PMC  :   PMC387885    
Abstract >>
Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular pathogen of amoebae, macrophages, and epithelial cells. The pathology of Legionella infections involves alveolar cell destruction, and several proteins of L. pneumophila are known to contribute to this ability. By screening a genomic library of L. pneumophila, we found an additional L. pneumophila gene, plaB, which coded for a hemolytic activity and contained a lipase consensus motif in its deduced protein sequence. Moreover, Escherichia coli harboring the L. pneumophila plaB gene showed increased activity in releasing fatty acids predominantly from diacylphospho- and lysophospholipids, demonstrating that it encodes a phospholipase A. It has been reported that culture supernatants and cell lysates of L. pneumophila possess phospholipase A activity; however, only the major secreted lysophospholipase A PlaA has been investigated on the molecular level. We therefore generated isogenic L. pneumophila plaB mutants and tested those for hemolysis, lipolytic activities, and intracellular survival in amoebae and macrophages. Compared to wild-type L. pneumophila, the plaB mutant showed reduced hemolysis of human red blood cells and almost completely lost its cell-associated lipolytic activity. We conclude that L. pneumophila plaB is the gene encoding the major cell-associated phospholipase A, possibly contributing to bacterial cytotoxicity due to its hemolytic activity. On the other hand, in view of the fact that the plaB mutant multiplied like the wild type both in U937 macrophages and in Acanthamoeba castellanii amoebae, plaB is not essential for intracellular survival of the pathogen.
KeywordMeSH Terms
Genes, Bacterial
65. De Buck  E, Lebeau  I, Maes  L, Geukens  N, Meyen  E, Van Mellaert  L, Anné  J, Lammertyn  E,     ( 2004 )

A putative twin-arginine translocation pathway in Legionella pneumophila.

Biochemical and biophysical research communications 317 (2)
PMID : 15063808  :   DOI  :   10.1016/j.bbrc.2004.03.091    
Abstract >>
Legionella pneumophila is a facultative intracellular human pathogen causing Legionnaires' disease, a severe form of pneumonia. Because of the importance of secretion pathways in virulence, we were interested in the possible presence of the twin-arginine translocation (Tat) pathway in L. pneumophila. This secretion pathway is used to transport folded proteins, characterized by two arginines in their signal peptide, across the cytoplasmic membrane. We describe here the presence of a putative Tat pathway in L. pneumophila. Three genes encoding Escherichia coli TatA, TatB, and TatC homologues were identified. The tatA and tatB genes were shown to constitute an operon while tatC is monocistronic. RT-PCR analysis revealed expression of the tat genes during both exponential and stationary growth as well as during intracellular growth in Acanthamoeba castellanii. A search for the conserved twin-arginine motif in predicted signal peptides resulted in a list of putative Tat substrates.
KeywordMeSH Terms
Sequence Homology, Amino Acid
66. Jacobi  S, Schade  R, Heuner  K,     ( 2004 )

Characterization of the alternative sigma factor sigma54 and the transcriptional regulator FleQ of Legionella pneumophila, which are both involved in the regulation cascade of flagellar gene expression.

Journal of bacteriology 186 (9)
PMID : 15090493  :   DOI  :   10.1128/jb.186.9.2540-2547.2004     PMC  :   PMC387802    
Abstract >>
We cloned and analyzed Legionella pneumophila Corby homologs of rpoN (encoding sigma(54)) and fleQ (encoding sigma(54) activator protein). Two other genes (fleR and pilR) whose products have a sigma(54) interaction domain were identified in the genome sequence of L. pneumophila. An rpoN mutant strain was nonflagellated and expressed very small amounts of the FlaA (flagellin) protein. Like the rpoN mutant, the fleQ mutant strain of L. pneumophila was also nonflagellated and expressed only small amounts of FlaA protein compared to the amounts expressed by the wild type. In this paper we show that the sigma(54) factor and the FleQ protein are involved in regulation of flagellar gene operons in L. pneumophila. RpoN and FleQ positively regulate the transcription of FliM and FleN, both of which have a sigma(54)-dependent promoter consensus sequence. However, they seemed to be dispensable for transcription of flaA, fliA, or icmR. Our results confirmed a recently described model of the flagellar gene regulation cascade in L. pneumophila (K. Heuner and M. Steinert, Int. J. Med. Microbiol. 293:133-145, 2003). Flagellar gene regulation was found to be different from that of Enterobacteriaceae but seems to be comparable to that described for Pseudomonas or Vibrio spp.
KeywordMeSH Terms
DNA-Binding Proteins
Gene Expression Regulation, Bacterial
67. Morozova  I, Qu  X, Shi  S, Asamani  G, Greenberg  JE, Shuman  HA, Russo  JJ,     ( 2004 )

Comparative sequence analysis of the icm/dot genes in Legionella.

Plasmid 51 (2)
PMID : 15003709  :   DOI  :   10.1016/j.plasmid.2003.12.004    
Abstract >>
The icm/dot genes in Legionella pneumophila are essential for the ability of the bacteria to survive within macrophages in lung infections such as Legionnaires' disease, or amoebae in nature. The 22 genes of the complex, thought to encode a transport apparatus for transfer of effector molecules into the host cell cytoplasm, are located in two chromosomal loci. We demonstrate that these genes are present in all the L. pneumophila strains examined herein, but display a wide range of sequence variation among the different strains, none of which are clearly associated with virulence potential. The strains fall within seven phylogenetic groups, but discrepancies among the gene trees indicate a complicated evolutionary history for the icm/dot loci, with perhaps two independent gene acquisition events and subsequent genomic rearrangements. Significant findings include a probable t-SNARE domain in IcmG that may indicate a direct role for this putative inner membrane protein in altering the host's membrane fusion machinery, a potential functional domain in the central hydrophobic portion of IcmK that may allow it to participate in forming the pore of the secretion complex, and strict conservation of the amino acid physicochemical characteristics in the IcmP region corresponding to the trbA domain that could play a role in molecular transfer.
KeywordMeSH Terms
68. Chen  J, de Felipe  KS, Clarke  M, Lu  H, Anderson  OR, Segal  G, Shuman  HA,     ( 2004 )

Legionella effectors that promote nonlytic release from protozoa.

Science (New York, N.Y.) 303 (5662)
PMID : 14988561  :   DOI  :   10.1126/science.1094226    
Abstract >>
Legionella pneumophila, the bacterial agent of legionnaires' disease, replicates intracellularly within a specialized vacuole of mammalian and protozoan host cells. Little is known about the specialized vacuole except that the Icm/Dot type IV secretion system is essential for its formation and maintenance. The Legionella genome database contains two open reading frames encoding polypeptides (LepA and LepB) with predicted coiled-coil regions and weak homology to SNAREs; these are delivered to host cells by an Icm/Dot-dependent mechanism. Analysis of mutant strains suggests that the Lep proteins may enable the Legionella to commandeer a protozoan exocytic pathway for dissemination of the pathogen.
KeywordMeSH Terms
69. 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
70. Luo  ZQ, Isberg  RR,     ( 2004 )

Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer.

Proceedings of the National Academy of Sciences of the United States of America 101 (3)
PMID : 14715899  :   DOI  :   10.1073/pnas.0304916101     PMC  :   PMC321768    
Abstract >>
Legionella pneumophila is an intracellular pathogen that multiplies in a specialized vacuole within host cells. Biogenesis of this vacuole requires the Dot/Icm type IV protein translocation system. By using a Cre/loxP-based protein translocation assay, we found that proteins translocated by the Dot/Icm complex across the host phagosomal membrane can also be transferred from one bacterial cell to another. The flexibility of this system allowed the identification of several families of proteins translocated by the Dot/Icm complex. When analyzed by immunofluorescence microscopy, a protein identified by this procedure, SidC, was shown to translocate across the phagosomal membranes to the cytoplasmic face of the L. pneumophila phagosome. The identification of large numbers of these substrates, and the fact that the absence of any one substrate rarely results in strong defects in intracellular growth, indicate that there is significant functional redundancy among the Dot/Icm translocation targets.
KeywordMeSH Terms
71. Rossier  O, Starkenburg  SR, Cianciotto  NP,     ( 2004 )

Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires' disease pneumonia.

Infection and immunity 72 (1)
PMID : 14688110  :   DOI  :   10.1128/iai.72.1.310-321.2004     PMC  :   PMC344012    
Abstract >>
Legionella pneumophila, the gram-negative agent of Legionnaires' disease, possesses type IV pili and a type II protein secretion (Lsp) system, both of which are dependent upon the PilD prepilin peptidase. By analyzing multiple pilD mutants and various types of Lsp mutants as well as performing trans-complementation of these mutants, we have confirmed that PilD and type II secretion genes are required for L. pneumophila infection of both amoebae and human macrophages. Based upon a complete analysis of lspDE, lspF, and lspG mutants, we found that the type II system controls the secretion of protease, RNase, lipase, phospholipase A, phospholipase C, lysophospholipase A, and tartrate-sensitive and tartrate-resistant acid phosphatase activities and influences the appearance of colonies. Examination of the developing L. pneumophila genome database indicated that the organism has two other loci (lspC and lspLM) that are predicted to promote secretion and thus a set of genes that is comparable to the type II secretion genes in other gram-negative bacteria. In contrast to lsp mutants, L. pneumophila pilus mutants lacking either the PilQ secretin, the PspA pseudopilin, or pilin were not defective for colonial growth, secreted activities, or intracellular replication. L. pneumophila dot/icm mutants were also not impaired for type II-dependent exoenzymes. Upon intratracheal inoculation into A/J mice, lspDE, lspF, and pilD mutants, but not pilus mutants, exhibited a reduced ability to grow in the lung, as measured by competition assays. The lspF mutant was also defective in an in vivo kinetic assay. Examination of infected mouse sera revealed that type II secreted proteins are expressed in vivo. Thus, the L. pneumophila Lsp system is a virulence factor and the only type II secretion system linked to intracellular infection.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
72. Lim  YH, Relus Kek  YL, Lim  PY, Yap  HM, Vivien Goh  TL, Ng  LC,     ( 2011 )

Environmental surveillance and molecular characterization of Legionella in tropical Singapore.

Tropical biomedicine 28 (1)
PMID : 21602781  :  
Abstract >>
Legionnaires' disease is often acquired by inhalation of legionellae from a contaminated environmental source. In recent years, Singapore has seen an increase in the use of aerosol-generating fixtures such as mist fans and spa pools. Poorly maintained and designed water fixtures could pose a public health threat to the community. In this study, we provided an update on the prevalence of Legionella in mist fans (N=28), household water heaters with storage tanks (N=19) and instantaneous heaters (N=30); and extended the survey to spa pools (N=29) and aerosol-generating fixtures in nursing homes (N=116). The prevalence of Legionella were 21.1% in water heaters with storage tanks, 24.1% in spa pools, 14.2% in mist fans and 3.3% in instantaneous heaters. Legionella was not detected in nursing homes. A total of 37 isolates were subjected to molecular characterization using Sequence-Based Typing (SBT) protocol from the European Working Group on Legionella Infections (EWGLI). This is the first study on the use of SBT protocol on environmental strains isolated from tropical South East Asia. The Legionella flora was very heterogenous. The overall diversity of the allelic profile was found to be 0.970 (95% CI 0.946 - 0.994). All known STs of our isolates have been associated with clinical cases in EWGLI database. The phylogenetic analysis showed that our novel environmental isolates were clustered with clinical STs that were previously reported in Europe, Japan, United Kingdom and United States etc. (in EWGLI database), suggesting that Legionella found in the environment of Singapore may potentially cause human disease.
KeywordMeSH Terms
Water Microbiology
73. Fong  DH, Xiong  B, Hwang  J, Berghuis  AM,     ( 2011 )

Crystal structures of two aminoglycoside kinases bound with a eukaryotic protein kinase inhibitor.

PloS one 6 (5)
PMID : 21573013  :   DOI  :   10.1371/journal.pone.0019589     PMC  :   PMC3090406    
Abstract >>
Antibiotic resistance is recognized as a growing healthcare problem. To address this issue, one strategy is to thwart the causal mechanism using an adjuvant in partner with the antibiotic. Aminoglycosides are a class of clinically important antibiotics used for the treatment of serious infections. Their usefulness has been compromised predominantly due to drug inactivation by aminoglycoside-modifying enzymes, such as aminoglycoside phosphotransferases or kinases. These kinases are structurally homologous to eukaryotic Ser/Thr and Tyr protein kinases and it has been shown that some can be inhibited by select protein kinase inhibitors. The aminoglycoside kinase, APH(3')-IIIa, can be inhibited by CKI-7, an ATP-competitive inhibitor for the casein kinase 1. We have determined that CKI-7 is also a moderate inhibitor for the atypical APH(9)-Ia. Here we present the crystal structures of CKI-7-bound APH(3')-IIIa and APH(9)-Ia, the first structures of a eukaryotic protein kinase inhibitor in complex with bacterial kinases. CKI-7 binds to the nucleotide-binding pocket of the enzymes and its binding alters the conformation of the nucleotide-binding loop, the segment homologous to the glycine-rich loop in eukaryotic protein kinases. Comparison of these structures with the CKI-7-bound casein kinase 1 reveals features in the binding pockets that are distinct in the bacterial kinases and could be exploited for the design of a bacterial kinase specific inhibitor. Our results provide evidence that an inhibitor for a subset of APHs can be developed in order to curtail resistance to aminoglycosides.
KeywordMeSH Terms
74. Duncan  C, Prashar  A, So  J, Tang  P, Low  DE, Terebiznik  M, Guyard  C,     ( 2011 )

Lcl of Legionella pneumophila is an immunogenic GAG binding adhesin that promotes interactions with lung epithelial cells and plays a crucial role in biofilm formation.

Infection and immunity 79 (6)
PMID : 21422183  :   DOI  :   10.1128/IAI.01304-10     PMC  :   PMC3125840    
Abstract >>
Legionellosis is mostly caused by Legionella pneumophila and is defined by a severe respiratory illness with a case fatality rate ranging from 5 to 80%. In vitro and in vivo, interactions of L. pneumophila with lung epithelial cells are mediated by the sulfated glycosaminoglycans (GAGs) of the host extracellular matrix. In this study, we have identified several Legionella heparin binding proteins. We have shown that one of these proteins, designated Lcl, is a polymorphic adhesin of L. pneumophila that is produced during legionellosis. Homologues of Lcl are ubiquitous in L. pneumophila serogroups but are undetected in other Legionella species. Recombinant Lcl binds to GAGs, and a �Glpg2644 mutant demonstrated reduced binding to GAGs and human lung epithelial cells. Importantly, we showed that the �Glpg2644 strain is dramatically impaired in biofilm formation. These data delineate the role of Lcl in the GAG binding properties of L. pneumophila and provide molecular evidence regarding its role in L. pneumophila adherence and biofilm formation.
KeywordMeSH Terms
75. Zhou  G, Wen  S, Liu  Y, Li  R, Zhong  X, Feng  L, Wang  L, Cao  B,     ( 2011 )

Development of a DNA microarray for detection and identification of Legionella pneumophila and ten other pathogens in drinking water.

International journal of food microbiology 145 (1)
PMID : 21276629  :   DOI  :   10.1016/j.ijfoodmicro.2011.01.014    
Abstract >>
The safety and accessibility of drinking water are major concerns throughout the world. Consumption of water contaminated with infectious agents, toxic chemicals or radiological hazards represents a significant health risk and is strongly associated with mortality. Therefore, we have developed an oligonucleotide-based microarray using the sequences of 16S-23S rDNA internal transcribed spacer regions (ITS) and the gyrase subunit B gene (gyrB) found in the most prevalent and devastating waterborne pathogenic agents. This new diagnostic contains 26 specific probes and can simultaneously detect Aeromonas hydrophila, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Salmonella spp., Shigella spp., Staphylococcus aureus, Vibrio choleraeo, Vibrio parahaemolyticus, Yersinia enterocolitica and Leptospira interrogans. Testing was carried out against a total of 218 bacterial strains, including 53 representative strains, 103 clinical isolates and 62 strains of other bacterial species belonging to 10 genera and 48 species. The results were specific and reproducible, with a detection sensitivity of 0.1 ng DNA or 10(4)CFU/ml achieved for pure cultures of each target organism. The diluted cultures and real drinking water samples were tested by the microarray with 100% accuracy. This novel diagnostic method is superior in time- and labor-efficiency to conventional bacterial culture and antiserum agglutination, and can be readily applied to epidemiological surveillance and other food safety applications.
KeywordMeSH Terms
Water Microbiology
76. Zhao  X, Dreyfus  LA,     ( 1990 )

Expression and nucleotide sequence analysis of the Legionella pneumophila recA gene.

FEMS microbiology letters 58 (2)
PMID : 2121588  :   DOI  :   10.1111/j.1574-6968.1990.tb13983.x    
Abstract >>
The nucleotide sequence of the L pneumophila recA gene was determined. The coding region was 1044 nucleotides (348 codons), specifying a 37,934 Da protein. Preceding the recA gene was a tandem set of transcription regulatory sequences and putative LexA binding sites. When expressed in E. coli, the cloned recA gene yield two proteins with molecular weights of approximately 38,000 and 35,500 Da. The larger of these two proteins shared 70.4% and 74.6% identity with the E. coli and Pseudomonas aeruginosa RecA proteins, respectively. The 35,500 Da recA encoded protein was presumed to be the product of translation from Met26 which was preceded by an alternate ribosomal binding site.
KeywordMeSH Terms
Genes, Bacterial
77. Black  WJ, Quinn  FD, Tompkins  LS,     ( 1990 )

Legionella pneumophila zinc metalloprotease is structurally and functionally homologous to Pseudomonas aeruginosa elastase.

Journal of bacteriology 172 (5)
PMID : 2110146  :   DOI  :   10.1128/jb.172.5.2608-2613.1990     PMC  :   PMC208904    
Abstract >>
The sequence of the structural gene encoding the Legionella pneumophila extracellular zinc metalloprotease has been determined and was found to possess a single large open reading frame (ORF) of 1,629 nucleotides (nt). This ORF was preceded by consensus promoter (TTAACT . . . 17 nt . . . TATAAC) and ribosome-binding (TAAGGAG) sequences. The deduced polypeptide contained a putative signal sequence and a total of 543 amino acid residues with a computed molecular size of 60,775 daltons, substantially larger than the observed 38,000 daltons of the native and recombinant proteins. A homology search revealed extensive amino acid identity with Pseudomonas aeruginosa elastase, a protein that is also encoded by an ORF substantially larger than that predicted for the mature size of the protein. The structural identity between the L. pneumophila protease and P. aeruginosa elastase was most pronounced in the regions forming the enzymatic active site of elastase. Amino acid residues constituting the active-site cleft of elastase were greater than 75% conserved. Elastase residues that interact with and mediate proteolysis of substrate peptides were 100% conserved. Competitive inhibitors of elastase and the structurally and functionally related thermolysin (phosphoramidon and a phosphoramidate analog, Z-GlyP(O)Leu-Ala), were shown to be equally potent at inhibiting the proteolytic activity of the L. pneumophila protease. These inhibitor studies along with the amino acid sequence similarities provide strong evidence that the L. pneumophila protease and P. aeruginosa elastase share a similar molecular mechanism of proteolysis.
KeywordMeSH Terms
Bacterial Proteins
Metalloendopeptidases
78. Müller  MP, Peters  H, Blümer  J, Blankenfeldt  W, Goody  RS, Itzen  A,     ( 2010 )

The Legionella effector protein DrrA AMPylates the membrane traffic regulator Rab1b.

Science (New York, N.Y.) 329 (5994)
PMID : 20651120  :   DOI  :   10.1126/science.1192276    
Abstract >>
In the course of Legionnaires' disease, the bacterium Legionella pneumophila affects the intracellular vesicular trafficking of infected eukaryotic cells by recruiting the small guanosine triphosphatase (GTPase) Rab1 to the cytosolic face of the Legionella-containing vacuole. In order to accomplish this, the Legionella protein DrrA contains a specific guanine nucleotide exchange activity for Rab1 activation that exchanges guanosine triphosphate (GTP) for guanosine diphosphate on Rab1. We found that the amino-terminal domain of DrrA possesses adenosine monophosphorylation (AMPylation) activity toward the switch II region of Rab1b, leading to posttranslational covalent modification of tyrosine 77. AMPylation of switch II by DrrA restricts the access of GTPase activating proteins, thereby rendering Rab1b constitutively active.
KeywordMeSH Terms
79. Palusinska-Szysz  M, Janczarek  M, Kalitynski  R, Dawidowicz  AL, Russa  R,     ( 2011 )

Legionella bozemanae synthesizes phosphatidylcholine from exogenous choline.

Microbiological research 166 (2)
PMID : 20338739  :   DOI  :   10.1016/j.micres.2010.02.004    
Abstract >>
The phospholipid class and fatty acid composition of Legionella bozemanae were determined using thin-layer chromatography, gas-liquid chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were the predominant phospholipids, while phosphatidyl-N-monomethylethanolamine, phosphatidylglycerol, and phosphatidyl-N,N-dimethylethanolamine were present at low concentrations. With the use of the LC/MS technique, PC16:0/15:0, PC17:/15:0, and PE16:1/15:0 were shown to be the dominant phospholipid constituents, which may be taxonomically significant. Two independent phosphatidylcholine synthesis pathways (the three-step methylation and the one-step CDP-choline pathway) were present and functional in L. bozemanae. In the genome of L. bozemanae, genes encoding two potential phosphatidylcholine forming enzymes, phospholipid N-methyl transferase (PmtA) and phosphatidylcholine synthase (Pcs), homologous to L. longbeachae, L. drancourtii, and L. pneumophila pmtA and pcs genes were identified. Genes pmtA and pcs from L. bozemanae were sequenced and analyzed on nucleotide and amino acid levels. Bacteria grown on an artificial medium with labelled choline synthesized phosphatidylcholine predominantly via the phosphatidylcholine synthase pathway, which indicates that L. bozemanae phosphatidylcholine, similarly as in other bacteria associated with eukaryotes, is an important determinant of host-microbe interactions.
KeywordMeSH Terms
80. Fong  DH, Lemke  CT, Hwang  J, Xiong  B, Berghuis  AM,     ( 2010 )

Structure of the antibiotic resistance factor spectinomycin phosphotransferase from Legionella pneumophila.

The Journal of biological chemistry 285 (13)
PMID : 20089863  :   DOI  :   10.1074/jbc.M109.038364     PMC  :   PMC2843205    
Abstract >>
Aminoglycoside phosphotransferases (APHs) constitute a diverse group of enzymes that are often the underlying cause of aminoglycoside resistance in the clinical setting. Several APHs have been extensively characterized, including the elucidation of the three-dimensional structure of two APH(3') isozymes and an APH(2'') enzyme. Although many APHs are plasmid-encoded and are capable of inactivating numerous 2-deoxystreptmaine aminoglycosides with multiple regiospecificity, APH(9)-Ia, isolated from Legionella pneumophila, is an unusual enzyme among the APH family for its chromosomal origin and its specificity for a single non-2-deoxystreptamine aminoglycoside substrate, spectinomycin. We describe here the crystal structures of APH(9)-Ia in its apo form, its binary complex with the nucleotide, AMP, and its ternary complex bound with ADP and spectinomycin. The structures reveal that APH(9)-Ia adopts the bilobal protein kinase-fold, analogous to the APH(3') and APH(2'') enzymes. However, APH(9)-Ia differs significantly from the other two types of APH enzymes in its substrate binding area and that it undergoes a conformation change upon ligand binding. Moreover, kinetic assay experiments indicate that APH(9)-Ia has stringent substrate specificity as it is unable to phosphorylate substrates of choline kinase or methylthioribose kinase despite high structural resemblance. The crystal structures of APH(9)-Ia demonstrate and expand our understanding of the diversity of the APH family, which in turn will facilitate the development of new antibiotics and inhibitors.
KeywordMeSH Terms
Drug Resistance, Microbial
81. Kozak  NA, Benson  RF, Brown  E, Alexander  NT, Taylor  TH, Shelton  BG, Fields  BS,     ( 2009 )

Distribution of lag-1 alleles and sequence-based types among Legionella pneumophila serogroup 1 clinical and environmental isolates in the United States.

Journal of clinical microbiology 47 (8)
PMID : 19553574  :   DOI  :   10.1128/JCM.02410-08     PMC  :   PMC2725700    
Abstract >>
Approximately 84% of legionellosis cases are due to Legionella pneumophila serogroup 1. Moreover, a majority of L. pneumophila serogroup 1 clinical isolates react positively with monoclonal antibody 2 (MAb2) of the international standard panel. Over 94% of the legionellosis outbreaks investigated by the Centers for Disease Control and Prevention are due to this subset of L. pneumophila serogroup 1. To date, there is no complete explanation for the enhanced ability of these strains to cause disease. To better characterize these organisms, we subtyped 100 clinical L. pneumophila serogroup 1 isolates and 50 environmental L. pneumophila serogroup 1 isolates from the United States by (i) reactivity with MAb2, (ii) presence of a lag-1 gene required for the MAb2 epitope, and (iii) sequence-based typing analysis. Our results showed that the MAb2 epitope and lag-1 gene are overrepresented in clinical L. pneumophila serogroup 1 isolates. MAb2 recognized 75% of clinical isolates but only 6% of environmental isolates. Similarly, 75% of clinical isolates but only 8% of environmental isolates harbored lag-1. We identified three distinct lag-1 alleles, referred to as Philadelphia, Arizona, and Lens alleles, among 79 isolates carrying this gene. The Arizona allele is described for the first time in this study. We identified 59 different sequence types (STs), and 34 STs (58%) were unique to the United States. Our results support the hypothesis that a select group of STs may have an enhanced ability to cause legionellosis. Combining sequence typing and lag-1 analysis shows that STs tend to associate with a single lag-1 allele type, suggesting a hierarchy of virulence genotypes. Further analysis of ST and lag-1 profiles may identify genotypes of L. pneumophila serogroup 1 that warrant immediate intervention.
KeywordMeSH Terms
Bacterial Typing Techniques
Environmental Microbiology
82. Coscollá  M, González-Candelas  F,     ( 2009 )

Comparison of clinical and environmental samples of Legionella pneumophila at the nucleotide sequence level.

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 9 (5)
PMID : 19465160  :   DOI  :   10.1016/j.meegid.2009.05.013    
Abstract >>
Legionella pneumophila serogroup 1 is the most common etiological agent of legionellosis. We have used clinical and environmental isolates from different sources to compare their genetic variability. We have obtained the nucleotide sequence for six protein-coding loci, included in the SBT scheme for L. pneumophila, and three intergenic regions from 127 samples, 47 of environmental origin and 80 from clinical samples. Levels of genetic variability were found to be higher in the environmental than in the clinical samples, but these did not represent a mere subset of the former. Not a single case of full identity between clinical and environmental isolates was found, which raises the possibility that only a specific subset of environmental isolates is actually capable of producing infection in humans. A phylogenetic analysis of the concatenate alignment of the nine loci sequences showed four main groups, each including clinical and environmental isolates, although their distribution was not uniform among them. The comparison of each individual gene tree with the others revealed several cases of incongruence involving samples from both origins, thus suggesting the presence of recombination in the two groups.
KeywordMeSH Terms
Environmental Microbiology
83. Thürmer  A, Helbig  JH, Jacobs  E, Lück  PC,     ( 2009 )

PCR-based 'serotyping' of Legionella pneumophila.

Journal of medical microbiology 58 (Pt 5)
PMID : 19369520  :   DOI  :   10.1099/jmm.0.008508-0    
Abstract >>
Currently, several PCR assays based on 16S rRNA and virulence-associated genes are available for detection of Legionella pneumophila. So far, no genotyping method has been published that can discriminate between serogroups and monoclonal subgroups of the most common L. pneumophila serogroup 1. Our first approach was to analyse LPS-associated genes of seven L. pneumophila serogroup 1 strains, and we developed two PCR-based methods specific for serogroup 1. Specific DNA fragments could be amplified from all the serogroup 1 strains (n=43) including the strains from the American Type Culture Collection. In contrast, none of the strains from serogroups 2-15 (n=41) contained these specific gene regions. In a second approach, primers specific for the lag-1 gene, encoding an O-acetyltransferase, which is responsible for the presence of the LPS epitope recognized by mAb 3/1, were designed and tested for their ability to differentiate between mAb 3/1-positive and -negative strains. All mAb 3/1-positive strains (n=30) contained the lag-1 gene, but in turn 4 of 13 tested mAb 3/1-negative strains were also positive in the PCR. Thus, the discrimination between mAb 3/1-positive and mAb 3/1-negative subgroups could not be achieved for all strains. In a third approach, two intergenic regions expected to be specific for monoclonal subgroup Knoxville and closely related subgroups Benidorm/Bellingham were identified and used for selective genotyping. These intergenic regions could not only be amplified in every tested strain belonging to the subgroups Knoxville, Benidorm and Bellingham, but also in some strains of other unrelated subgroups. The two PCR approaches with primers specific for serogroup 1 genes definitely represent a valuable tool in outbreak investigations and for risk assessment. They also might be used for culture-independent diagnosis of legionellosis caused by L. pneumophila serogroup 1.
KeywordMeSH Terms
84. Brombacher  E, Urwyler  S, Ragaz  C, Weber  SS, Kami  K, Overduin  M, Hilbi  H,     ( 2009 )

Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.

The Journal of biological chemistry 284 (8)
PMID : 19095644  :   DOI  :   10.1074/jbc.M807505200     PMC  :   PMC2643517    
Abstract >>
The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the alpha-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIbeta. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.
KeywordMeSH Terms
85. Carvalho  FR, Nastasi  FR, Gamba  RC, Foronda  AS, Pellizari  VH,     ( 2008 )

Occurrence and diversity of Legionellaceae in polar lakes of the Antarctic peninsula.

Current microbiology 57 (4)
PMID : 18587615  :   DOI  :   10.1007/s00284-008-9192-y    
Abstract >>
Legionellaceae is a family of gram-negative, mesophilic, and facultative intracellular parasitic bacteria that inhabits freshwater environments. In this article, the Legionella population of water samples from the North and South Lake, located close to the Brazilian Scientific Station on King George Island, Keller Peninsula, Antarctica has been characterized. Culture onto selective medium and a independent-culture method were applied to the samples. In our attempt to isolate Legionella species from Antarctic lakes, we were able to obtain one L. pneumophila colony by an amoebic coculture procedure followed by plate culture onto a selective medium. In addition, results obtained from phylogenetic inference showed the presence of noncharacterized specimens of Legionella spp. These findings indicated the presence of legionellae in Antarctica and suggest that these bacteria can adapt to extreme conditions and open new possibilities for understanding the survival strategies of mesophilic Legionellaceae living in Antarctic environments. Furthermore, the isolation of these symbiotic bacteria in Antarctic lakes will allow future studies on cold-resistant mechanisms of legionellae in polar environments.
KeywordMeSH Terms
Biodiversity
Legionellaceae
86. Ludwig  B, Schmid  A, Marre  R, Hacker  J,     ( 1991 )

Cloning, genetic analysis, and nucleotide sequence of a determinant coding for a 19-kilodalton peptidoglycan-associated protein (Ppl) of Legionella pneumophila.

Infection and immunity 59 (8)
PMID : 1855972  :   PMC  :   PMC258049    
Abstract >>
A genomic library of Legionella pneumophila, the causative agent of Legionnaires disease in humans, was constructed in Escherichia coli K-12, and the recombinant clones were screened by immuno-colony blots with an antiserum raised against heat-killed L. pneumophila. Twenty-three clones coding for a Legionella-specific protein of 19 kDa were isolated. The 19-kDa protein, which represents an outer membrane protein, was found to be associated with the peptidoglycan layer both in L. pneumophila and in the recombinant E. coli clones. This was shown by electrophoresis and Western immunoblot analysis of bacterial cell membrane fractions with a monospecific polyclonal 19-kDa protein-specific antiserum. The protein was termed peptidoglycan-associated protein of L. pneumophila (Ppl). The corresponding genetic determinant, ppl, was subcloned on a 1.8-kb ClaI fragment. DNA sequence studies revealed that two open reading frames, pplA and pplB, coding for putative proteins of 18.9 and 16.8 kDa, respectively, were located on the ClaI fragment. Exonuclease III digestion studies confirmed that pplA is the gene coding for the peptidoglycan-associated 19-kDa protein of L. pneumophila. The amino acid sequence of PplA exhibits a high degree of homology to the sequences of the Pal lipoproteins of E. coli K-12 and Haemophilus influenzae.
KeywordMeSH Terms
87. Huston  WM, Naylor  J, Cianciotto  NP, Jennings  MP, McEwan  AG,     ( 2008 )

Functional analysis of the multi-copper oxidase from Legionella pneumophila.

Microbes and infection 10 (5)
PMID : 18403241  :   DOI  :   10.1016/j.micinf.2008.01.011    
Abstract >>
Multicopper oxidases have been described to have functions in copper tolerance, manganese oxidation, and iron oxidation in a range of bacteria. The putative cytoplasmic membrane multicopper oxidase from Legionella pneumophila was investigated. The mcoL gene was found to be critical for aerobic extracellular growth under either iron-limiting conditions or in the presence of ferrous Fe(II) iron, as a sole source of this essential metal. The mcoL mutants showed minor growth defects when grown in the presence of Fe(III) as the iron source. In contrast, intracellular growth and survival was not affected by the absence of the mcoL gene regardless of available iron concentration. The evidence presented here could indicate a possible role for mcoL in prevention of the toxic effects of ferrous iron during aerobic conditions. However, a function in high-affinity acquisition of iron could also be possible given the inability of the McoL mutants to grow aerobically under iron-limiting conditions.
KeywordMeSH Terms
88. Cazalet  C, Jarraud  S, Ghavi-Helm  Y, Kunst  F, Glaser  P, Etienne  J, Buchrieser  C,     ( 2008 )

Multigenome analysis identifies a worldwide distributed epidemic Legionella pneumophila clone that emerged within a highly diverse species.

Genome research 18 (3)
PMID : 18256241  :   DOI  :   10.1101/gr.7229808     PMC  :   PMC2259107    
Abstract >>
Genomics can provide the basis for understanding the evolution of emerging, lethal human pathogens such as Legionella pneumophila, the causative agent of Legionnaires' disease. This bacterium replicates within amoebae and persists in the environment as a free-living microbe. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease and within the 15 serogroups (Sg), L. pneumophila Sg1 causes over 84% of Legionnaires' disease worldwide. Why L. pneumophila Sg1 is so predominant is unknown. Here, we report the first comprehensive screen of the gene content of 217 L. pneumophila and 32 non-L. pneumophila strains isolated from humans and the environment using a Legionella DNA-array. Strikingly, we uncovered a high conservation of virulence- and eukaryotic-like genes, indicating strong environmental selection pressures for their preservation. No specific hybridization profile differentiated clinical and environmental strains or strains of different serogroups. Surprisingly, the gene cluster coding the determinants of the core and the O side-chain synthesis of the lipopolysaccaride (LPS cluster) determining Sg1 was present in diverse genomic backgrounds, strongly implicating the LPS of Sg1 itself as a principal cause of the high prevalence of Sg1 strains in human disease and suggesting that the LPS cluster can be transferred horizontally. Genomic analysis also revealed that L. pneumophila is a genetically diverse species, in part due to horizontal gene transfer of mobile genetic elements among L. pneumophila strains, but also between different Legionella species. However, the genomic background also plays a role in disease causation as demonstrated by the identification of a globally distributed epidemic strain exhibiting the genotype of the sequenced L. pneumophila strain Paris.
KeywordMeSH Terms
Genetic Variation
89. D'Auria  G, Jiménez  N, Peris-Bondia  F, Pelaz  C, Latorre  A, Moya  A,     ( 2008 )

Virulence factor rtx in Legionella pneumophila, evidence suggesting it is a modular multifunctional protein.

BMC genomics 9 (N/A)
PMID : 18194518  :   DOI  :   10.1186/1471-2164-9-14     PMC  :   PMC2257941    
Abstract >>
The repeats in toxin (Rtx) are an important pathogenicity factor involved in host cells invasion of Legionella pneumophila and other pathogenic bacteria. Its role in escaping the host immune system and cytotoxic activity is well known. Its repeated motives and modularity make Rtx a multifunctional factor in pathogenicity. The comparative analysis of rtx gene among 6 strains of L. pneumophila showed modularity in their structures. Among compared genomes, the N-terminal region of the protein presents highly dissimilar repeats with functionally similar domains. On the contrary, the C-terminal region is maintained with a fashionable modular configuration, which gives support to its proposed role in adhesion and pore formation. Despite the variability of rtx among the considered strains, the flanking genes are maintained in synteny and similarity. In contrast to the extracellular bacteria Vibrio cholerae, in which the rtx gene is highly conserved and flanking genes have lost synteny and similarity, the gene region coding for the Rtx toxin in the intracellular pathogen L. pneumophila shows a rapid evolution. Changes in the rtx could play a role in pathogenicity. The interplay of the Rtx toxin with host membranes might lead to the evolution of new variants that are able to escape host cell defences.
KeywordMeSH Terms
90. Belyi  Y, Tabakova  I, Stahl  M, Aktories  K,     ( 2008 )

Lgt: a family of cytotoxic glucosyltransferases produced by Legionella pneumophila.

Journal of bacteriology 190 (8)
PMID : 18281405  :   DOI  :   10.1128/JB.01798-07     PMC  :   PMC2293231    
Abstract >>
Legionella pneumophila is a facultative intracellular pathogen responsible for severe lung disease in humans, known as legionellosis or Legionnaires' disease. Previously, we reported on the approximately 60-kDa glucosyltransferase (Lgt1) from Legionella pneumophila, which modified eukaryotic elongation factor 1A. In the present study, using L. pneumophila Philadelphia-1, Lens, Paris, and Corby genome databases, we identified several genes coding for proteins with considerable sequence homology to Lgt1. These new enzymes form three subfamilies, termed Lgt1 to -3, glucosylate mammalian elongation factor eEF1A at serine-53, inhibit its activity, and subsequently kill target eukaryotic cells. Expression studies on L. pneumophila grown in broth medium or in Acanthamoeba castellanii revealed that production of Lgt1 was maximal at stationary phase of broth culture or during the late phase of Legionella-host cell interaction, respectively. In contrast, synthesis of Lgt3 peaked during the lag phase of liquid culture and at early steps of bacterium-amoeba interaction. Thus, the data indicate that members of the L. pneumophila glucosyltransferase family are differentially regulated, affect protein synthesis of host cells, and represent potential virulence factors of Legionella.
KeywordMeSH Terms
91. Ingmundson  A, Delprato  A, Lambright  DG, Roy  CR,     ( 2007 )

Legionella pneumophila proteins that regulate Rab1 membrane cycling.

Nature 450 (7168)
PMID : 17952054  :   DOI  :   10.1038/nature06336    
Abstract >>
Rab1 is a GTPase that regulates the transport of endoplasmic-reticulum-derived vesicles in eukaryotic cells. The intracellular pathogen Legionella pneumophila subverts Rab1 function to create a vacuole that supports bacterial replication by a mechanism that is not well understood. Here we describe L. pneumophila proteins that control Rab1 activity directly. We show that a region in the DrrA (defect in Rab1 recruitment A) protein required for recruitment of Rab1 to membranes functions as a guanine nucleotide dissociation inhibitor displacement factor. A second region of the DrrA protein stimulated Rab1 activation by functioning as a guanine nucleotide exchange factor. The LepB protein was found to inactivate Rab1 by stimulating GTP hydrolysis, indicating that LepB has GTPase-activating protein activity that regulates removal of Rab proteins from membranes. Thus, L. pneumophila encodes proteins that regulate three distinct biochemical reactions critical for Rab GTPase membrane cycling to redirect Rab1 to the pathogen-occupied vacuole and to control Rab1 function.
KeywordMeSH Terms
92. Pinotsis  N, Waksman  G,     ( 2017 )

Structure of the WipA protein reveals a novel tyrosine protein phosphatase effector from Legionella pneumophila.

The Journal of biological chemistry 292 (22)
PMID : 28389563  :   DOI  :   10.1074/jbc.M117.781948     PMC  :   PMC5454105    
Abstract >>
Legionnaires' disease is a severe form of pneumonia caused by the bacterium Legionella pneumophila. L. pneumophila pathogenicity relies on secretion of more than 300 effector proteins by a type IVb secretion system. Among these Legionella effectors, WipA has been primarily studied because of its dependence on a chaperone complex, IcmSW, for translocation through the secretion system, but its role in pathogenicity has remained unknown. In this study, we present the crystal structure of a large fragment of WipA, WipA435. Surprisingly, this structure revealed a serine/threonine phosphatase fold that unexpectedly targets tyrosine-phosphorylated peptides. The structure also revealed a sequence insertion that folds into an �\-helical hairpin, the tip of which adopts a canonical coiled-coil structure. The purified protein was a dimer whose dimer interface involves interactions between the coiled coil of one WipA molecule and the phosphatase domain of another. Given the ubiquity of protein-protein interaction mediated by interactions between coiled-coils, we hypothesize that WipA can thereby transition from a homodimeric state to a heterodimeric state in which the coiled-coil region of WipA is engaged in a protein-protein interaction with a tyrosine-phosphorylated host target. In conclusion, these findings help advance our understanding of the molecular mechanisms of an effector involved in Legionella virulence and may inform approaches to elucidate the function of other effectors.
KeywordMeSH Terms
Legionella effector
Michaelis-Menten
coiled-coil
crystal structure
enzyme kinetics
phosphatase
phosphoesterase fold
tyrosine phosphatase
tyrosine-protein phosphatase (tyrosine phosphatase)
Legionella effector
Michaelis-Menten
coiled-coil
crystal structure
enzyme kinetics
phosphatase
phosphoesterase fold
tyrosine phosphatase
tyrosine-protein phosphatase (tyrosine phosphatase)
93. Nishida  T, Watanabe  K, Tachibana  M, Shimizu  T, Watarai  M,     ( 2017 )

Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector.

Plasmid 90 (N/A)
PMID : 28259635  :   DOI  :   10.1016/j.plasmid.2017.02.004    
Abstract >>
In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584bp with a GC content of 37.3% and contained three putative open reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the putative protein shared 39% sequence identity with a putative plasmid replication protein RepL. ORF1 was needed for replication in L. pneumophila but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded putative hypothetical proteins of 114 amino acids and 78 amino acids, respectively, but the functions of the putative proteins ORF2 and OFR3 are not clear. The transfer mechanism for pOfk55 was independent on the type IVB secretion system in the original host. A L. pneumophila-E. coli shuttle vector, pNT562 (5058bp, KmR), was constructed by In-Fusion Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites from pBluescript SK(+) as well as the tac promoter region and lacI gene from pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation efficiency of pNT562 in L. pneumophila strains ranged from 1.6��101 to 1.0��105CFU/ng. The relative number of pNT562 was estimated at 5.7��1.0 copies and 73.6% of cells maintained the plasmid after 1week in liquid culture without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563, was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563 was introduced into L. pneumophila Lp02 and E. coli DH5�\, and both strains expressed GFP successfully. These results suggest that the shuttle vector is useful for genetic studies in L. pneumophila.
KeywordMeSH Terms
Intracellular bacteria
Legionella pneumophila
Plasmid
Shuttle vector
Intracellular bacteria
Legionella pneumophila
Plasmid
Shuttle vector
94. Rao  C, Guyard  C, Pelaz  C, Wasserscheid  J, Bondy-Denomy  J, Dewar  K, Ensminger  AW,     ( 2016 )

Active and adaptive Legionella CRISPR-Cas reveals a recurrent challenge to the pathogen.

Cellular microbiology 18 (10)
PMID : 26936325  :   DOI  :   10.1111/cmi.12586     PMC  :   PMC5071653    
Abstract >>
Clustered regularly interspaced short palindromic repeats with CRISPR-associated gene (CRISPR-Cas) systems are widely recognized as critical genome defense systems that protect microbes from external threats such as bacteriophage infection. Several isolates of the intracellular pathogen Legionella pneumophila possess multiple CRISPR-Cas systems (type I-C, type I-F and type II-B), yet the targets of these systems remain unknown. With the recent observation that at least one of these systems (II-B) plays a non-canonical role in supporting intracellular replication, the possibility remained that these systems are vestigial genome defense systems co-opted for other purposes. Our data indicate that this is not the case. Using an established plasmid transformation assay, we demonstrate that type I-C, I-F and II-B CRISPR-Cas provide protection against spacer targets. We observe efficient laboratory acquisition of new spacers under 'priming' conditions, in which initially incomplete target elimination leads to the generation of new spacers and ultimate loss of the invasive DNA. Critically, we identify the first known target of L. pneumophila CRISPR-Cas: a 30 kb episome of unknown function whose interbacterial transfer is guarded against by CRISPR-Cas. We provide evidence that the element can subvert CRISPR-Cas by mutating its targeted sequences - but that primed spacer acquisition may limit this mechanism of escape. Rather than generally impinging on bacterial fitness, this element drives a host specialization event - with improved fitness in Acanthamoeba but a reduced ability to replicate in other hosts and conditions. These observations add to a growing body of evidence that host range restriction can serve as an existential threat to L. pneumophila in the wild.
KeywordMeSH Terms
Clustered Regularly Interspaced Short Palindromic Repeats
95. Sheedlo  MJ, Qiu  J, Tan  Y, Paul  LN, Luo  ZQ, Das  C,     ( 2015 )

Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination.

Proceedings of the National Academy of Sciences of the United States of America 112 (49)
PMID : 26598703  :   DOI  :   10.1073/pnas.1514568112     PMC  :   PMC4679006    
Abstract >>
Manipulation of the host's ubiquitin network is emerging as an important strategy for counteracting and repurposing the posttranslational modification machineries of the host by pathogens. Ubiquitin E3 ligases encoded by infectious agents are well known, as are a variety of viral deubiquitinases (DUBs). Bacterial DUBs have been discovered, but little is known about the structure and mechanism underlying their ubiquitin recognition. In this report, we found that members of the Legionella pneumophila SidE effector family harbor a DUB module important for ubiquitin dynamics on the bacterial phagosome. Structural analysis of this domain alone and in complex with ubiquitin vinyl methyl ester (Ub-VME) reveals unique molecular contacts used in ubiquitin recognition. Instead of relying on the Ile44 patch of ubiquitin, as commonly used in eukaryotic counterparts, the SdeADub module engages Gln40 of ubiquitin. The architecture of the active-site cleft presents an open arrangement with conformational plasticity, permitting deubiquitination of three of the most abundant polyubiquitin chains, with a distinct preference for Lys63 linkages. We have shown that this preference enables efficient removal of Lys63 linkages from the phagosomal surface. Remarkably, the structure reveals by far the most parsimonious use of molecular contacts to achieve deubiquitination, with less than 1,000 ?(2) of accessible surface area buried upon complex formation with ubiquitin. This type of molecular recognition appears to enable dual specificity toward ubiquitin and the ubiquitin-like modifier NEDD8.
KeywordMeSH Terms
Legionella
deubiquitinase
phagosome
type IV secretion
ubiquitination
Legionella
deubiquitinase
phagosome
type IV secretion
ubiquitination
Ubiquitination
96. Cao  B, Tian  Z, Wang  S, Zhu  Z, Sun  Y, Feng  L, Wang  L,     ( 2015 )

Structural comparison of O-antigen gene clusters of Legionella pneumophila and its application of a serogroup-specific multiplex PCR assay.

Antonie van Leeuwenhoek 108 (6)
PMID : 26415652  :   DOI  :   10.1007/s10482-015-0594-0    
Abstract >>
The Legionella pneumophila serogroups O1, O4, O6, O7, O10 and O13 are pathogenic strains associated with pneumonia. The surface O-antigen gene clusters of L. pneumophila serogroups O4, O6, O7, O10 and O13 were sequenced and analyzed, with the function annotated on the basis of homology to that of the genes of L. pneumophila serogroup O1 (L. pneumophila subsp. pneumophila str. Philadelphia 1). The gene locus of the six L. pneumophila serogroups contains genes of yvfE, neuABCD, pseA-like for nucleotide sugar biosynthesis, wecA for sugar transfer, and wzm as well as wzt for O-antigen processing. The detection of O-antigen genes allows the fine differentiation at species and serogroup level without the neccessity of nucleotide sequencing. The O-antigen-processing genes wzm and wzt, which were found to be distinctive for different for different serogroups, have been used as the target genes for the detection and identification of L. pneumophila strains of different O serogroups. In this report, a multiplex PCR assay based on wzm or wzt that diferentiates all the six serogroups by amplicon size was developed with the newly designed specific primer pairs for O1 and O7, and the specific primer pairs for O4, O6, O10, and O13 reported previously. The array was validated by analysis of 34 strains including 15 L. pneumophila O-standard reference strains, eight reference strains of other Legionella non-pneumophila species, six other bacterial species, and five L. pneumophila environmental isolates. The detection sensitivity was one ng genomic DNA. The accurate and sensitive assay is suitable for the identification and detection of strains of these serogroups in environmental and clinical samples.
KeywordMeSH Terms
Gene clusters
Legionella pneumophila
Multiplex PCR
O-antigen
Serogroups
Gene clusters
Legionella pneumophila
Multiplex PCR
O-antigen
Serogroups
Multigene Family
97. Luo  X, Wasilko  DJ, Liu  Y, Sun  J, Wu  X, Luo  ZQ, Mao  Y,     ( 2015 )

Structure of the Legionella Virulence Factor, SidC Reveals a Unique PI(4)P-Specific Binding Domain Essential for Its Targeting to the Bacterial Phagosome.

PLoS pathogens 11 (6)
PMID : 26067986  :   DOI  :   10.1371/journal.ppat.1004965     PMC  :   PMC4467491    
Abstract >>
The opportunistic intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires' disease. L. pneumophila delivers nearly 300 effector proteins into host cells for the establishment of a replication-permissive compartment known as the Legionella-containing vacuole (LCV). SidC and its paralog SdcA are two effectors that have been shown to anchor on the LCV via binding to phosphatidylinositol-4-phosphate [PI(4)P] to facilitate the recruitment of ER proteins to the LCV. We recently reported that the N-terminal SNL (SidC N-terminal E3 Ligase) domain of SidC is a ubiquitin E3 ligase, and its activity is required for the recruitment of ER proteins to the LCV. Here we report the crystal structure of SidC (1-871). The structure reveals that SidC contains four domains that are packed into an arch-like shape. The P4C domain (PI(4)P binding of SidC) comprises a four �\-helix bundle and covers the ubiquitin ligase catalytic site of the SNL domain. Strikingly, a pocket with characteristic positive electrostatic potentials is formed at one end of this bundle. Liposome binding assays of the P4C domain further identified the determinants of phosphoinositide recognition and membrane interaction. Interestingly, we also found that binding with PI(4)P stimulates the E3 ligase activity, presumably due to a conformational switch induced by PI(4)P from a closed form to an open active form. Mutations of key residues involved in PI(4)P binding significantly reduced the association of SidC with the LCV and abolished its activity in the recruitment of ER proteins and ubiquitin signals, highlighting that PI(4)P-mediated targeting of SidC is critical to its function in the remodeling of the bacterial phagosome membrane. Finally, a GFP-fusion with the P4C domain was demonstrated to be specifically localized to PI(4)P-enriched compartments in mammalian cells. This domain shows the potential to be developed into a sensitive and accurate PI(4)P probe in living cells.
KeywordMeSH Terms
98. Folly-Klan  M, Sancerne  B, Alix  E, Roy  CR, Cherfils  J, Campanacci  V,     ( 2015 )

On the use of Legionella/Rickettsia chimeras to investigate the structure and regulation of Rickettsia effector RalF.

Journal of structural biology 189 (2)
PMID : 25498244  :   DOI  :   10.1016/j.jsb.2014.12.001    
Abstract >>
A convenient strategy to interrogate the biology of regulatory proteins is to replace individual domains by an equivalent domain from a related protein of the same species or from an ortholog of another species. It is generally assumed that the overall properties of the native protein are retained in the chimera, and that functional differences reflect only the specific determinants contained in the swapped domains. Here we used this strategy to circumvent the difficulty in obtaining crystals of Rickettsia prowazekii RalF, a bacterial protein that functions as a guanine nucleotide exchange factor for eukaryotic Arf GTPases. A RalF homolog is encoded by Legionella pneumophila, in which a C-terminal capping domain auto-inhibits the catalytic Sec7 domain and localizes the protein to the Legionella-containing vacuole. The crystal structures of domain-swapped chimeras were determined and used to construct a model of Legionella RalF with a RMSD of less than 1? with the crystal structure, which validated the use of this approach to build a model of Rickettsia RalF. In the Rickettsia RalF model, sequence differences in the capping domain that target it to specific membranes are accommodated by a shift of the entire domain with respect to the Sec7 domain. However, local sequence changes also give rise to an artifactual salt bridge in one of the chimeras, which likely explains why this chimera is recalcitrant to activation. These findings highlight the structural plasticity whereby chimeras can be engineered, but also underline that unpredictable differences can modify their biochemical responses.
KeywordMeSH Terms
Bacterial pathogens
Chimera
Guanine nucleotide exchange factor
Small GTPases
Bacterial pathogens
Chimera
Guanine nucleotide exchange factor
Small GTPases
Rickettsia prowazekii
99. Kuroda  T, Kubori  T, Thanh Bui  X, Hyakutake  A, Uchida  Y, Imada  K, Nagai  H,     ( 2015 )

Molecular and structural analysis of Legionella DotI gives insights into an inner membrane complex essential for type IV secretion.

Scientific reports 5 (N/A)
PMID : 26039110  :   DOI  :   10.1038/srep10912     PMC  :   PMC4454188    
Abstract >>
The human pathogen Legionella pneumophila delivers a large array of the effector proteins into host cells using the Dot/Icm type IVB secretion system. Among the proteins composing the Dot/Icm system, an inner membrane protein DotI is known to be crucial for the secretion function but its structure and role in type IV secretion had not been elucidated. We report here the crystal structures of the periplasmic domains of DotI and its ortholog in the conjugation system of plasmid R64, TraM. These structures reveal a striking similarity to VirB8, a component of type IVA secretion systems, suggesting that DotI/TraM is the type IVB counterpart of VirB8. We further show that DotI and its partial paralog DotJ form a stable heterocomplex. R64 TraM, encoded by the conjugative plasmid lacking DotJ ortholog, forms a homo-hexamer. The DotI-DotJ complex is distinct from the core complex, which spans both inner and outer membranes to form a substrate conduit, and seems not to stably associate with the core complex. These results give insight into VirB8-family inner membrane proteins essential for type IV secretion and aid towards understanding the molecular basis of secretion systems essential for bacterial pathogenesis.
KeywordMeSH Terms
Type IV Secretion Systems
100. Hsu  F, Luo  X, Qiu  J, Teng  YB, Jin  J, Smolka  MB, Luo  ZQ, Mao  Y,     ( 2014 )

The Legionella effector SidC defines a unique family of ubiquitin ligases important for bacterial phagosomal remodeling.

Proceedings of the National Academy of Sciences of the United States of America 111 (29)
PMID : 25006264  :   DOI  :   10.1073/pnas.1402605111     PMC  :   PMC4115505    
Abstract >>
The activity of proteins delivered into host cells by the Dot/Icm injection apparatus allows Legionella pneumophila to establish a niche called the Legionella-containing vacuole (LCV), which is permissive for intracellular bacterial propagation. Among these proteins, substrate of Icm/Dot transporter (SidC) anchors to the cytoplasmic surface of the LCV and is important for the recruitment of host endoplasmic reticulum (ER) proteins to this organelle. However, the biochemical function underlying this activity is unknown. Here, we determined the structure of the N-terminal domain of SidC, which has no structural homology to any protein. Sequence homology analysis revealed a potential canonical catalytic triad formed by Cys46, His444, and Asp446 on the surface of SidC. Unexpectedly, we found that SidC is an E3 ubiquitin ligase that uses the C-H-D triad to catalyze the formation of high-molecular-weight polyubiquitin chains through multiple ubiquitin lysine residues. A C46A mutation completely abolished the E3 ligase activity and the ability of the protein to recruit host ER proteins as well as polyubiquitin conjugates to the LCV. Thus, SidC represents a unique E3 ubiquitin ligase family important for phagosomal membrane remodeling by L. pneumophila.
KeywordMeSH Terms
ER vesicle
membrane trafficking
pathogen infection
type IV secretion system
ubiquitination
ER vesicle
membrane trafficking
pathogen infection
type IV secretion system
ubiquitination
101. Folly-Klan  M, Alix  E, Stalder  D, Ray  P, Duarte  LV, Delprato  A, Zeghouf  M, Antonny  B, Campanacci  V, Roy  CR, Cherfils  J,     ( 2013 )

A novel membrane sensor controls the localization and ArfGEF activity of bacterial RalF.

PLoS pathogens 9 (11)
PMID : 24244168  :   DOI  :   10.1371/journal.ppat.1003747     PMC  :   PMC3828167    
Abstract >>
The intracellular bacterial pathogen Legionella pneumophila (Lp) evades destruction in macrophages by camouflaging in a specialized organelle, the Legionella-containing vacuole (LCV), where it replicates. The LCV maturates by incorporating ER vesicles, which are diverted by effectors that Lp injects to take control of host cell membrane transport processes. One of these effectors, RalF, recruits the trafficking small GTPase Arf1 to the LCV. LpRalF has a Sec7 domain related to host ArfGEFs, followed by a capping domain that intimately associates with the Sec7 domain to inhibit GEF activity. How RalF is activated to function as a LCV-specific ArfGEF is unknown. We combined the reconstitution of Arf activation on artificial membranes with cellular expression and Lp infection assays, to analyze how auto-inhibition is relieved for LpRalF to function in vivo. We find that membranes activate LpRalF by about 1000 fold, and identify the membrane-binding region as the region that inhibits the Sec7 active site. It is enriched in aromatic and positively charged residues, which establish a membrane sensor to control the GEF activity in accordance with specific lipid environments. A similar mechanism of activation is found in RalF from Rickettsia prowazekii (Rp), with a different aromatic/charged residues ratio that results in divergent membrane preferences. The membrane sensor is the primary determinant of the localization of LpRalF on the LCV, and drives the timing of Arf activation during infection. Finally, we identify a conserved motif in the capping domain, remote from the membrane sensor, which is critical for RalF activity presumably by organizing its active conformation. These data demonstrate that RalF proteins are regulated by a membrane sensor that functions as a binary switch to derepress ArfGEF activity when RalF encounters a favorable lipid environment, thus establishing a regulatory paradigm to ensure that Arf GTPases are efficiently activated at specific membrane locations.
KeywordMeSH Terms
102. Qin  T, Zhou  H, Ren  H, Guan  H, Li  M, Zhu  B, Shao  Z,     ( 2014 )

Distribution of sequence-based types of legionella pneumophila serogroup 1 strains isolated from cooling towers, hot springs, and potable water systems in China.

Applied and environmental microbiology 80 (7)
PMID : 24463975  :   DOI  :   10.1128/AEM.03844-13     PMC  :   PMC3993137    
Abstract >>
Legionella pneumophila serogroup 1 causes Legionnaires' disease. Water systems contaminated with Legionella are the implicated sources of Legionnaires' disease. This study analyzed L. pneumophila serogroup 1 strains in China using sequence-based typing. Strains were isolated from cooling towers (n = 96), hot springs (n = 42), and potable water systems (n = 26). Isolates from cooling towers, hot springs, and potable water systems were divided into 25 sequence types (STs; index of discrimination [IOD], 0.711), 19 STs (IOD, 0.934), and 3 STs (IOD, 0.151), respectively. The genetic variation among the potable water isolates was lower than that among cooling tower and hot spring isolates. ST1 was the predominant type, accounting for 49.4% of analyzed strains (n = 81), followed by ST154. With the exception of two strains, all potable water isolates (92.3%) belonged to ST1. In contrast, 53.1% (51/96) and only 14.3% (6/42) of cooling tower and hot spring, respectively, isolates belonged to ST1. There were differences in the distributions of clone groups among the water sources. The comparisons among L. pneumophila strains isolated in China, Japan, and South Korea revealed that similar clones (ST1 complex and ST154 complex) exist in these countries. In conclusion, in China, STs had several unique allelic profiles, and ST1 was the most prevalent sequence type of environmental L. pneumophila serogroup 1 isolates, similar to its prevalence in Japan and South Korea.
KeywordMeSH Terms
Genetic Variation
Molecular Typing
103. Paveenkittiporn  W, Dejsirilert  S, Kalambaheti  T,     ( 2012 )

Genetic speciation of environmental Legionella isolates in Thailand.

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 12 (7)
PMID : 22504352  :   DOI  :   10.1016/j.meegid.2012.03.025    
Abstract >>
Legionella-like organisms were isolated during 2003-2007 from various water resources by culturing on selective media of Wadowsky-Yee-Okuda agar. The 256 isolates were identified as belonging to the Legionella genus based on detection of 108 bp PCR product of the 5S rRNA gene, while the inclusion as Legionella pneumophila were confirmed by PCR detection of a specific mip gene region of 168 bp. The 50 isolates, identified as non-pneumophila, were then subjected to DNA tree analysis, based on mip gene of ~650 bp and rnpB genes product ranged from 304 to 354 bp. Phylogenetic tree was constructed to predict their species in relative to the available database. The isolates of which their speciation, based on those two genes were inconclusive, were then investigated for the almost full-length of 16S rRNA sequences. The isolates were assigned as 16 known Legionella species, and proposed seven novel species based on their unique 16S rRNA sequence.
KeywordMeSH Terms
Genetic Speciation
Water Microbiology
104. Cao  B, Yao  F, Liu  X, Feng  L, Wang  L,     ( 2013 )

Development of a DNA microarray method for detection and identification of all 15 distinct O-antigen forms of Legionella pneumophila.

Applied and environmental microbiology 79 (21)
PMID : 23974134  :   DOI  :   10.1128/AEM.01957-13     PMC  :   PMC3811488    
Abstract >>
Legionella is ubiquitous in many environments. At least 50 species and 70 serogroups of the Gram-negative bacterium have been identified. Of the 50 species, 20 are pathogenic, and Legionella pneumophila is responsible for the great majority (approximately 90%) of the Legionnaires' disease cases that occur. Furthermore, of the 15 L. pneumophila serogroups identified, O1 alone causes more than 84% of the Legionnaires' disease cases that occur worldwide. Rapid and reliable assays for the detection and identification of L. pneumophila in water, environmental, and clinical samples are in great demand. L. pneumophila bacteria are traditionally identified by their O antigens by immunological methods. We have recently developed an O serogroup-specific DNA microarray for the detection of all 15 distinct O-antigen forms of L. pneumophila, including serogroups O1 to O15. A total of 35 strains were used to verify the specificity of the microarray, including 15 L. pneumophila O-antigen standard reference strains and seven L. pneumophila clinical isolates as target strains, seven reference strains of other non-pneumophila Legionella species as closely related strains, and six non-Legionella bacterial species as nonrelated strains. The detection sensitivity was 1 ng of genomic DNA or 0.4 CFU/ml in water samples with filter enrichment and plate culturing. This study demonstrated that the microarray allows specific, sensitive, and reproducible detection of L. pneumophila serogroups. To the best of our knowledge, this is the first report of a microarray serotyping method for all 15 distinct O-antigen forms of L. pneumophila.
KeywordMeSH Terms
Phylogeny
105. Hoffman  PS, Houston  L, Butler  CA,     ( 1990 )

Legionella pneumophila htpAB heat shock operon: nucleotide sequence and expression of the 60-kilodalton antigen in L. pneumophila-infected HeLa cells.

Infection and immunity 58 (10)
PMID : 2205580  :   PMC  :   PMC313664    
Abstract >>
A 60-kilodalton (kDa) immunodominant antigen of Legionella pneumophila is a heat shock protein (HSP) of the GroEL class of HSPs. The gene (htpB) coding the 60-kDa protein was localized to a 3.2-kilobase DNA fragment of L. pneumophila cloned into pUC19 (pSH16) (P. S. Hoffman, C. A. Butler, and F. D. Quinn, Infect. Immun. 57:1731-1739, 1989). The nucleotide sequence of the DNA fragment cloned into M13 confirmed two open reading frames, htpA and htpB, that code for proteins of 96 and 548 amino acids, respectively. A consensus heat shock promoter sequence upstream of the start of htpA was identified, and no obvious promoter sequences were detected upstream of htpB. Amino acid sequence comparison studies revealed that the L. pneumophila HtpB protein exhibited 76% homology with the 65-kDa protein of Mycobacterium tuberculosis and 85% homology with both GroEL of Escherichia coli and HtpB of Coxiella burnetii. A comparison of the amino acid sequences among these proteins revealed several regions of nearly absolute sequence conservation, with the variable regions occurring in common areas. The purified L. pneumophila 60-kDa protein was antigenic for human T lymphocytes. Indirect fluorescent antibody studies indicated that the 60-kDa protein may be located in the periplasm or expressed on the surface by intracellular bacteria, suggesting that a stress-related mechanism may be involved in the expression of this immunodominant antigen.
KeywordMeSH Terms
DNA, Bacterial
Operon
106. Costa  J, d'Avó  AF, da Costa  MS, Veríssimo  A,     ( 2012 )

Molecular evolution of key genes for type II secretion in Legionella pneumophila.

Environmental microbiology 14 (8)
PMID : 22118294  :   DOI  :   10.1111/j.1462-2920.2011.02646.x    
Abstract >>
Given the role of type II protein secretion system (T2S) in the ecology and pathogenesis of Legionella pneumophila, it is possible that this system is a target for adaptive evolution. The population genetic structure of L.pneumophila was inferred from the partial sequences of rpoB and from the complete sequence of three T2S structural components (lspD, lspE and pilD) and from two T2S effectors critical for intracellular infection of protozoa (proA and srnA) of 37 strains isolated from natural and man-made environments and disease-related from worldwide sources. A phylogenetic analysis was obtained for the concatenated alignment and for each individual locus. Seven main groups were identified containing the same L.pneumophila strains, suggesting an ancient divergence for each cluster and indicating that the operating selective pressures have equally affected the evolution of the five genes. Although linkage disequilibrium analysis indicate a clonal nature for population structure in this sample, our results indicate that recombination is a common phenomenon among T2S-related genes on this species, as 24 of the 37 L.pneumophila isolates contained at least one locus in which recombination was identified. Furthermore, neutral selection acting on the analysed T2S-related genes emerged as a clear result, namely on T2S effectors, ProA and SrnA, indicating that they are probably implicated in conserved virulence mechanisms through legionellae hosts.
KeywordMeSH Terms
Evolution, Molecular
107.     ( 1997 )

Isolation of a gene encoding a novel spectinomycin phosphotransferase from Legionella pneumophila.

Antimicrobial agents and chemotherapy 41 (6)
PMID : 9174205  :   PMC  :   PMC163921    
Abstract >>
A gene capable of conferring spectinomycin resistance was isolated from Legionella pneumophila, the agent of Legionnaires' disease. The gene (aph) encoded a 36-kDa protein which has similarity to aminoglycoside phosphotransferases. Biochemical analysis confirmed that aph encodes a phosphotransferase which modifies spectinomycin but not hygromycin, kanamycin, or streptomycin. The strain that was the source of aph demonstrated resistance to spectinomycin, and Southern hybridizations determined that aph also exists in other legionellae.
KeywordMeSH Terms
Genes, Bacterial
108.     ( 1997 )

Cloning and sequencing of the dnaK and grpE genes of Legionella pneumophila.

Gene 197 (1��2��)
PMID : 9332363  :   DOI  :   10.1016/s0378-1119(97)00257-6    
Abstract >>
A 4.4-kb DNA fragment from Legionella pneumophila (Lp) was isolated, which could complement an Escherichia coli (Ec) dnaK ts mutant, HC4102. Nucleotide sequence analysis of the region revealed two complete open reading frames (ORFs) encoding both a predicted DnaK protein of 644 aa and a predicted GrpE protein of 199 aa, and also the 5'-end of the predicted dnaJ gene organized in the order of grpE-dnaK-dnaJ. Consensus heat shock (HS) promoter sequences were identified upstream of the start of both grpE and dnaK transcripts. However, no obvious promoter sequences were detected upstream of dnaJ. The transcription start points of grpE and dnaK were determined by primer extension analysis and the amount of each of the transcripts increased four- to eightfold after HS.
KeywordMeSH Terms
Escherichia coli Proteins
109.     ( 1996 )

Phylogenetic analysis of tmRNA secondary structure.

RNA (New York, N.Y.) 2 (12)
PMID : 8972778  :   PMC  :   PMC1369456    
Abstract >>
The bacterial tmRNA acts with dual tRNA-like and mRNA-like character to tag incomplete translation products for degradation. Comparative analysis of 17 tmRNA genes (including eight new sequences) has allowed us to deduce conserved features of the tmRNA secondary structure. Except in a segment that includes the first codon of the tag reading frame, tmRNA is highly structured, with four pseudoknots and a total of 11 conserved base pairing regions. The previously identified tRNA minihelix structure is connected by a long base paired region to a large structured domain composed of a pseudoknot, followed by the tag reading frame and a string of three rather similar pseudoknots. The conservation of numerous structural elements among diverse eubacterial species indicates that these elements have important function beyond simply forming an endonuclease-resistant link between the reading frame and the tRNA-like domain.
KeywordMeSH Terms
Nucleic Acid Conformation
110.     ( 1997 )

The alternative sigma factor sigma28 of Legionella pneumophila restores flagellation and motility to an Escherichia coli fliA mutant.

Journal of bacteriology 179 (1)
PMID : 8981975  :   DOI  :   10.1128/jb.179.1.17-23.1997     PMC  :   PMC178656    
Abstract >>
Gene expression in Legionella pneumophila, the etiological agent of Legionnaires' disease, can be controlled by alternative forms of RNA polymerase programmed by distinct sigma factors. To understand the regulation of L. pneumophila flagellin expression, we cloned the sigma factor (FliA) of RNA polymerase responsible for the transcription of the flagellin gene, flaA. FliA is a member of the sigma28 class of alternative sigma factors identified in several bacterial genera. The gene fliA has been isolated from an expression library of L. pneumophila isolate Corby in Escherichia coli K-12. This library was transformed into a fliA mutant of E. coli K-12 containing a plasmid carrying the L. pneumophila-specific flaA promoter fused to the reporter gene luxAB. Screening the obtained transformants for luciferase activity, we isolated the major part of the fliA gene on a 1.64-kb fragment. This fragment was sequenced and used for reverse PCR in order to recover the complete fliA gene. The resulting 1.03-kb fragment was shown to contain the entire fliA gene. L. pneumophila FliA has 55 and 43% amino acid identity with the homologous sequences of Pseudomonas aeruginosa and E. coli. Furthermore, the L. pneumophila fliA gene was able to restore the flagellation and the motility defect of an E. coli fliA mutant. This result suggests that the L. pneumophila sigma28 protein can bind to the E. coli core RNA polymerase to direct transcription initiation from the flaA-specific promoter.
KeywordMeSH Terms
111.     ( 1996 )

An oxaloacetate decarboxylase homologue protein influences the intracellular survival of Legionella pneumophila.

FEMS microbiology letters 145 (2)
PMID : 8961567  :   DOI  :   10.1111/j.1574-6968.1996.tb08589.x    
Abstract >>
Legionella pneumophila is a facultative intracellular parasite which is able to survive in various eukaryotic cells. We characterised a Tn5-mutant of the L. pneumophila Corby strain and were able to identify the insertion site of the transposon. It is localised within an open reading frame which shows high homology to the alpha-subunit of the oxaloacetate decarboxylase (OadA) of Klebsiella pneumoniae. The OadA homologous protein of L. pneumophila was detected in the wild-type strain by Western blotting. Since the intracellular multiplication of the oadA- mutant strain is reduced in guinea pig alveolar macrophages and human monocytes, it is concluded that the oadA gene product has an effect on the intracellular survival of L. pneumophila.
KeywordMeSH Terms
112.     ( 1996 )

A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila--characterization, molecular cloning and overexpression.

Molecular microbiology 21 (6)
PMID : 8898384  :   DOI  :   10.1046/j.1365-2958.1996.00061.x    
Abstract >>
Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires' disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPlase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPlases. The Icy gene (Legionella cyclophilin) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17968 Da called L. pneumophila cyclophilin 18 (L.p.Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coli with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histidine tag and an enterokinase cleavage site exhibits PPlase activity when produced at high levels in E. coli K-12. After removal of the extension by enterokinase, the properties of the recombinant Cyp18 were indistinguishable from those of the authentic enzyme. In order to investigate the influence of Cyp18 on intracellular survival of L. pneumophila an Icy-negative L. pneumophila strain was constructed. Compared with the wild-type strain, the mutant did not exhibit a significant phenotype but was 10-fold less invasive for Acanthamoeba castellanii. Like human cyclophilin, the L. p. Cyp18 exhibits nuclease activity, but this enzymatic activity does not appear to be linked with the native structure of the protein.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
113.     ( 1993 )

Cloning, nucleotide sequence and expression in Escherichia coli of a gene (ompM) encoding a 25 kDa major outer-membrane protein (MOMP) of legionella pneumophila.

Journal of general microbiology 139 (8)
PMID : 8409914  :   DOI  :   10.1099/00221287-139-8-1715    
Abstract >>
A genomic library derived from a virulent isolate of Legionella pneumophila was constructed in Escherichia coli JM 83 using the cloning vector pUC19. The clones were screened by filter immunoassay using L. pneumophila rabbit polyclonal antisera and in the absence of in situ bacterial lysis one such clone, LP 116, expressed L. pneumophila-specific antigens on the surface of E. coli. Restriction endonuclease digest analysis and agarose gel electrophoresis revealed a fragment measuring approximately 750 bp. Southern hybridization confirmed that the fragment was L. pneumophila DNA. Sequencing data showed that the fragment was 810 bp in length with an open reading frame (ORF) of 678 bp. The outer-membrane profiles of the E. coli parent, the L. pneumophila DNA-contributing strain and clone LP 116 were compared by SDS-PAGE. A protein of 25 kDa was found in outer-membrane preparations of both the clone LP 116 and L. pneumophila but not in E. coli JM 83. This was in agreement with the molecular mass of the deduced peptide of the mature protein. Immunoblots using L. pneumophila-specific polyclonal antiserum confirmed that this 25 kDa outer-membrane protein (OMP) was a L. pneumophila polypeptide. Both direct immunofluorescence assay and immunoblots using the commercially produced monoclonal antibody specific for the common antigen of the major outer-membrane protein (MOMP) confirmed that the 25 kDa protein produced by LP 116 was involved with the MOMP complex. The gene encoding this protein has been designated ompM.(ABSTRACT TRUNCATED AT 250 WORDS)
KeywordMeSH Terms
Bacterial Proteins
114.     ( 1997 )

An iron- and fur-repressed Legionella pneumophila gene that promotes intracellular infection and encodes a protein with similarity to the Escherichia coli aerobactin synthetases.

Infection and immunity 65 (1)
PMID : 8975903  :   PMC  :   PMC174567    
Abstract >>
Legionella pneumophila, a parasite of alveolar macrophages, requires iron for intra- and extracellular growth. Although its mechanisms for iron assimilation are poorly understood, this bacterium produces Fur, a protein that can repress gene transcription in response to iron concentration. Because iron- and Fur-regulated genes are important for infection in other bacteria, the identification of similar genes in L. pneumophila was undertaken. A wild-type strain of L. pneumophila was randomly mutated with a mini-Tn10' lacZ transposon, and the resulting gene fusions were tested for iron regulation by assessing beta-galactosidase production in the presence and absence of iron chelators. Of the initial six mutants with iron-repressed lacZ fusions, two strains, NU229 and NU232, possessed fusions that were stably iron regulated. To assay for Fur regulation, the levels of beta-galactosidase were measured in strains no longer producing Fur. As in a number of pathogenic bacteria, L. pneumophila fur could not be insertionally inactivated, but spontaneous Fur- derivatives were generated by selecting for manganese resistance. Strain NU229 contained a Fur-repressed fusion based on derepression of lacZ expression in its manganese-resistant derivative. Extracellular growth of NU229 in bacteriological media was similar to that of wild-type strain 130b. To assess the role of an iron- and Fur-regulated (frgA) gene in intracellular infection, the ability of NU229 to grow within U937 cell monolayers was tested. Quantitative infection assays demonstrated that intracellular growth of NU229 was impaired as much as 80-fold. Reconstruction of the mutant by allelic exchange proved that the infectivity defect in NU229 was due to the inactivation of frgA and not to a second-site mutation. Subsequently, complementation of the interrupted gene by an intact plasmid-encoded gene demonstrated that the infectivity defect was due to the loss of frgA and not to a polar effect. Nucleotide sequence analysis revealed that the 63-kDa FrgA protein has homology with the aerobactin synthetases IucA and IucC of Escherichia coli, raising the possibility that L. pneumophila encodes a siderophore which is required for optimal intracellular replication. Southern hybridization analysis determined that frgA is specific to L. pneumophila.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
115.     ( 1996 )

A Legionella pneumophila gene that promotes hemin binding.

Infection and immunity 64 (3)
PMID : 8641790  :   PMC  :   PMC173846    
Abstract >>
The ability to bind and utilize hemin is a trait common to many human pathogens. Nevertheless, the relationship between Legionella pneumophila, the agent of Legionnaires' disease, and hemin has received little attention. Thus, we explored the capacity of a virulent, serogroup 1 strain of L. pneumophila to bind hemin and use it as an iron source. Hemin, but not protoporphyrin IX, restored bacterial growth in iron-limiting media, indicating that it can serve as an iron source for L. pneumophila. In support of this idea, we observed that wildtype legionellae were able to bind 50 to 60% of added hemin, a binding capacity that was comparable to those of other pathogens. To begin to identify proteins involved in hemin acquisition, we identified a Legionella locus that conferred hemin binding upon Escherichia coli. Subcloning and nucleotide sequence analysis determined that a single open reading frame, which was designated hbp for hemin-binding promotion, was responsible for this binding activity. The hbp gene was predicted to encode a secreted, 15.5-kDa protein. To ascertain the importance of this gene in L. pneumophila biology, we used allelic exchange to construct an hbp mutant. Importantly, the mutant displayed a 42% reduction in hemin binding, confirming that hbp potentiates hemin acquisition by L. pneumophila. However, the strain was unaltered in its ability to grow within macrophage-like cells and freshwater amoebae, indicating that hbp is not required for intracellular infection. Despite this, Southern hybridization analysis and database searches demonstrated that hbp is nearly exclusive to the L. pneumophila species.
KeywordMeSH Terms
Genes, Bacterial
116.     ( 1996 )

The Legionella pneumophila hel locus encodes intracellularly induced homologs of heavy-metal ion transporters of Alcaligenes spp.

Infection and immunity 64 (5)
PMID : 8613357  :   PMC  :   PMC173958    
Abstract >>
We continued characterization of the Legionella pneumophila hel locus. Mutagenesis and DNA sequencing identified three genes similar to the czc and cnr loci of Alcaligenes eutrophus and the ncc locus of Alcaligenes xylosoxidans. On the basis of their similarity to these loci, we designated the L. pneumophila genes helC, helB, and helA. Mutations in the hel genes led to reduced cytopathicity towards U937 cells, although the mutant strains did not appear defective in other assays of virulence. Transcription of the hel locus was induced by the intracellular environment but was not induced by any of a variety of in vitro stress conditions. The function of the hel gene products remains to be determined.
KeywordMeSH Terms
117.     ( 1996 )

De novo synthesis of Legionella pneumophila antigens during intracellular growth in phagocytic cells.

Infection and immunity 64 (5)
PMID : 8613378  :   PMC  :   PMC173979    
Abstract >>
Legionella pneumophilia is a gram-negative rod which is able to multiply within phagocytic cells. The process of phagocytosis leads to a rapid environmental change that might require a coordinate regulation of gene expression to ensure intracellular survival. Since there is little information on up- and downregulation of genes during the early phases of phagocytosis, we radiolabeled intracellular L. pneumophila at different times after phagocytosis by macrophages of the Mono Mac 6 cell line and immunoprecipitated antigens with antilegionella sera or monoclonal antibodies. We could identify two antigens which were upregulated, one of which was the Mip protein, three antigens which were downregulated, and three antigens which were not detectable in extracellularly grown L. pneumophila. The Mip protein was stained most intensively 4 to 8 h after intracellular infection, suggesting that it is needed during intracellular multiplication rather than initiation of infection. A 44-kDa antigen which was not detectable during extracellular growth was most prominent from 2 to 4 h postinfection when Mono Mac 6 cells were used as phagocytic cells. The 44-kDa antigen was also expressed during growth with Acanthamoeba castelanii, MRC-5, and U937 cells but with different kinetics. Synthesis of this antigen was not dependent on protein synthesis of the host cell. Since the 44-kDa antigen could be precipitated by an antiserum produced against a recombinant Escherichia coli harboring a plasmid with an L. pneumophila insert which also codes for the mip gene, we believe that the corresponding gene is within the vicinity of the mip gene. We named this protein legionella intracellular growth antigen (LIGA), since it could be found exclusively in intracellularly grown L. pneumophila.
KeywordMeSH Terms
Immunophilins
Peptidylprolyl Isomerase
118.     ( 1996 )

Periplasmic copper-zinc superoxide dismutase of Legionella pneumophila: role in stationary-phase survival.

Journal of bacteriology 178 (6)
PMID : 8626284  :   DOI  :   10.1128/jb.178.6.1578-1584.1996     PMC  :   PMC177841    
Abstract >>
Copper-zinc superoxide dismutases (CuZnSODs) are infrequently found in bacteria although widespread in eukaryotes. Legionella pneumophila, the causative organism of Legionnaires' disease, is one of a small number of bacterial species that contain a CuZnSOD, residing in the periplasm, in addition to an iron SOD (FeSOD) in their cytoplasm. To investigate CuZnSOD function, we purified the enzyme from wild-type L. pneumophila, obtained amino acid sequence data from isolated peptides, cloned and sequenced the gene from a L. pneumophila library, and then constructed and characterized a CuZnSOD null mutant. In contrast to the cytoplasmic FeSOD, the CuZnSOD of L. pneumophila is not essential for viability. However, CuZnSOD is critical for survival during the stationary phase of growth. The CuZnSOD null mutant survived 10(4)- to 10(6)-fold less than wild-type L. pneumophila. In wild-type L. pneumophila, the specific activity of CuZnSOD increased during the transition from exponential to stationary-phase growth while the FeSOD activity was constant. These data support a role of periplasmic CuZnSOD in survival of L. pneumophila during stationary phase. Since L. pneumophila survives extensive periods of dormancy between growth within hosts. CuZnSOD may contribute to the ability of this bacterium to be a pathogen. In exponential phase, wild-type and CuZnSOD null strains grew with comparable doubling times. In cultured HL-60 and THP-1 macrophage-like cell lines and in primary cultures of human monocytes, multiplication of the CuZnSOD null mutant was comparable to that of wild type. This indicated that CuZnSOD is not essential for intracellular growth within macrophages or for killing of macrophages in those systems.
KeywordMeSH Terms
119.     ( 1993 )

Phenotypic modulation by Legionella pneumophila upon infection of macrophages.

Infection and immunity 61 (4)
PMID : 8454334  :   PMC  :   PMC281365    
Abstract >>
Since many pathogenic bacteria manifest a coordinate regulation of gene expression in response to different environmental stimuli, we examined the phenotypic response of Legionella pneumophila to infection of macrophage-like U937 cells. Intracellular L. pneumophila was radiolabeled, and cell extracts were subjected to two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At least 35 Legionella proteins were selectively induced during infection of macrophages, and one of these proteins was not detected in organisms grown in vitro. Expression of at least 32 proteins was selectively repressed during infection of macrophages, and 9 of these proteins were undetectable in intracellularly grown organisms. Thirteen of the macrophage-induced proteins were also induced by one or more of several stress conditions in vitro, and two of these proteins were the heat shock GroEL- and GroES-like proteins. Nineteen of the macrophage-repressed proteins were also repressed by one or more of the stress conditions in vitro. Our data showed that intracellular L. pneumophila manifested a phenotypic modulation and a global stress response to the intracellular environment of the macrophage. The data suggested that multiple regulons are involved in this modulation, which may contribute to the survival of L. pneumophila within alveolar macrophages.
KeywordMeSH Terms
120.     ( 1994 )

Cloning and molecular characterization of a Legionella pneumophila gene induced by intracellular infection and by various in vitro stress conditions.

Molecular microbiology 13 (2)
PMID : 7984104  :   DOI  :   10.1111/j.1365-2958.1994.tb00419.x    
Abstract >>
The synthesis of a global stress protein (GspA) of Legionella pneumophila is induced in the intracellular environment of the phagocytic cell and by various in vitro stress stimuli. We used techniques of reverse genetics to isolate the gspA gene from a genomic library of L. pneumophila. Sequence analysis of approximately 1700 bp of a representative clone (pBSP1) showed the presence of two open reading frames (ORFs). ORF1 encoded for a polypeptide with an inferred molecular mass of 19 kDa and an isoelectric point of 6.1. These predictions correlated with the migration of the GspA protein on two-dimensional SDS-polyacrylamide gels. The predicted amino acid sequence of the GspA protein was identical to 22/23 residues of the N-terminal amino acid sequence derived by Edman degradation of the purified protein. The GspA protein was 41.3% and 36.5% identical to the 16 kDa IbpA and IbpB heat-shock proteins, respectively, of Escherichia coli. Primer extension from mRNA isolated from L. pneumophila showed that transcription of the gspA gene was controlled by two overlapping promoters. One of the promoters was a sigma 70 promoter, while the other was a heat-shock promoter and was regulated by the sigma 32 transcription factor in E. coli. Northern blot analysis showed that the level of gspA mRNA was elevated 3.4-, 5.0-, and 6.7-fold after exposure of L. pneumophila to heat shock, oxidative stress and osmotic shock, respectively. The gspA gene was conserved among 13 serogroups of L. pneumophila. Our data showed that the gspA gene of L. pneumophila, which is induced by intracellular infection and by various stress stimuli, is controlled transcriptionally by two overlapping and separately regulated promoters.
KeywordMeSH Terms
Genes, Bacterial
Transcription Factors
121.     ( 1994 )

A putative regulatory gene downstream of recA is conserved in gram-negative and gram-positive bacteria.

Nucleic acids research 22 (7)
PMID : 8165147  :   DOI  :   10.1093/nar/22.7.1313     PMC  :   PMC523658    
Abstract >>
N/A
KeywordMeSH Terms
Conserved Sequence
Genes, Bacterial
Genes, Regulator
122.     ( 1993 )

Protein profiles of Legionella pneumophila Philadelphia-1 grown in macrophages and characterization of a gene encoding a novel 24 kDa Legionella protein.

Microbial pathogenesis 15 (6)
PMID : 8007818  :  
Abstract >>
Legionella pneumophila Philadelphia-1 strain was grown in cultured macrophages of guinea pigs, hamsters, and A/J mice and the bacteria were purified by Percoll density gradient centrifugation without any detergent. Patterns of the bacterial proteins were compared by SDS-PAGE with those of organisms cultured in vitro. A 24 kDa protein was a major protein of intracellularly grown bacteria: its expression was about four times as much as a 24 kDa protein of agar-grown bacteria. At least three novel proteins (100, 65, and 16 kDa) that were not found on agar-grown bacteria were also observed. In this paper, we focused on the 24 kDa major Legionella protein expressed within macrophages. Western blot and N-terminal amino acid analysis revealed that this protein is a novel protein different from Legionella proteins previously reported, including a 24 kDa macrophage infectivity potentiator protein (Mip). On the basis of amino acid sequence (MQRIKKI and IANAQGK), two kinds of oligonucleotides were synthesized and radiolabeled. Using these oligonucleotides as DNA probes, a 7.2 kb EcoRI-digested DNA fragment encoding the 24 kDa protein was cloned into lambda ZAP II phage vector in Escherichia coli XL1-Blue. A 0.9 kb DNA fragment from the 7.2 kb fragment was further subcloned into pUC118 in E. coli CSR603 for maxicell analysis or XL1-Blue for DNA sequencing. Maxicells which carry recombinant plasmids consisting of the 0.9 kb DNA fragment and vector plasmid pUC118 expressed the 24 kDa protein. When the DNA fragment encoding the protein was sequenced, an open reading frame of 555 base pairs was identified. The inferred polypeptide had a molecular weight of 20 kDa and an estimated isoelectric point of 8.14. Both the nucleotide sequence and the deduced amino acid sequence were distinct from those of bacterial proteins reported previously, suggesting that the protein is a novel Legionella protein.
KeywordMeSH Terms
123.     ( 1994 )

Sequence determination and mutational analysis of the lly locus of Legionella pneumophila.

Infection and immunity 62 (3)
PMID : 8112844  :   PMC  :   PMC186230    
Abstract >>
The lly (legiolysin) locus codes for a 39-kDa protein which confers hemolysis, pigment production, and fluorescence on recombinant Escherichia coli K-12 clones carrying the lly gene. The nucleotide sequences of the lly genes from two Legionella pneumophila isolates were determined. The lly loci exhibited identical nucleotide sequences. They contained open reading frames of 348 amino acid residues, encoding proteins with a deduced molecular mass of 38.9 kDa. N-terminal amino acid sequencing further confirmed that the Lly protein corresponds to the open reading frame sequenced. The amino acid sequence of the Lly protein exhibits a high degree of homology with the sequences of the MelA protein responsible for melanin production in the freshwater bacterium Shewanella colwelliana and the 4-hydroxyphenylpyruvate dioxygenase of Pseudomonas spp. 4-Hydroxyphenylpyruvate dioxygenase is involved in the degradation of aromatic amino acids in various organisms. An Lly-negative mutant of L. pneumophila Philadelphia I derivative JR32 and an Lly-positive transcomplementant were constructed. The Lly-negative mutant lost the ability to produce brown pigment and to confer fluorescence but retained hemolysis. Introduction of a plasmid carrying the lly locus restored pigment production and fluorescence. Intracellular survival of L. pneumophila in U937 macrophage-like cells and in Acanthamoeba castellanii was not affected by mutagenization of the lly locus.
KeywordMeSH Terms
Chromosome Mapping
Genes, Bacterial
124.     ( 1994 )

Shuttle mutagenesis of Legionella pneumophila: identification of a gene associated with host cell cytopathicity.

Infection and immunity 62 (9)
PMID : 8063428  :   PMC  :   PMC303072    
Abstract >>
We performed shuttle mutagenesis of Legionella pneumophila. Mutants were screened for reduced cellular infectivity. Approximately 10% of the mutants had decreased cytopathicity. The DNA sequence of one locus was determined; the inferred amino acid sequence revealed homology with transport proteins including Escherichia coli TolC, Bordetella pertussis CyaE, and Alcaligenes eutrophus CzcC and CnrC.
KeywordMeSH Terms
Genes, Bacterial
Mutagenesis
125.     ( 1994 )

Cloning and sequencing of the Legionella pneumophila fur gene.

Gene 143 (1)
PMID : 8200525  :   DOI  :   10.1016/0378-1119(94)90615-7    
Abstract >>
Iron is required for the intracellular and extracellular growth of Legionella pneumophila (Lp). In addition, variations in iron levels may serve as a signal for changes in gene expression. In a number of bacterial pathogens, the regulation of gene expression by iron is usually mediated by the Fur (ferric uptake regulation) repressor protein. Through complementation of an Escherichia coli fur mutation and nucleotide sequence analysis, we have cloned and characterized the Lp fur gene. Lp fur encoded a 15.0-kDa protein whose repressive activity was, as expected, highest in bacteria grown in iron-rich media. Computer analysis determined that Lp Fur had an amino-acid identity of over 54% and a similarity of over 72% to the Fur of E. coli, Yersinia pestis, Vibrio species and Pseudomonas aeruginosa. The promoter region of Lp fur contained sequences homologous to the Fur-binding site, suggesting that fur is autoregulated in Lp. Finally, Southern blot hybridizations demonstrated that fur is conserved among Lp strains and Legionella species.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
126. Berger  KH, Merriam  JJ, Isberg  RR,     ( 1994 )

Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene.

Molecular microbiology 14 (4)
PMID : 7891566  :   DOI  :   10.1111/j.1365-2958.1994.tb01317.x    
Abstract >>
Legionella pneumophila dot mutations cause defects in intracellular targeting of the microorganism within cultured macrophages. Each of the previously characterized dot mutations was shown to be complemented by a single open reading frame designated dotA. The defects caused by the mutations appear to be due to disrupted function of the predicted 1048-amino-acid residue DotA protein, and not by polarity effects on a downstream gene. Complementation studies indicated that the product of the dotA53 mutation results in a partially functional DotA protein, consistent with a stable N-terminal fragment having biological activity.
KeywordMeSH Terms
Genes, Bacterial
Mutation
127. Heuner  K, Bender-Beck  L, Brand  BC, Lück  PC, Mann  KH, Marre  R, Ott  M, Hacker  J,     ( 1995 )

Cloning and genetic characterization of the flagellum subunit gene (flaA) of Legionella pneumophila serogroup 1.

Infection and immunity 63 (7)
PMID : 7790062  :   PMC  :   PMC173334    
Abstract >>
The gene flaA, encoding the flagellum subunit protein of Legionella pneumophila serogroup 1, has been isolated from an expression library of L. pneumophila isolate Corby in Escherichia coli K-12 by using an antiflagellin specific polyclonal antiserum. DNA sequence analysis of the flaA gene revealed the presence of a 1,428-bp open reading frame encoding a protein of 475 amino acids with an apparent molecular mass of 48 kDa that is expressed independently of an E. coli vector promoter. Peptide sequencing of the N terminus of the isolated flagellum subunit protein confirmed that this open reading frame encodes the flagellin. By comparing the FlaA amino acid sequence with those of flagellins of various other bacteria, high degrees of homology in the N-terminal and C-terminal amino acids could be observed. The flaA-specific mRNA was determined to be 1.6 kb in size, the expected size of a monocistronic mRNA. Temperature-dependent expression of flagellin was found to be regulated at the transcriptional level. Sequence analysis and primer extension experiments indicated that the transcription of the gene flaA is directed by a sigma 28-like RpoF-FliA factor. By using fliA and fliA+ E. coli K-12 mutants, it was shown that flaA expression in E. coli required the sigma 28 factor. A flaA-specific DNA probe hybridizes with genomic DNA isolated from L. pneumophila and with most of the genomic DNAs from non-L. pneumophila Legionella strains. Two L. pneumophila strains and isolates of Legionella bozemanii and Legionella feeleii (serogroup 1) carry flaA-specific sequences but were not able to produce flagella.
KeywordMeSH Terms
128. Brand  BC, Sadosky  AB, Shuman  HA,     ( 1994 )

The Legionella pneumophila icm locus: a set of genes required for intracellular multiplication in human macrophages.

Molecular microbiology 14 (4)
PMID : 7891565  :   DOI  :   10.1111/j.1365-2958.1994.tb01316.x    
Abstract >>
Legionella pneumophila, the causative agent of Legionnaires' disease and related pneumonias, infects, replicates within and eventually kills human macrophages. A key feature of the intracellular life-style is the ability of the organism to replicate within a specialized phagosome which does not fuse with lysosomes or acidify. Avirulent mutants that are defective in intracellular multiplication and host-cell killing are unable to prevent phagosome-lysosome fusion. In a previous study, a 12 kb fragment of the L. pneumophila genome containing the icm locus (intracellular multiplication) was found to enable the mutant bacteria to prevent phagosome-lysosome fusion, to multiply intracellularly and to kill human macrophages. The complemented mutant also regained the ability to produce lethal pneumonia in guinea-pigs. In order to gain information about how L. pneumophila prevents phagosome-lysosome fusion and alters other intracellular events, we have studied the region containing the icm locus. This locus contains four genes, icmWXYZ, which appear to be transcribed from a single promoter to produce a 2.1-2.4 kb mRNA. The deduced amino acid sequences of the Icm proteins do not exhibit significant similarity to other proteins of known sequence, suggesting that they may carry out novel functions. The icmX gene encodes a product with an apparent signal sequence suggesting that it is a secreted protein. The icmWXYZ genes are located adjacent to and on the opposite strand from the dot gene, which is also required for intracellular multiplication and the ability of L. pneumophila to modify organelle traffic in human macrophages. Five L. pneumophila Icm mutants that had been generated with transposon Tn903dIIlacZ were found to have inserted the transposon within the icmX, icmY, icmZ and dot genes, confirming their role in the ability of the organism to multiply intracellularly.
KeywordMeSH Terms
Genes, Bacterial
129. Wüthrich  D, Gautsch  S, Spieler-Denz  R, Dubuis  O, Gaia  V, Moran-Gilad  J, Hinic  V, Seth-Smith  HM, Nickel  CH, Tschudin-Sutter  S, Bassetti  S, Haenggi  M, Brodmann  P, Fuchs  S, Egli  A,     ( 2019 )

Air-conditioner cooling towers as complex reservoirs and continuous source of Legionella pneumophila infection evidenced by a genomic analysis study in 2017, Switzerland.

Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin 24 (4)
PMID : 30696527  :   DOI  :   10.2807/1560-7917.ES.2019.24.4.1800192     PMC  :   PMC6351994    
Abstract >>
IntroductionWater supply and air-conditioner cooling towers (ACCT) are potential sources of Legionella pneumophila infection in people. During outbreaks, traditional typing methods cannot sufficiently segregate L. pneumophila strains to reliably trace back transmissions to these artificial water systems. Moreover, because multiple L. pneumophila strains may be present within these systems, methods to adequately distinguish strains are needed. Whole genome sequencing (WGS) and core genome multilocus sequence typing (cgMLST), with their higher resolution are helpful in this respect. In summer 2017, the health administration of the city of Basel detected an increase of L. pneumophila infections compared with previous months, signalling an outbreak.AimWe aimed to identify L. pneumophila strains populating suspected environmental sources of the outbreak, and to assess the relations between these strains and clinical outbreak strains.MethodsAn epidemiological and WGS-based microbiological investigation was performed, involving isolates from the local water supply and two ACCTs (n = 60), clinical outbreak and non-outbreak related isolates from 2017 (n = 8) and historic isolates from 2003-2016 (n = 26).ResultsIn both ACCTs, multiple strains were found. Phylogenetic analysis of the ACCT isolates showed a diversity of a few hundred allelic differences in cgMLST. Furthermore, two isolates from one ACCT showed no allelic differences to three clinical isolates from 2017. Five clinical isolates collected in the Basel area in the last decade were also identical in cgMLST to recent isolates from the two ACCTs.ConclusionCurrent outbreak-related and historic isolates were linked to ACCTs, which form a complex environmental habitat where strains are conserved over years.
KeywordMeSH Terms
L. pneumophila
Legionella pneumophila
Legionnaires’ disease
Switzerland
WGS
cooling tower
outbreak
whole genome sequencing
L. pneumophila
Legionella pneumophila
Legionnaires’ disease
Switzerland
WGS
cooling tower
outbreak
whole genome sequencing
130. Engleberg  NC, Carter  C, Weber  DR, Cianciotto  NP, Eisenstein  BI,     ( 1989 )

DNA sequence of mip, a Legionella pneumophila gene associated with macrophage infectivity.

Infection and immunity 57 (4)
PMID : 2925252  :   PMC  :   PMC313259    
Abstract >>
In a previous study, a 24-kilodalton (kDa) protein surface antigen of Legionella pneumophila was cloned into Escherichia coli and found to be expressed on the host cell surface. Subsequently, a site-directed mutation in this gene (designated mip) in L. pneumophila was found to impair the capacity of this bacterium to initiate intracellular infection in human macrophages. The work presented here indicates that the antigenic gene product is distinct from the 24- to 29-kDa major outer membrane protein of L. pneumophila. In addition, the antigen was identified as a highly basic protein on two-dimensional nonequilibrium polyacrylamide gels and on two-dimensional monoclonal antibody immunoblots. When the DNA fragment encoding this protein was sequenced, a long open reading frame of 699 base pairs was identified within a region to which antigen expression was previously mapped. mip mRNA isolated from both L. pneumophila and transformed E. coli had the same 5' end, as determined by primer extension analysis, indicating that the same promoter sequences are used in both species. A likely factor-independent transcriptional terminator was found 20 residues downstream of the stop codon, suggesting that mip is encoded on a monocistronic message. The inferred polypeptide began with a possible 20- to 24-residue signal sequence, and, as predicted by two-dimensional electrophoresis, had a molecular weight of 24,868 and was a potent polycation with an estimated pI of 9.8.
KeywordMeSH Terms
Genes, Bacterial
131. Sousa  PS, Silva  IN, Moreira  LM, Veríssimo  A, Costa  J,     ( 2018 )

Differences in Virulence Between Legionella pneumophila Isolates From Human and Non-human Sources Determined in Galleria mellonella Infection Model.

Frontiers in cellular and infection microbiology 8 (N/A)
PMID : 29670859  :   DOI  :   10.3389/fcimb.2018.00097     PMC  :   PMC5893783    
Abstract >>
Legionella pneumophila is a ubiquitous bacterium in freshwater environments and in many man-made water systems capable of inducing pneumonia in humans. Despite its ubiquitous character most studies on L. pneumophila virulence focused on clinical strains and isolates from man-made environments, so little is known about the nature and extent of virulence variation in strains isolated from natural environments. It has been established that clinical isolates are less diverse than man-made and natural environmental strains, suggesting that only a subset of environmental isolates is specially adapted to infect humans. In this work we intended to determine if unrelated L. pneumophila strains, isolated from different environments and with distinct virulence-related genetic backgrounds, displayed differences in virulence, using the Wax Moth Galleria mellonella infection model. We found that all tested strains were pathogenic in G. mellonella, regardless of their origin. Indeed, a panoply of virulence-related phenotypes was observed sustaining the existence of significant differences on the ability of L. pneumophila strains to induce disease. Taken together our results suggest that the occurrence of human infection is not related with the increased capability of some strains to induce disease since we also found a concentration threshold above which L. pneumophila strains are equally able to cause disease. In addition, no link could be established between the sequence-type (ST) and L. pneumophila pathogenicity. We envision that in man-made water distribution systems environmental filtering selection and biotic competition acts structuring L. pneumophila populations by selecting more resilient and adapted strains that can rise to high concentration if no control measures are implemented. Therefore, public health strategies based on the sequence based typing (STB) scheme analysis should take into account that the major disease-associated clones of L. pneumophila were not related with higher virulence in G. mellonella infection model, and that potential variability of virulence-related phenotypes was found within the same ST.
KeywordMeSH Terms
Galleria mellonella
Legionella pneumophila
disease
environmental selection
man-made environments
natural reservoirs
virulence
Galleria mellonella
Legionella pneumophila
disease
environmental selection
man-made environments
natural reservoirs
virulence
132. Wang  Y, Shi  M, Feng  H, Zhu  Y, Liu  S, Gao  A, Gao  P,     ( 2018 )

Structural Insights into Non-canonical Ubiquitination Catalyzed by SidE.

Cell 173 (5)
PMID : 29731171  :   DOI  :   10.1016/j.cell.2018.04.023    
Abstract >>
Ubiquitination constitutes one of the most important signaling mechanisms in eukaryotes. Conventional ubiquitination is catalyzed by the universally conserved E1-E2-E3 three-enzyme cascade in an ATP-dependent manner. The newly identified SidE family effectors of the pathogen Legionella pneumophila ubiquitinate several human proteins by a different mechanism without engaging any of the conventional ubiquitination machinery. We now report the crystal structures of SidE alone and in complex with ubiquitin, NAD, and ADP-ribose, thereby capturing different conformations of SidE before and after ubiquitin and ligand binding. The structures of ubiquitin bound to both mART and PDE domains reveal several unique features of the two reaction steps catalyzed by SidE. Further, the structural and biochemical results demonstrate that SidE family members do not recognize specific structural folds of the substrate proteins. Our studies provide both structural explanations for the functional observations and new insights into the molecular mechanisms of this non-canonical ubiquitination machinery.
KeywordMeSH Terms
ADP-ribosylation
ADP-ribosyltransferase
Legionella pneumophila
SdeA
SidE
host-pathogen interaction
phosphodiesterase
structure
ubiquitin
ubiquitination
ADP-ribosylation
ADP-ribosyltransferase
Legionella pneumophila
SdeA
SidE
host-pathogen interaction
phosphodiesterase
structure
ubiquitin
ubiquitination
133. Akturk  A, Wasilko  DJ, Wu  X, Liu  Y, Zhang  Y, Qiu  J, Luo  ZQ, Reiter  KH, Brzovic  PS, Klevit  RE, Mao  Y,     ( 2018 )

Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector.

Nature 557 (7707)
PMID : 29795346  :   DOI  :   10.1038/s41586-018-0147-6     PMC  :   PMC5980775    
Abstract >>
Ubiquitination is a post-translational modification that regulates many cellular processes in eukaryotes1-4. The conventional ubiquitination cascade culminates in a covalent linkage between the C terminus of ubiquitin (Ub) and a target protein, usually on a lysine side chain1,5. Recent studies of the Legionella pneumophila SidE family of effector proteins revealed a ubiquitination method in which a phosphoribosyl ubiquitin (PR-Ub) is conjugated to a serine residue on substrates via a phosphodiester bond6-8. Here we present the crystal structure of a fragment of the SidE family member SdeA that retains ubiquitination activity, and determine the mechanism of this unique post-translational modification. The structure reveals that the catalytic module contains two distinct functional units: a phosphodiesterase domain and a mono-ADP-ribosyltransferase domain. Biochemical analysis shows that the mono-ADP-ribosyltransferase domain-mediated conversion of Ub to ADP-ribosylated Ub (ADPR-Ub) and the phosphodiesterase domain-mediated ligation of PR-Ub to substrates are two independent activities of SdeA. Furthermore, we present two crystal structures of a homologous phosphodiesterase domain from the SidE family member SdeD 9 in complexes with Ub and ADPR-Ub. The structures suggest a mechanism for how SdeA processes ADPR-Ub to PR-Ub and AMP, and conjugates PR-Ub to a serine residue in substrates. Our study establishes the molecular mechanism of phosphoribosyl-linked ubiquitination and will enable future studies of this unusual type of ubiquitination in eukaryotes.
KeywordMeSH Terms
Ubiquitination
134. Lin  YH, Lucas  M, Evans  TR, Abascal-Palacios  G, Doms  AG, Beauchene  NA, Rojas  AL, Hierro  A, Machner  MP,     ( 2018 )

RavN is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases.

PLoS pathogens 14 (2)
PMID : 29415051  :   DOI  :   10.1371/journal.ppat.1006897     PMC  :   PMC5819833    
Abstract >>
The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway.
KeywordMeSH Terms
135. Prevost  MS, Waksman  G,     ( 2018 )

X-ray crystal structures of the type IVb secretion system DotB ATPases.

Protein science : a publication of the Protein Society 27 (8)
PMID : 29770512  :   DOI  :   10.1002/pro.3439     PMC  :   PMC6153414    
Abstract >>
Human infections by the intracellular bacterial pathogen Legionella pneumophila result in a severe form of pneumonia, the Legionnaire's disease. L. pneumophila utilizes a Type IVb secretion (T4bS) system termed "dot/icm" to secrete protein effectors to the host cytoplasm. The dot/icm system is powered at least in part by a functionally critical AAA+ ATPase, a protein called DotB, thought to belong to the VirB11 family of proteins. Here we present the crystal structure of DotB at 3.19 ? resolution, in its hexameric form. We observe that DotB is in fact a structural intermediate between VirB11 and PilT family proteins, with a PAS-like N-terminal domain coupled to a RecA-like C-terminal domain. It also shares critical structural elements only found in PilT. The structure also reveals two conformers, termed �\ and �], with an �\�]�\�]�\�] configuration. The existence of �\ and �] conformers in this class of proteins was confirmed by solving the structure of DotB from another bacterial pathogen, Yersinia, where, intriguingly, we observed an �\�\�]�\�\�] configuration. The two conformers co-exist regardless of the nucleotide-bound states of the proteins. Our investigation therefore reveals that these ATPases can adopt a wider range of conformational states than was known before, shedding new light on the extraordinary spectrum of conformations these ATPases can access to carry out their function. Overall, the structure of DotB provides a template for further rational drug design to develop more specific antibiotics to tackle Legionnaire's disease. PDB Code(s): Will; be; provided.
KeywordMeSH Terms
ATPase
DotB
Legionella pneumophila
Type IV secretion system
crystal structure
ATPase
DotB
Legionella pneumophila
Type IV secretion system
crystal structure
136. Dong  Y, Mu  Y, Xie  Y, Zhang  Y, Han  Y, Zhou  Y, Wang  W, Liu  Z, Wu  M, Wang  H, Pan  M, Xu  N, Xu  CQ, Yang  M, Fan  S, Deng  H, Tan  T, Liu  X, Liu  L, Li  J, Wang  J, Fang  X, Feng  Y,     ( 2018 )

Structural basis of ubiquitin modification by the Legionella effector SdeA.

Nature 557 (7707)
PMID : 29795342  :   DOI  :   10.1038/s41586-018-0146-7    
Abstract >>
Protein ubiquitination is a multifaceted post-translational modification that controls almost every process in eukaryotic cells. Recently, the Legionella effector SdeA was reported to mediate a unique phosphoribosyl-linked ubiquitination through successive modifications of the Arg42 of ubiquitin (Ub) by its mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains. However, the mechanisms of SdeA-mediated Ub modification and phosphoribosyl-linked ubiquitination remain unknown. Here we report the structures of SdeA in its ligand-free, Ub-bound and Ub-NADH-bound states. The structures reveal that the mART and PDE domains of SdeA form a catalytic domain over its C-terminal region. Upon Ub binding, the canonical ADP-ribosyltransferase toxin turn-turn (ARTT) and phosphate-nicotinamide (PN) loops in the mART domain of SdeA undergo marked conformational changes. The Ub Arg72 might act as a 'probe' that interacts with the mART domain first, and then movements may occur in the side chains of Arg72 and Arg42 during the ADP-ribosylation of Ub. Our study reveals the mechanism of SdeA-mediated Ub modification and provides a framework for further investigations into the phosphoribosyl-linked ubiquitination process.
KeywordMeSH Terms
Ubiquitination
137. Wasilko  DJ, Huang  Q, Mao  Y,     ( 2018 )

Insights into the ubiquitin transfer cascade catalyzed by the Legionella effector SidC.

eLife 7 (N/A)
PMID : 30015617  :   DOI  :   10.7554/eLife.36154     PMC  :   PMC6063727    
Abstract >>
The causative agent of Legionnaires' disease, Legionella pneumophila, delivers more than 330 virulent effectors to its host to establish an intracellular membrane-bound organelle called the Legionella containing vacuole. Among the army of Legionella effectors, SidC and its paralog SdcA have been identified as novel bacterial ubiquitin (Ub) E3 ligases. To gain insight into the molecular mechanism of SidC/SdcA as Ub ligases, we determined the crystal structures of a binary complex of the N-terminal catalytic SNL domain of SdcA with its cognate E2 UbcH5C and a ternary complex consisting of the SNL domain of SidC with the Ub-linked E2 UbcH7. These two structures reveal the molecular determinants governing the Ub transfer cascade catalyzed by SidC. Together, our data support a common mechanism in the Ub transfer cascade in which the donor Ub is immobilized with its C-terminal tail locked in an extended conformation, priming the donor Ub for catalysis.
KeywordMeSH Terms
HECT E3 ligase
UbcH5
UbcH7
legionella pneumophila
molecular biophysics
structural biology
HECT E3 ligase
UbcH5
UbcH7
legionella pneumophila
molecular biophysics
structural biology
Biocatalysis
138. Jia  Q, Lin  Y, Gou  X, He  L, Shen  D, Chen  D, Xie  W, Lu  Y,     ( 2018 )

Legionella pneumophila effector WipA, a bacterial PPP protein phosphatase with PTP activity.

Acta biochimica et biophysica Sinica 50 (6)
PMID : 29701815  :   DOI  :   10.1093/abbs/gmy042    
Abstract >>
The gram-negative bacterium Legionella pneumophila invades human's lung and causes Legionnaires' disease. To benefit its survival and replication in cellular milieu, L. pneumophila secrets at least 330 effector proteins into host cells. We found that the effector WipA has the protein tyrosine phosphatase (PTP) activity but does not depend on the classical CX5R motif for activity, suggesting that WipA is an unconventional PTP. Meanwhile, the presence of three other highly conserved motifs typically seen in protein serine/threonine phosphatases and the poor inhibition of WipA activity by okadaic acid led us to propose that WipA is a bacterial protein phosphatase. In addition, the determination of the 2.55-? crystal structure of WipA revealed that WipA resembles cold-active protein tyrosine phosphatase (CAPTPase), and therefore very likely shares the same catalytic mechanism.
KeywordMeSH Terms
139. Prevost  MS, Pinotsis  N, Dumoux  M, Hayward  RD, Waksman  G,     ( 2017 )

The Legionella effector WipB is a translocated Ser/Thr phosphatase that targets the host lysosomal nutrient sensing machinery.

Scientific reports 7 (1)
PMID : 28842705  :   DOI  :   10.1038/s41598-017-10249-6     PMC  :   PMC5572681    
Abstract >>
Legionella pneumophila infects human alveolar macrophages and is responsible for Legionnaire's disease, a severe form of pneumonia. L. pneumophila encodes more than 300 putative effectors, which are translocated into the host cell via the Dot/Icm type IV secretion system. These effectors highjack the host's cellular processes to allow bacterial intracellular growth and replication. Here we adopted a multidisciplinary approach to investigate WipB, a Dot/Icm effector of unknown function. The crystal structure of the N-terminal domain at 1.7 ? resolution comprising residues 25 to 344 revealed that WipB harbours a Ser/Thr phosphatase domain related to the eukaryotic phospho-protein phosphatase (PPP) family. The C-terminal domain (residues 365-524) is sufficient to pilot the effector to acidified LAMP1-positive lysosomal compartments, where WipB interacts with the v-ATPase and the associated LAMTOR1 phosphoprotein, key components of the lysosomal nutrient sensing (LYNUS) apparatus that controls the mammalian target of rapamycin (mTORC1) kinase complex at the lysosomal surface. We propose that WipB is a lysosome-targeted phosphatase that modulates cellular nutrient sensing and the control of energy metabolism during Legionella infection.
KeywordMeSH Terms
140.     ( 2013 )

Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP.

Cell research 23 (6)
PMID : 23588383  :   DOI  :   10.1038/cr.2013.54     PMC  :   PMC3674391    
Abstract >>
Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.
KeywordMeSH Terms
141.     ( 2013 )

Genetic Characterization of Legionella pneumophila Isolated from a Common Watershed in Comunidad Valenciana, Spain.

PloS one 8 (4)
PMID : 23634210  :   DOI  :   10.1371/journal.pone.0061564     PMC  :   PMC3636276    
Abstract >>
Legionella pneumophila infects humans to produce legionellosis and Pontiac fever only from environmental sources. In order to establish control measures and study the sources of outbreaks it is essential to know extent and distribution of strain variants of this bacterium in the environment. Sporadic and outbreak-related cases of legionellosis have been historically frequent in the Comunidad Valenciana region (CV, Spain), with a high prevalence in its Southeastern-most part (BV). Environmental investigations for the detection of Legionella pneumophila are performed in this area routinely. We present a population genetics study of 87 L. pneumophila strains isolated in 13 different localities of the BV area irrigated from the same watershed and compare them to a dataset of 46 strains isolated in different points of the whole CV. Our goal was to compare environmental genetic variation at two different geographic scales, at county and regional levels. Genetic diversity, recombination and population structure were analyzed with Sequence-Based Typing data and three intergenic regions. The results obtained reveal a low, but detectable, level of genetic differentiation between both datasets, mainly, but not only, attributed to the occurrence of unusual variants of the neuA locus present in the BV populations. This differentiation is still detectable when the 10 loci considered are analyzed independently, despite the relatively high incidence of the most common genetic variant in this species, sequence type 1 (ST-1). However, when the genetic data are considered without their associated geographic information, four major groups could be inferred at the genetic level which did not show any correlation with sampling locations. The overall results indicate that the population structure of these environmental samples results from the joint action of a global, widespread ST-1 along with genetic differentiation at shorter geographic distances, which in this case are related to the common watershed for the BV localities.
KeywordMeSH Terms
Water Microbiology
142.     ( 1998 )

Legionella pneumophila catalase-peroxidases: cloning of the katB gene and studies of KatB function.

Journal of bacteriology 180 (20)
PMID : 9765568  :   PMC  :   PMC107585    
Abstract >>
Legionella pneumophila, the causative organism of Legionnaires' pneumonia, is spread by aerosolization from man-made reservoirs, e.g. , water cooling towers and air conditioning ducts, whose nutrient-poor conditions are conducive to entrance into stationary phase. Exposure to starvation conditions is known to induce several virulence traits in L. pneumophila. Since catalase-peroxidases have been extremely useful markers of the stationary-phase response in many bacterial species and may be an avenue for identifying virulence genes in L. pneumophila, an investigation of these enzymes was initiated. L. pneumophila was shown to contain two bifunctional catalase-peroxidases and to lack monofunctional catalase and peroxidase. The gene encoding the KatB catalase-peroxidase was cloned and sequenced, and lacZ fusion and null mutant strains were constructed. Null mutants in katB are delayed in the infection and lysis of cultured macrophage-like cell lines. KatB is similar to the KatG catalase-peroxidase of Escherichia coli in its 20-fold induction during exponential growth and in playing a role in resistance to hydrogen peroxide. Analysis of the changes in katB expression and in the total catalase and peroxidase activity during growth indicates that the 8- to 10-fold induction of peroxidase activity that occurs in stationary phase is attributable to KatA, the second L. pneumophila catalase-peroxidase.
KeywordMeSH Terms
Bacterial Proteins
143.     ( 1998 )

Sequence-based classification scheme for the genus Legionella targeting the mip gene.

Journal of clinical microbiology 36 (6)
PMID : 9620377  :   PMC  :   PMC104877    
Abstract >>
The identification and speciation of strains of Legionella is often difficult, and even the more successful chromatographic classification techniques have struggled to discriminate newly described species. A sequence-based genotypic classification scheme is reported, targeting approximately 700 nucleotide bases of the mip gene and utilizing gene amplification and direct amplicon sequencing. With the exception of Legionella geestiana, for which an amplicon was not produced, the scheme clearly and unambiguously discriminated among the remaining 39 Legionella species and correctly grouped 26 additional serogroup and reference strains within those species. Additionally, the genotypic classification of approximately 150 wild strains from several continents was consistent with their phenotypic classification, with the exception of a few strains where serological cross-reactivity was complex, potentially confusing the latter classification. Strains thought to represent currently uncharacterized species were also found to be genotypically unique. The scheme is technically simple for a laboratory with even basic molecular capabilities and equipment, if access to a sequencing laboratory is available.
KeywordMeSH Terms
Bacterial Typing Techniques
Immunophilins
Peptidylprolyl Isomerase
144.     ( 1998 )

Identification of the aspartate-beta-semialdehyde dehydrogenase gene of Legionella pneumophila and characterization of a null mutant.

Infection and immunity 66 (5)
PMID : 9573067  :   PMC  :   PMC108141    
Abstract >>
The ability of Legionella pneumophila to cause Legionnaires' disease is dependent on its capacity to survive in the intracellular environment of its host cells. Furthermore, outbreaks of this disease have been associated with contaminated water sources where L. pneumophila survives as a parasite of protozoa. In this study, we determined the effect of nutritional auxotrophy on the ability of L. pneumophila to survive in the intracellular environment of its host cells. We generated a diaminopimelic acid (DAP) auxotroph (AA400) of L. pneumophila by disruption of the aspartate-beta-semialdehyde (asd) gene. The ability of AA400 to survive within macrophages and protozoa was found to be defective. This defect was due solely to the asd disruption since complementation of the mutant with the wild-type asd gene restored its capacity for intracellular survival. Furthermore, the defect was not completely complemented by DAP supplementation to the culture media. Thus, our results suggest that disruption of the asd gene may prove to be useful in the design of attenuated vaccines against Legionnaires' disease.
KeywordMeSH Terms
Genes, Bacterial
145.     ( 1998 )

Induced expression of the Legionella pneumophila gene encoding a 20-kilodalton protein during intracellular infection.

Infection and immunity 66 (1)
PMID : 9423859  :   PMC  :   PMC107878    
Abstract >>
The eukaryotic protein synthesis inhibitor cycloheximid has been used by many investigators to selectively radiolabel intracellular bacteria. Although cycloheximide has no direct effect on bacterial gene expression, there are concerns that long-term inhibition of the host cell protein synthesis may have secondary effects on bacterial gene expression. Therefore, prior to further identification and cloning of the macrophage-induced (MI) genes of Legionella pneumophila, the effects of cycloheximide on L. pneumophila-infected U937 cells were evaluated by transmission electron microscopy. Inhibition of protein synthesis of the host cell for 6 h had no major effect on the ultrastructure of the host cell, on the formation of rough endoplasmic reticulum-surrounded replicative phagosome, or on initiation of intracellular bacterial replication. In contrast, by 15 h of cycloheximide treatment, there was profound deterioration in the host cell as well as in the phagosome. To examine protein synthesis by L. pneumophila during the intracellular infection, U937 macrophage-like cells were infected with L. pneumophila, and intracellular bacteria were radiolabeled during a 2-h cycloheximide treatment or following 12 h of cycloheximide treatment. Comparison by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein profile of radiolabeled in vitro-grown L. pneumophila to that of intracellularly radiolabeled bacteria showed that 23 proteins were induced in response to the intracellular environment during 2 h of inhibition of host cell protein biosynthesis. Twelve MI proteins of L. pneumophila were artifactually induced due to prolonged inhibition of the host cell protein synthesis. The gene encoding a 20-kDa MI protein was cloned by a reverse genetics technique. Sequence analysis showed that the cloned gene encoded a protein that was 80% similar to the enzyme inorganic pyrophosphatase. Studies of promoter fusion to a promoterless lacZ gene showed that compared to in vitro-grown bacteria, expression of the pyrophosphatase gene (ppa) was induced fourfold throughout the intracellular infection. There was no detectable induction in transcription of the ppa promoter during exposure to stress stimuli in vitro. The ppa gene of L. pneumophila is the first example of a regulated ppa gene which is selectively induced during intracellular infection and which may reflect enhanced capabilities of macromolecular biosynthesis by intracellular L. pneumophila. The data indicate caution in the long-term use of inhibition of host cell protein synthesis to selectively examine gene expression by intracellular bacteria.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
146.     ( 1998 )

Conjugative transfer by the virulence system of Legionella pneumophila.

Science (New York, N.Y.) 279 (5352)
PMID : 9452389  :   DOI  :   10.1126/science.279.5352.873    
Abstract >>
Legionella pneumophila, the causative agent of Legionnaires' pneumonia, replicates within alveolar macrophages by preventing phagosome-lysosome fusion. Here, a large number of mutants called dot (defective for organelle trafficking) that were unable to replicate intracellularly because of an inability of the bacteria to alter the endocytic pathway of macrophages were isolated. The dot virulence genes encoded a large putative membrane complex that functioned as a secretion system that was able to transfer plasmid DNA from one cell to another.
KeywordMeSH Terms
Conjugation, Genetic
147.     ( 1997 )

Characterization of a new region required for macrophage killing by Legionella pneumophila.

Infection and immunity 65 (12)
PMID : 9393796  :   PMC  :   PMC175729    
Abstract >>
In a previous study, a collection of 55 Legionella pneumophila mutants defective for macrophage killing was isolated by transposon mutagenesis. In this study, nine of these mutants that belong to the same DNA hybridization group (group 3) were characterized. A wild-type DNA fragment that covers this DNA hybridization group was cloned and sequenced. This region was found to contain six new genes (designated icmT, icmS, icmR, icmQ, icmP, and icmO), five of which contain at least one transposon insertion. No transposon insertion was found in icmS. However, this gene was found to be required for macrophage killing, since a kanamycin resistance cassette introduced into icmS by gene replacement resulted in a mutant that was attenuated for macrophage killing. A plasmid containing the DNA fragment that covers this region complements all the mutants for macrophage killing, although various levels of complementation were observed for mutants in different genes. Complementation tests were also performed with plasmids containing one or two of these genes, as well as with plasmids containing nonpolar in-frame deletions. The results from these complementation tests indicated that all six genes located in this region are needed for macrophage killing and that they are probably arranged as two transcriptional units (icmTS and icmPO) and two genes (icmR and icmQ). A region upstream of the coding sequence of several icm genes may contain a potential promoter and/or regulatory site. Homology searches show that icmP and icmO bear significant homology to the trbA and trbC genes from the Salmonella R64 plasmid, respectively. The sequences of the other four genes do not show significant homology with any entries in sequence databases.
KeywordMeSH Terms
Mutation
148.     ( 1998 )

Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome.

Proceedings of the National Academy of Sciences of the United States of America 95 (4)
PMID : 9465074  :   DOI  :   10.1073/pnas.95.4.1669     PMC  :   PMC19142    
Abstract >>
A 22-kb DNA locus of Legionella pneumophila is described that contains 18 genes, 16 of which are required for macrophage killing (icm genes). In this paper two previously described icm loci were linked by the discovery of five genes located between the two loci. Four of the newly described genes are required for macrophage killing (icmMLKE) and one is dispensable. The 16 icm genes appeared to be organized as six individual genes (icmR, icmQ, icmG, icmC, icmD, and icmF), and four operons (icmTS, icmPO, icmMLKE, and icmJB). Four icm genes (icmP, icmO, icmL, and icmE) show significant sequence similarity to plasmid genes involved in conjugation, whereas the other icm genes were found not to bear any sequence similarity to database entries. We found that L. pneumophila can mediate plasmid DNA transfer at a frequency of 10(-3) to 10(-4) per donor. Strains containing null mutations in two icm genes (icmT and icmR) showed a severe reduction in conjugation frequency and macrophage killing. Strains containing an insertion in four other icm genes (icmF, icmE, icmC, and dotA) were shown to have a less severe defect in conjugation. Mutations in the other 11 icm genes had no effect on conjugation frequency. We currently do not know whether conjugation itself plays a role in macrophage killing. It is possible either that small plasmids can take advantage of an existing secretion system to be mobilized or that DNA transfer is required for human macrophage killing by L. pneumophila.
KeywordMeSH Terms
Genes, Bacterial
149.     ( 1998 )

The Legionella pneumophila icmGCDJBF genes are required for killing of human macrophages.

Infection and immunity 66 (5)
PMID : 9573114  :   PMC  :   PMC108188    
Abstract >>
Previously, a collection of mutants of Legionella pneumophila that had lost the ability to multiply within and kill human macrophages was generated by Tn903dIIlacZ transposon mutagenesis and classified into DNA hybridization groups. A subset of these mutants was complemented by a plasmid, pMW100, containing a 13.5-kb genomic DNA insert. This plasmid restored the ability to multiply within and produce cytopathic effects on human macrophages to members of DNA hybridization groups II, IV, VI, and XVII. A region of the genomic insert of pMW100 was sequenced, and eight potential genes were identified and named icmE, icmG, icmC, icmD, icmJ, icmB, icmF, and tphA. None of the genes encode potential protein products with significant homology to previously characterized proteins, except for tphA, whose product has significant homology to a family of metabolite/H+ symport proteins from gram-negative bacteria. The positions of the Tn903dIIlacZ insertions within the genes were determined by nucleotide sequencing. No Tn903dIIlacZ insertions mapped to icmG, icmJ, or tphA; therefore, these loci were mutated to test whether they were required for macrophage killing. Complementation analysis was used to evaluate the roles of the potential gene products and provide information on the organization of transcriptional units within the region. The results indicate that all identified open reading frames except tphA are required for killing of human macrophages.
KeywordMeSH Terms
Genes, Bacterial
150.     ( 1998 )

Identification and temperature regulation of Legionella pneumophila genes involved in type IV pilus biogenesis and type II protein secretion.

Infection and immunity 66 (4)
PMID : 9529113  :   PMC  :   PMC108120    
Abstract >>
Previously, we had isolated by transposon mutagenesis a Legionella pneumophila mutant that appeared defective for intracellular iron acquisition. While sequencing in the proximity of the mini-Tn10 insertion, we found a locus that had a predicted protein product with strong similarity to PilB from Pseudomonas aeruginosa. PilB is a component of the type II secretory pathway, which is required for the assembly of type IV pili. Consequently, the locus was cloned and sequenced. Within this 4-kb region were three genes that appeared to be organized in an operon and encoded homologs of P. aeruginosa PilB, PilC, and PilD, proteins essential for pilus production and type II protein secretion. Northern blot analysis identified a transcript large enough to include all three genes and showed a substantial increase in expression of this operon when L. pneumophila was grown at 30 degrees C as opposed to 37 degrees C. The latter observation was then correlated with an increase in piliation when bacteria were grown at the lower temperature. Southern hybridization analysis indicated that the pilB locus was conserved within L. pneumophila serogroups and other Legionella species. These data represent the first isolation of type II secretory genes from an intracellular parasite and indicate that the legionellae express temperature-regulated type IV pili.
KeywordMeSH Terms
Genes, Bacterial
151.     ( 1998 )

Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells.

Infection and immunity 66 (4)
PMID : 9529112  :   PMC  :   PMC108119    
Abstract >>
Legionella pneumophila expresses pili of variable lengths, either long (0.8 to 1.5 microm) or short (0.1 to 0.6 microm), that can be observed by transmission electron microscopy. We have identified a gene in L. pneumophila with homology to the type IV pilin genes (pilEL). An insertion mutation was constructed in pilEL and introduced into the L. pneumophila wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The L. pneumophila pilEL mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and Acanthamoeba polyphaga but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that the pilEL locus is present in L. pneumophila serogroups 1 through 13 but is variable in 16 other Legionella species. The presence of a type IV pilin gene and its expression by L. pneumophila may provide an advantage for colonization of lung tissues during Legionnaires' disease and invasion of amoebas in the environment.
KeywordMeSH Terms
Bacterial Adhesion
Genes, Bacterial
152.     ( 1998 )

Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway.

Infection and immunity 66 (3)
PMID : 9488381  :   PMC  :   PMC108001    
Abstract >>
Legionella pneumophila replicates within a specialized phagosome in cultured cells, a function necessary for its pathogenicity. The replicative phagosome lacks membrane marker proteins, such as the glycoprotein LAMP-1, that are indicators of the normal endocytic pathway. We describe the isolation of several Legionella genes essential for intracellular growth and evasion of the endocytic pathway, using a genetic and cell biological approach. We screened 4,960 ethyl methanesulfonate-mutagenized colonies for defects in intracellular growth and trafficking to the replicative phagosome. Six mutant strains of L. pneumophila that had severe intracellular growth defects in mouse bone marrow-derived macrophages were identified. All six mutants were found in phagosomes that colocalized with LAMP-1, indicating defects in intracellular trafficking. The growth defects of two of these strains were complemented by molecular clones from a bank constructed from a wild-type L. pneumophila strain. The inserts from these clones are located in a region of the chromosome contiguous with several other genes essential for intracellular growth. Three mutants could be complemented by single open reading frames placed in trans, one mutant by a gene termed dotH and two additional mutants by a gene termed dotO. A deletion mutation was created in a third gene, dotI, which is located directly upstream of dotH. The delta dotI strain was also defective for intracellular growth in macrophages, and this defect was complemented by a single open reading frame in trans. Based on sequence analysis and structural predictions, possible roles of dotH, dotI, and dotO in intracellular growth are discussed.
KeywordMeSH Terms
Endocytosis
Genes, Bacterial

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