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1.
2.
Examination of cell-free culture supernatants revealed that Legionella pneumophila strains secrete an endoglucanase activity. Legionella pneumophila lspF mutants were deficient for this activity, indicating that the endoglucanase is secreted by the bacterium's type II protein secretion (T2S) system. Inactivation of celA , encoding a member of the family-5 of glycosyl hydrolases, abolished the endoglucanase activity in L. pneumophila culture supernatants. The cloned celA gene conferred activity upon recombinant Escherichia coli . Thus, CelA is the major secreted endoglucanase of L. pneumophila . Mutants inactivated for celA grew normally in protozoa and macrophage, indicating that CelA is not required for the intracellular phase of L. pneumophila . The CelA endoglucanase is one of at least 25 proteins secreted by the type II system of L. pneumophil a and the 17th type of enzyme effector associated with this pathway. Only a subset of the other Legionella species tested expressed secreted endoglucanase activity, suggesting that the T2S output differs among the different legionellae. Overall, this study represents the first documentation of an endoglucanase (EC 3.2.1.4) being produced by a strain of Legionella . 相似文献
3.
When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent "sliding" motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment. 相似文献
4.
Multiple roles of the pilus biogenesis protein pilD: involvement of pilD in excretion of enzymes from Pseudomonas aeruginosa. 总被引:21,自引:5,他引:16
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In Pseudomonas aeruginosa, the genes pilB, pilC, and pilD encode proteins necessary for posttranslational modification and assembly of pilin monomers into pilus organelles (D. Nunn, S. Bergman, and S. Lory, J. Bacteriol. 172:2911-2919, 1990). We show that PilD, encoding a putative pilin-specific leader peptidase, also controls export of alkaline phosphatase, phospholipase C, elastase, and exotoxin A. pilD mutants accumulate these proteins in the periplasmic space, while secretion of periplasmic and outer membrane proteins appears to be normal. The periplasmic form of exotoxin A was fully mature in size, contained all cysteines in disulfide bonds, and was toxic in a tissue culture cytotoxicity assay, suggesting that in pilD mutants, exotoxin A was folded into its native conformation. The function of the other two accessory proteins, PilB and PilC, appears to be restricted to pilus biogenesis, and strains carrying mutations in their respective genes do not show an export defect. These studies show that in addition to cleaving the leader sequence from prepilin, PilD has an additional role in secretion of proteins that are released from P. aeruginosa into the surrounding media. PilD most likely functions as a protease that is involved in processing and assembly of one or more components of the membrane machinery necessary for the later stages of protein extracellular localization. 相似文献
5.
Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD , was identified by its ability to complement the pilD mutation in P. aeruginosa . Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes ( tapABCD ) that are homologous to P. aeruginosa type IV pilus biogenesis genes ( pilABCD ). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N -methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila 相似文献
6.
Legionella pneumophila, the causative agent of Legionnaires' disease, survives in macrophages by altering the endocytic pathway of its host cell. To accomplish this, the bacterium utilizes a type IVB secretion system to deliver effector molecules into the host cell cytoplasm. In a previous report, we performed an extensive characterization of the L. pneumophila type IVB secretion system that resulted in the identification of a critical five-protein subcomplex that forms the core of the secretion apparatus. Here we describe a second Dot/Icm protein subassembly composed of the type IV coupling protein DotL, the apparatus proteins DotM and DotN, and the secretion adaptor proteins IcmS and IcmW. In the absence of IcmS or IcmW, DotL becomes destabilized at the transition from the exponential to stationary phases of growth, concurrent with the expression of many secreted substrates. Loss of DotL is dependent on ClpA, a regulator of the cytoplasmic protease ClpP. The resulting decreased levels of DotL in the icmS and icmW mutants exacerbates the intracellular defects of these strains and can be partially suppressed by overproduction of DotL. Thus, in addition to their role as chaperones for Legionella type IV secretion system substrates, IcmS and IcmW perform a second function as part of the Dot/Icm type IV coupling protein subcomplex. 相似文献
7.
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. 相似文献
8.
Legionella pneumophila is the Gram-negative bacterial agent of Legionnaires' disease, an acute, often fatal pneumonia. L. pneumophila infects alveolar macrophages, evading the antimicrobial defences of the phagocyte by preventing fusion of the phagosome with lysosomes and avoiding phagosome acidification. The bacteria then modulate the composition of the vacuole so that it takes on the characteristics of the endoplasmic reticulum. Similar events occur when the bacteria infect unicellular protozoa. It is thought that replication in fresh water protozoa provides an environmental reservoir for the organism. Several effector proteins are delivered to the host by the Icm/Dot type IV secretion system (TFSS). Some of these have been shown to participate in the trafficking of the Legionella phagosome. Here we describe the ability of the Icm/Dot TFSS to translocate two effectors, LepA and LepB, that play a role in the non-lytic release of Legionella from protozoa. We report that translocation of the Lep proteins is inhibited by agents that depolymerize actin filaments and that effectors may be secreted into the extracellular medium upon cell contact. Depletion of the Lep proteins by deletion of their genes results in increased ability to lyse red blood cells. In contrast, overexpression of Lep-containing hybrid proteins appears to specifically inhibit the activity of the Icm/Dot TFSS and may prevent the delivery of other effectors that are critical for intracellular multiplication. 相似文献
9.
Flieger A Gong S Faigle M Stevanovic S Cianciotto NP Neumeister B 《Journal of bacteriology》2001,183(6):2121-2124
We show that Legionella pneumophila possesses lysophospholipase A activity, which releases fatty acids from lysophosphatidylcholine. The NH2-terminal sequence of the enzyme contained FGDSLS, corresponding to a catalytic domain in a recently described group of lipolytic enzymes. Culture supernatants of a L. pneumophila pilD mutant lost the ability to cleave lysophosphatidylcholine. 相似文献
10.
The intracellular pathogen Legionella pneumophila has evolved sophisticated mechanisms that enable it to subvert host functions, enter, survive and replicate in amoebae or alveolar macrophages, and to finally evade these hosts. Protozoa are essential for the growth of Legionella and the interaction with amoeba seems to be the driving force in the evolution of its pathogenicity. This is reflected in the genome of this pathogen, which encodes a high number and variety of eukaryotic-like proteins that are able to interfere in the various steps of the infectious cycle by mimicking functions of eukaryotic proteins. Central to the pathogenicity of L. pneumophila are the many secretion systems delivering these and other effectors to the host cell. Recent studies have highlighted the multi-functional role of these factors secreted by L. pneumophila, in host-pathogen interactions. 相似文献
11.
Zusman T Aloni G Halperin E Kotzer H Degtyar E Feldman M Segal G 《Molecular microbiology》2007,63(5):1508-1523
Legionella pneumophila and Coxiella burnetii have been shown to utilize the icm/dot type IV secretion system for pathogenesis and recently a large number of icm/dot-translocated substrates were identified in L. pneumophila. Bioinformatic analysis has revealed that 13 of the genes encoding for L. pneumophila-translocated substrates and five of the C. burnetii icm/dot genes, contain a conserved regulatory element that resembles the target sequence of the PmrA response regulator. Experimental analysis which included the construction of a L. pneumophila pmrA deletion mutant, intracellular growth analysis, comparison of gene expression between L. pneumophila wild type and the pmrA mutant, construction of mutations in the PmrA conserved regulatory element, controlled expression studies as well as mobility shift assays, demonstrated the direct relation between the PmrA regulator and the expression of L. pneumophila icm/dot-translocated substrates and several C. burnetii icm/dot genes. Furthermore, genomic analysis identified 35 L. pneumophila and 68 C. burnetii unique genes that contain the PmrA regulatory element and few of these genes from L. pneumophila were found to be new icm/dot-translocated substrates. Our results establish the PmrA regulator as a fundamental regulator of the icm/dot type IV secretion system in these two bacteria. 相似文献
12.
Legionella pneumophila infects alveolar macrophages and protozoa through establishment of an intracellular replication niche. This process is mediated by bacterial effectors translocated into the host cell via the Icm/Dot type IV secretion system. Most of the effectors identified so far are unique to L. pneumophila ; however, some of the effectors are homologous to eukaryotic proteins. We performed a distribution analysis of many known L. pneumophila effectors and found that several of them, mostly eukaryotic homologous proteins, are present in different Legionella species. In-depth analysis of LegS2, a L. pneumophila homologue of the highly conserved eukaryotic enzyme sphingosine-1-phosphate lyase (SPL), revealed that it was most likely acquired from a protozoan organism early during Legionella evolution. The LegS2 protein was found to translocate into host cells using a C-terminal translocation domain absent in its eukaryotic homologues. LegS2 was found to complement the sphingosine-sensitive phenotype of a Saccharomyces serevisia SPL-null mutant and this complementation depended on evolutionary conserved residues in the LegS2 catalytic domain. Interestingly, unlike the eukaryotic SPL that localizes to the endoplasmic reticulum, LegS2 was found to be targeted mainly to host cell mitochondria. Collectively, our results demonstrate the remarkable adaptations of a eukaryotic protein to the L. pneumophila pathogenesis system. 相似文献
13.
The Dot/Icm type IV secretion system of Legionella pneumophila translocates numerous bacterial effectors into the host cell and is essential for bacterial proliferation within macrophages and protozoa. We have recently shown that L. pneumophila strain AA100/130b harbours 11 genes encoding eukaryotic-like ankyrin (Ank) proteins, a family of proteins involved in various essential eukaryotic cellular processes. In contrast to most Dot/Icm-exported substrates, which have little or no detectable role in intracellular proliferation, a mutation in ankB results in a severe growth defect in intracellular replication within human monocyte-derived macrophages (hMDMs), U937 macrophages and Acanthamoeba polyphaga. Single cell analyses of coinfections of hMDMs have shown that the intracellular growth defect of the ankB mutant is totally rescued in cis within communal phagosomes harbouring the wild type strain. Interestingly, distinct from dot/icm structural mutants, the ankB mutant is also rescued in trans within cells harbouring the wild type strain in a different phagosome, indicating that AnkB is a trans-acting secreted effector. Using adenylate cyclase fusions to AnkB, we show that AnkB is translocated into the host cell via the Dot/Icm secretion system in an IcmSW-dependent manner and that the last three C-terminal amino acid residues are essential for translocation. Distinct from the dot/icm structural mutants, the ankB mutant-containing phagosomes exclude late endosomal and lysosomal markers and their phagosomes are remodelled by the rough endoplasmic reticulum. We show that at the postexponential phase of growth, the LetA/S and PmrA/B Two Component Systems confer a positive regulation on expression of the ankB gene, whereas RpoS, LetE and RelA suppress its expression. Our data show that the eukaryotic-like AnkB protein is a Dot/Icm-exported effector that plays a major role in intracellular replication of L. pneumophila within macrophages and protozoa, and its expression is temporally controlled by regulators of the postexponential phase of growth. 相似文献
14.
A plasmid-encoded prepilin peptidase gene from enteropathogenic Escherichia coli. 总被引:4,自引:1,他引:3
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Enteropathogenic Escherichia coli, a leading agent of infantile diarrhea worldwide, adheres to tissue culture cells in a pattern called "localized adherence." Localized adherence is associated with bundle-forming pili encoded by the plasmid bfpA gene, the product of which is homologous with the major structural subunit proteins of type IV fimbriae in other bacteria. Several of these proteins have been shown to be processed from a precursor by a specific prepilin peptidase. We cloned restriction fragments downstream of the bfpA gene into an E. coli-Pseudomonas aeruginosa shuttle vector and mobilized them into a P. aeruginosa prepilin peptidase (pilD) mutant. A plasmid containing a 1.3-kb PstI-BamHI fragment was able to complement the pilD mutation, as demonstrated by restoration of sensitivity to the pilus-specific bacteriophage PO4. The DNA sequence of this fragment revealed an open reading frame, designated bfpP, the predicted product of which is homologous to other prepilin peptidases, including TcpJ of Vibrio cholerae (30% identical amino acids), PulO of Klebsiella oxytoca (29%), and PilD of P. aeruginosa (28%). A bfpA::TnphoA mutant complemented with a bfpA-containing DNA fragment only partially processes the BfpA protein. When complemented with a larger fragment containing bfpP as well as bfpA, the mutant expresses the fully processed BfpA protein. P. aeruginosa PAK, but not a pilD mutant of PAK, expresses mature BfpA protein when the bfpA gene is mobilized into this strain. Thus, as in other type IV fimbria systems, enteropathogenic E. coli utilizes a specific prepilin peptidase to process the major subunit of the bundle-forming pilus. This prepilin petidase contains sequence and reciprocal functional homologies with the PilD protein of P. aeruginosa. 相似文献
15.
Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W. 总被引:9,自引:2,他引:7
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Four components of the apparatus of extracellular protein secretion of Pseudomonas aeruginosa, Xcpt, -U, -V, and -W (XcpT-W), are synthesized as precursors with short N-terminal leader peptides that share sequence similarity with the pilin subunit of this organism. A specialized leader peptidase/methylase, product of the pilD gene, has been shown to cleave the leader peptide from prepilin and to methylate the N-terminal phenylalanine of the mature pilin. Antibodies were prepared against XcpT-W and used to purify each of these proteins. Sequence analysis of XcpT-W has shown that these proteins, like mature pilin, contain N-methylphenylalanine as the N-terminal amino acid. Analysis of cellular fractions from wild-type and pilD mutant strains of P. aeruginosa showed that the precursor forms of XcpT-W are located predominantly in the bacterial inner membrane, and their localization is not altered after PilD-mediated removal of the leader sequence. These studies demonstrate that the biogenesis of the apparatus of extracellular protein secretion and that of type IV pili share a requirement for PilD. This bifunctional enzyme, acting in the inner membrane, cleaves the leader peptides from precursors of pilins and XcpT-W and subsequently methylates the amino group of the N-terminal phenylalanine of each of its substrates. 相似文献
16.
Lippmann J Müller HC Naujoks J Tabeling C Shin S Witzenrath M Hellwig K Kirschning CJ Taylor GA Barchet W Bauer S Suttorp N Roy CR Opitz B 《Cellular microbiology》2011,13(11):1668-1682
Defence mechanisms against intracellular bacterial pathogens are incompletely understood. Our study characterizes a type I IFN-dependent cell-autonomous defence pathway directed against Legionella pneumophila, an intracellular model organism and frequent cause of pneumonia. We show that macrophages infected with L. pneumophila produced IFNβ in a STING- and IRF3- dependent manner. Paracrine type I IFNs stimulated upregulation of IFN-stimulated genes and a cell-autonomous defence pathway acting on replicating and non-replicating Legionella within their specialized vacuole. Our infection experiments in mice lacking receptors for type I and/or II IFNs show that type I IFNs contribute to expression of IFN-stimulated genes and to bacterial clearance as well as resistance in L. pneumophila pneumonia in addition to type II IFN. Overall, our study shows that paracrine type I IFNs mediate defence against L. pneumophila, and demonstrates a protective role of type I IFNs in in vivo infections with intracellular bacteria. 相似文献
17.
To identify host proteins involved in Legionella pneumophila intracellular replication, the soil amoeba Dictyostelium discoideum was analysed. The absence of the amoebal RtoA protein is demonstrated here to depress L. pneumophila intracellular growth. Uptake of L. pneumophila into a D. discoideum rtoA(-) strain was marginally defective, but this effect was not sufficient to account for the defective intracellular growth of L. pneumophila. The rtoA mutant was also more resistant to high-multiplicity killing by the bacterium. A targeting assay testing the colocalization of L. pneumophila-containing vacuole with an endoplasmic reticulum/pre-Golgi intermediate compartment marker protein, GFP-HDEL, was used to analyse these defects. In parental D. discoideum, the L. pneumophila vacuole showed recruitment of GFP-HDEL within 40 min after introduction of bacteria to the amoebae. By 6 h after infection it was clear that the rtoA mutant acquired and retained the GFP-HDEL less efficiently than the parental strain, and that the mutant was defective for promoting the physical expansion of the membranous compartment surrounding the bacteria. Depressed intracellular growth of L. pneumophila in a D. discoideum rtoA(-) mutant therefore appeared to result from a lowered efficiency of vesicle trafficking events that are essential for the modification and expansion of the L. pneumophila-containing compartment. 相似文献
18.
The causative agent of Legionnaires' disease, Legionella pneumophila, employs the intracellular multiplication (Icm)/defective organelle trafficking (Dot) type IV secretion system (T4SS) to upregulate phagocytosis and to establish a replicative vacuole in amoebae and macrophages. Legionella-containing vacuoles (LCVs) do not fuse with endosomes but recruit early secretory vesicles. Here we analyze the role of host cell phosphoinositide (PI) metabolism during uptake and intracellular replication of L. pneumophila. Genetic and pharmacological evidence suggests that class I phosphatidylinositol(3) kinases (PI3Ks) are dispensable for phagocytosis of wild-type L. pneumophila but inhibit intracellular replication of the bacteria and participate in the modulation of the LCV. Uptake and degradation of an icmT mutant strain lacking a functional Icm/Dot transporter was promoted by PI3Ks. We identified Icm/Dot-secreted proteins which specifically bind to phosphatidylinositol(4) phosphate (PI(4)P) in vitro and preferentially localize to LCVs in the absence of functional PI3Ks. PI(4)P was found to be present on LCVs using as a probe either an antibody against PI(4)P or the PH domain of the PI(4)P-binding protein FAPP1 (phosphatidylinositol(4) phosphate adaptor protein-1). Moreover, the presence of PI(4)P on LCVs required a functional Icm/Dot T4SS. Our results indicate that L. pneumophila modulates host cell PI metabolism and exploits the Golgi lipid second messenger PI(4)P to anchor secreted effector proteins to the LCV. 相似文献
19.
嗜肺军团菌是引起军团菌肺炎以及庞蒂亚克热的革兰氏阴性胞内病原细菌,嗜肺军团菌侵染宿主的主要特点是可以通过其IVB型毒力分泌系统,向宿主细胞内分泌超过150种的底物效应蛋白。通过这些效应蛋白的作用,嗜肺军团菌能够调整宿主细胞的胞内运输途径,改变内外环境来伪装自己的吞噬泡,干扰宿主的细胞周期,抑制宿主细胞的凋亡,从而有效逃避宿主细胞的防御功能,创造出理想的胞内增殖环境。最后,效应蛋白还可以帮助军团菌从宿主细胞中逃逸。目前,嗜肺军团菌已经成为"病原菌-宿主相互作用"的重要研究模型,其毒力分泌系统及其底物效应蛋白的功能也成为细胞微生物学的研究热点。对嗜肺军团菌分泌系统及效应蛋白的研究不仅能够帮助阐明病原细菌的致病机理,还有助于推动对宿主免疫机制的更深层次的研究。文章主要针对嗜肺军团菌的毒力分泌系统,尤其是IVB型分泌系统的结构和功能,以及底物效应蛋白的研究进展进行了综述,向读者展示出一个小小的细菌所拥有的那令人惊叹的、如此狡猾的生存策略和它精致的杀伤武器。 相似文献
20.
Many bacterial pathogens require a functional type IV secretion system (T4SS) for virulence. Legionella pneumophila, the causative agent of Legionnaires' disease, employs the Dot/Icm T4SS to inject a large number of protein substrates into its host, thereby altering phagosome trafficking. The L. pneumophila T4SS substrate SdeA has been shown to require the accessory factor IcmS for its export. IcmS, defined as a type IV adaptor, exists as a heterodimer with IcmW and this complex functions in a manner similar to a type III secretion chaperone. Here we report an interaction between IcmS and the previously identified virulence factor LvgA. Similar to the icmS mutant, the lvgA mutant appears to assemble a fully functional Dot/Icm complex. Both LvgA and IcmS are small, acidic proteins localized to the cytoplasm and are not exported by the Dot/Icm system, suggesting they form a novel type IV adaptor complex. Inactivation of lvgA causes a minimal defect in growth in the human monocytic cell line U937 and the environmental host Acanthamoeba castellanii. However, the lvgA mutant was severely attenuated for intracellular growth of L. pneumophila in mouse macrophages, suggesting LvgA may be a critical factor that confers host specificity. 相似文献