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PubMed2003

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

Shigella deliver a subset of effector proteins such as IpaA, IpaB and IpaC via the type III secretion system (TTSS) into host cells during the infection of colonic epithelial cells. Many bacterial effectors including some from Shigella require specific chaperones for protection from degradation and targeting to the TTSS. In this study, we have investigated the role of the icsB gene located upstream of the ipaBCDA operon in Shigella infection because the role of IcsB as a virulence factor remains unknown. Here, we found that the IcsB protein is secreted via the TTSS of Shigella in vitro and in vivo. We show that IpgA protein encoded by ipgA, the gene immediately downstream of icsB, serves as the chaperone required for the stabilization and secretion of IcsB. We have shown that IcsB binds to IpgA in bacterial cytosol and the binding site is in the middle of the IcsB protein. Intriguingly, although its significance in Shigella pathogenicity is as yet unclear, the icsB gene can be read-through into the ipgA gene to create a translational fusion protein. Furthermore, the contribution of IcsB to the pathogenicity of Shigella was demonstrated by plaque-forming assay and the Sereny test. The ability of the icsB mutant to form plaques was greatly reduced compared with that of the wild type in MDCK cell monolayers. Furthermore, when guinea pig eyes were infected with a non-polar icsB mutant, the bacteria failed to provoke keratoconjunctivitis. These results suggest that IcsB is secreted via the TTSS, chaperoned by IpgA, and required at the post-invasion stage of Shigella pathogenicity

Animals↗

PubMed2006

Mutations in the Yersinia pseudotuberculosis type III secretion system needle protein, YscF, that specifically abrogate effector translocation into host cells.

The trafficking of effectors, termed Yops, from Yersinia spp. into host cells is a multistep process that requires the type III secretion system (TTSS). The TTSS has three main structural parts: a base, a needle, and a translocon, which work together to ensure the polarized movement of Yops directly from the bacterial cytosol into the host cell cytosol. To understand the interactions that take place at the interface between the tip of the TTSS needle and the translocon, we developed a screen to identify mutations in the needle protein YscF that separated its function in secretion from its role in translocation. We identified 25 translocation-defective (TD) yscF mutants, which fall into five phenotypic classes. Some classes exhibit aberrant needle structure and/or reduced levels of Yop secretion, consistent with known functions for YscF. Strikingly, two yscF TD classes formed needles and secreted Yops normally but displayed distinct translocation defects. Class I yscF TD mutants showed diminished pore formation, suggesting incomplete pore insertion and/or assembly. Class II yscF TD mutants formed pores but showed nonpolar translocation, suggesting unstable needle-translocon interactions. These results indicate that YscF functions in Yop secretion and translocation can be genetically separated. Furthermore, the identification of YscF residues that are required for the assembly of the translocon and/or productive interactions with the translocon has allowed us to initiate the mapping of the needle-translocon interface.

Bacterial Proteins↗

PubMed2004

RtsA coordinately regulates DsbA and the Salmonella pathogenicity island 1 type III secretion system.

Salmonella serovars cause a wide variety of diseases ranging from mild gastroenteritis to life-threatening systemic infections. An important step in Salmonella enterica serovar Typhimurium infection is the invasion of nonphagocytic epithelial cells, mediated by a type III secretion system (TTSS) encoded on Salmonella pathogenicity island 1 (SPI1). The SPI1 TTSS forms a needle complex through which effector proteins are injected into the cytosol of host cells, where they promote actin rearrangement and engulfment of the bacteria. We previously identified the Salmonella-specific regulatory protein RtsA, which induces expression of hilA and, thus, the SPI1 genes. Here we show that the hilA regulators RtsA, HilD, and HilC can each induce transcription of dsbA, which encodes a periplasmic disulfide bond isomerase. RtsA induces expression of dsbA independent of either the SPI1 TTSS or the only known regulator of dsbA, the CpxRA two-component system. We show that DsbA is required for both the SPI1 and SPI2 TTSS to translocate effector proteins into the cytosol of host cells. DsbA is also required for survival during the systemic stages of infection. We also present evidence that production of SPI1 effector proteins is coupled to assembly of the TTSS. This feedback regulation is mediated at either the transcriptional or posttranscriptional level, depending on the particular effector. Loss of DsbA leads to feedback inhibition, which is consistent with the hypothesis that disulfide bond formation plays a role in TTSS assembly or function.

Animals↗

PubMed2003

Protein secretion systems of Pseudomonas aeruginosa and P fluorescens.

Gram-negative bacteria have evolved numerous systems for the export of proteins across their dual-membrane envelopes. Three of these systems (types I, III and IV) secrete proteins across both membranes in a single energy-coupled step. Four systems (Sec, Tat, MscL and Holins) secrete only across the inner membrane, and four systems [the main terminal branch (MTB), fimbrial usher porin (FUP), autotransporter (AT) and two-partner secretion families (TPS)] secrete only across the outer membrane. We have examined the genome sequences of Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens Pf0-1 for these systems. All systems except type IV were found in P. aeruginosa, and all except types III and IV were found in P. fluorescens. The numbers of each such system were variable depending on the system and species examined. Biochemical and physiological functions were assigned to these systems when possible, and the structural constituents were analyzed. Available information regarding the mechanisms of transport and energy coupling as well as physiological functions is summarized. This report serves to identify and characterize protein secretion systems in two divergent pseudomonads, one an opportunistic human pathogen, the other a plant symbiont.

Bacterial Proteins↗

PubMed2006

Safety and immunogenicity of attenuated Salmonella enterica serovar Typhimurium delivering an HIV-1 Gag antigen via the Salmonella Type III secretion system.

BACKGROUND: CKS257 (Salmonella typhimurium SL1344 DeltaphoP/phoQDelta aroA Deltaasd DeltastrA/strB pSB2131) is a live oral vaccine vector expressing HIV Gag. METHODS: HIV Gag was expressed as a fusion protein of a Salmonella Type III secretion system protein SopE, from a balanced lethal asd-based plasmid. Eighteen healthy adults were given single escalating oral doses of 5 x 10(6) to 1 x 10(10)CFU of CKS257 and were monitored for clinical events, shedding and immune responses. RESULTS: Adverse events were mild except at the highest dose. Volunteers shed the organism an average of 5.1 days (range 0-13 days). Eighty-three percent (15/18) of subjects had a mucosal immune response to Salmonella LPS and flagella by IgA ELISPOT assay. Seventy-two percent (13/18) of subjects seroconverted to Salmonella antigens. No volunteer had a response to recombinant Gag as measured by serology, IgA ELISPOT, or immediate ex vivo gamma-interferon ELISPOT response to Gag peptide pools. Two volunteers responded to Gag peptides by IL-2 ELISPOT, and 4 of 10 volunteers receiving >or=5 x 10(8)CFU had a response to HIV peptides in a cultured gamma-interferon ELISPOT assay. CONCLUSIONS: Although immunogenicity of the HIV antigen needs augmentation, the attenuated Salmonella strain proved to be an excellent platform for vaccine development.

AIDS Vaccines↗

PubMed2006

Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle.

The obligate intracellular bacterium Chlamydia trachomatis possesses a biphasic developmental cycle that is manifested by differentiation of infectious, metabolically inert elementary bodies (EBs) to larger, metabolically active reticulate bodies (RBs). The cycle is completed by asynchronous differentiation of dividing RBs back to a population of dormant EBs that can initiate further rounds of infection upon lysis of the host cell. Chlamydiae express a type III secretion system (T3SS) that is presumably employed to establish and maintain the permissive intracellular niche by secretion of anti-host proteins. We hypothesize that T3SS activity is essential for chlamydial development and pathogenesis. However, the lack of a genetic system has confounded efforts to establish any role of the T3SS. We therefore employed the small molecule Yersinia T3SS inhibitor N'-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide, designated compound 1 (C1), to examine the interdependence of the chlamydial T3SS and development. C1 treatment inhibited C. trachomatis but not T4SS-expressing Coxiella burnetii development in a dose-dependent manner. Although chlamydiae remained viable and metabolically active, they failed to divide significantly and RB to EB differentiation was inhibited. These effects occurred in the absence of host cell cytotoxicity and were reversible by washing out C1. We further demonstrate that secretion of T3S substrates is perturbed in C1-treated chlamydial cultures. We have therefore provided evidence that C1 can inhibit C. trachomatis development and T3SS activity and present a model in which progression of the C. trachomatis developmental cycle requires a fully functional T3SS.

Bacterial Proteins↗

PubMed2004

"One size fits it all": translocation of foreign antigens by Yersinia type III secretion system (TTSS) leads to concomitant CD4 and CD8 T-cell priming.

Live replicating bacteria expressing heterologous antigens are vaccine candidates that are able to induce complex immune responses. Yersinia pseudotuberculosis employs a type III secretion system for translocation of several virulence factors directly to the cytosol of eukaryotic cells. Mice orally inoculated with an attenuated recombinant Yersinia strain translocating a chimeric Yersinia outer protein E (YopE) molecule reveal high numbers of foreign antigen-specific CD4 and CD8 T cells. Thus, cytosolic display of a single hybrid protein results in concomitant CD4 and CD8 T-cell priming. This "one-size-fits-it-all"-feature of Yersinia-translocated heterologous antigens might be advantageous to mount T-cellular immune responses against complex microbes and tumors.

Administration, Oral↗

PubMed2005

SepZ/EspZ is secreted and translocated into HeLa cells by the enteropathogenic Escherichia coli type III secretion system.

Enteropathogenic Escherichia coli (EPEC) is a major bacterial cause of infantile diarrhea in developing countries and is the prototype for a group of gastrointestinal pathogens causing characteristic attaching and effacing (A/E) histopathology on intestinal epithelia. A/E pathogens utilize a type III secretion system (TTSS), encoded by the locus of enterocyte effacement (LEE) pathogenicity island, to deliver effector proteins into host cells. Here, we investigate sequence divergence of the LEE-encoded SepZ protein and identify it as a TTSS-secreted and -translocated molecule. SepZ is hypervariable among A/E pathogens, with sequences sharing between 60 to 81% amino acid identity with SepZ of EPEC. A SepZ-CyaA fusion was secreted and translocated into HeLa cells in a TTSS-dependent manner. Additionally, we determined that the first 20 amino acids of SepZ were sufficient to direct its translocation. In contrast to previous studies suggesting a role in invasion and the structure and/or regulation of the TTSS, we found that SepZ does not mediate uptake of EPEC into host cells or affect translocation and tyrosine phosphorylation of the translocated intimin receptor. Immunohistochemistry reveals that, after an extended HeLa cell infection, accumulated SepZ can be detected beneath the site of bacterial attachment in a subset of pedestal regions. To indicate its newly identified status as a translocated effector protein, we propose to rename SepZ as EspZ.

Adenylate Cyclase Toxin↗

PubMed2003

Distribution of the secondary type III secretion system locus found in enterohemorrhagic Escherichia coli O157:H7 isolates among Shiga toxin-producing E. coli strains.

The ability of the complete genome sequence of enterohemorrhagic Escherichia coli O157 led to the identification of a 17-kb chromosomal region which contained a type III secretion system gene cluster at min 64.5. This locus contains open reading frames whose amino acid sequences show high degrees of similarity with those of proteins that make up the type III secretion apparatus, which is encoded by the inv-spa-prg locus on a Salmonella SPI-1 pathogenicity island. This locus was designated ETT2 (E. coli type III secretion 2) and consisted of the epr, epa, and eiv genes. ETT2 was found in enteropathogenic E. coli strains and also in some non-O157 Shiga toxin-producing E. coli (STEC) strains, but most of them contained a truncated portion of ETT2. Most O157 isolates had a complete collection of toxin-encoding genes eae and hlyA and the ETT2 locus, while most O26 strains had toxin-encoding genes eae and hlyA genes but an incomplete ETT2 locus. Thus, an intact copy of ETT2 might mark a pathogenic distinction for particular STEC strains. Therefore, the presence of the ETT2 locus can be used for identification of truly pathogenic STEC strains and for molecular fingerprinting of the epidemic strains in humans and animals.

Animals↗

PubMed2004

Cellular microbiology of intracellular Salmonella enterica: functions of the type III secretion system encoded by Salmonella pathogenicity island 2.

The facultative intracellular pathogen Salmonella enterica resides in a special membrane compartment of the host cell and modifies its host to achieve intracellular survival and proliferation. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has a central role in the interference of intracellular Salmonella with host cell functions. SPI2 function affects antimicrobial defense mechanisms of the host, intracellular transport processes, integrity and function of the cytoskeleton and host cell death. These modifications are mediated by translocation of a large number of effector proteins by the SPI2 system. In this review, we summarize recent work on the cellular phenotypes related to SPI2 function and contribution of SPI2 effector proteins to these manipulations. These studies reveal a complex set of pathogenic interferences between intracellular Salmonella and its host cells.

Animals↗

PubMed2004

The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells.

Bartonella henselae is an arthropod-borne zoonotic pathogen causing intraerythrocytic bacteraemia in the feline reservoir host and a broad range of clinical manifestations in incidentally infected humans. Remarkably, B. henselae can specifically colonize the human vascular endothelium, resulting in inflammation and the formation of vasoproliferative lesions known as bacillary angiomatosis and bacillary peliosis. Cultured human endothelial cells provide an in vitro system to study this intimate interaction of B. henselae with the vascular endothelium. However, little is known about the bacterial virulence factors required for this pathogenic process. Recently, we identified the type IV secretion system (T4SS) VirB as an essential pathogenicity factor in Bartonella, required to establish intraerythrocytic infection in the mammalian reservoir. Here, we demonstrate that the VirB T4SS also mediates most of the virulence attributes associated with the interaction of B. henselae during the interaction with human endothelial cells. These include: (i) massive rearrangements of the actin cytoskeleton, resulting in the formation of bacterial aggregates and their internalization by the invasome structure; (ii) nuclear factor kappaB-dependent proinflammatory activation, leading to cell adhesion molecule expression and chemokine secretion, and (iii) inhibition of apoptotic cell death, resulting in enhanced endothelial cell survival. Moreover, we show that the VirB system mediates cytostatic and cytotoxic effects at high bacterial titres, which interfere with a potent VirB-independent mitogenic activity. We conclude that the VirB T4SS is a major virulence determinant of B. henselae, required for targeting multiple endothelial cell functions exploited by this vasculotropic pathogen.

Actin Cytoskeleton↗

PubMed2005

Helicobacter pylori cag-type IV secretion system facilitates corpus colonization to induce precancerous conditions in Mongolian gerbils.

BACKGROUND & AIMS: Epidemiological studies suggest that atrophic corpus-dominant gastritis is an increased risk factor for gastric carcinogenesis. The role of the Helicobacter pylori type IV secretion system (T4SS) for pathogenesis in the Mongolian gerbil model was explored. METHODS: Mongolian gerbils were infected for 32 weeks either with H. pylori type I strain B128 or with isogenic mutant strain B128delta cytotoxin-associated gene (cagY) or B128delta cagA , defective in T4SS or in the production of its effector protein CagA, respectively. Quantitative H. pylori reisolation was performed from the gastric antrum and corpus separately, cytokines were measured by quantitative reverse-transcription polymerase chain reaction, and gastric pH and hormones were determined. RESULTS: B128-infected gerbils harbored high numbers of bacteria in the gastric antrum and corpus, whereas B128delta cagY and B128delta cagA colonized the antrum more densely than the corpus. All infected animals showed a strong antral inflammation and epithelial cell proliferation. B128-infected, rather than mutant-infected, gerbils presented a severe transmural inflammation with huge lymph aggregates, increased proliferation, significant atrophy, and mucous gland metaplasia in the corpus. Plasma gastrin levels and gastric pH values were significantly increased only in B128-infected gerbils. In all infected animals, the expression of the proinflammatory cytokines interleukin 1beta, interferon gamma, and growth-regulated protein was considerably increased in the antrum, but only in wild type-infected animals was an increase seen in the corpus mucosa. CONCLUSIONS: The presence of an intact T4SS allows H. pylori to colonize the gastric corpus. This results in atrophic corpus-dominant gastritis, a severe precancerous condition, thus highlighting T4SS and CagA as major risk factors for gastric cancer development.

Achlorhydria↗

PubMed2001

Structural studies of the Hrp secretion system: expression, purification, crystallization and preliminary X-ray analysis of the C-terminal domain of the HrcQB protein from Pseudomonas syringae pv. phaseolicola.

The C-terminal domain of the HrcQ(B) protein from the Hrp secretion system of the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola has been crystallized from MPD using the hanging-drop vapour-diffusion method. The crystals belong to space group P2(1), with unit-cell parameters a = 51.6, b = 27.3, c = 97.2 A and beta = 99.8 degrees. A complete native data set extending to 3.0 A resolution was collected from a single cryoprotected crystal. The crystal solvent content and calculation of self-rotation functions showing non-crystallographic twofold symmetry axes are consistent with the presence of an oligomeric assembly in the asymmetric unit.

Amino Acid Sequence↗

PubMed2004

The type II protein secretion system of Legionella pneumophila promotes growth at low temperatures.

The gram-negative bacterium Legionella pneumophila grows in both natural and man-made water systems and in the mammalian lung as a facultative intracellular parasite. The PilD prepilin peptidase of L. pneumophila promotes type IV pilus biogenesis and type II protein secretion. Whereas pili enhance adherence, Legionella type II secretion is critical for intracellular growth and virulence. Previously, we observed that pilD transcript levels are greater in legionellae grown at 30 versus 37 degrees C. Using a new pilD::lacZ fusion strain, we now show that pilD transcriptional initiation increases progressively as L. pneumophila is grown at 30, 25, and 17 degrees C. Legionella pilD mutants also had a dramatically reduced ability to grow in broth and to form colonies on agar at the lower temperatures. Whereas strains specifically lacking type IV pili were not defective for low-temperature growth, mutations in type II secretion (lsp) genes greatly impaired the capacity of L. pneumophila to form colonies at 25, 17, and 12 degrees C. Indeed, the lsp mutants were completely unable to grow at 12 degrees C. The growth defect of the pilD and lsp mutants was complemented by reintroduction of the corresponding intact gene. Interestingly, the lsp mutants displayed improved growth at 25 degrees C when plated next to a streak of wild-type but not mutant bacteria, implying that a secreted, diffusible factor promotes low-temperature growth. Mutants lacking either the known secreted acid phosphatases, lipases, phospholipase C, lysophospholipase A, or protease grew normally at 25 degrees C, suggesting the existence of a critical, yet-to-be-defined exoprotein(s). In summary, these data document, for the first time, that L. pneumophila replicates at temperatures below 20 degrees C and that a bacterial type II protein secretion system facilitates growth at low temperatures.

Bacterial Proteins↗

PubMed2005

Type III secretion system cluster 3 is required for maximal virulence of Burkholderia pseudomallei in a hamster infection model.

Burkholderia pseudomallei, the etiological agent of melioidosis, is an animal pathogen capable of inducing a highly fatal septicemia. B. pseudomallei possesses three type III secretion system (TTSS) clusters, two of which (TTSS1 and TTSS2) are homologous to the TTSS of the plant pathogen Ralstonia solanacearum, and one (TTSS3) is homologous to the Salmonella SPI-1 mammalian pathogenicity island. We have demonstrated that TTSS3 is required for the full virulence of B. pseudomallei in a hamster model of infection. We have also examined the virulence of B. pseudomallei mutants deficient in several putative TTSS3 effector molecules, and found no significant attenuation of B. pseudomallei virulence in the hamster model.

Animals↗

PubMed1999

Interaction of Salmonella with host cells through the centisome 63 type III secretion system.

Salmonella enterica engages host cells in a complex two-way biochemical interaction that results in a variety of responses from both the bacteria and the host cell. Central to this interaction is the function of a type III protein secretion system that delivers effector proteins into the host cell. During the past year we have seen major advances in our knowledge of both the bacterial determinants and the host-signal transduction pathways involved in these interactions. A coherent picture of the mechanisms by which Salmonella engages the host cell is now beginning to emerge.

Adhesins, Bacterial↗

PubMed2004

Inventory and functional analysis of the large Hrp regulon in Ralstonia solanacearum: identification of novel effector proteins translocated to plant host cells through the type III secretion system.

The ability of Ralstonia solanacearum strain GMI1000 to cause disease on a wide range of host plants (including most Solanaceae and Arabidopsis thaliana) depends on genes activated by the regulatory gene hrpB. HrpB controls the expression of the type III secretion system (TTSS) and pathogenicity effectors transiting through this pathway. In order to establish the complete repertoire of TTSS-dependent effectors belonging to the Hrp regulon and to start their functional analysis, we developed a rapid method for insertional mutagenesis, which was used to monitor the expression of 71 candidate genes and disrupt 56 of them. This analysis yielded a total of 48 novel hrpB-regulated genes. Using the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter fusion system, we provide direct biochemical evidence that five R. solanacearum effector proteins are translocated into plant host cells through the TTSS. Among these novel TTSS effectors, RipA and RipG both belong to multigenic families, RipG defining a novel class of leucine-rich-repeats harbouring proteins. The members of these multigenic families are differentially regulated, being composed of genes expressed in either an hrpB-dependent or an hrpB-independent manner. Pathogenicity assays of the 56 mutant strains on two host plants indicate that, with two exceptions, mutations in individual effectors have no effect on virulence, a probable consequence of genetic and functional redundancy. This large repertoire of HrpB-regulated genes, which comprises > 20 probable TTSS effector genes with no counterparts in other bacterial species, represents an important step towards a full-genome understanding of R. solanacearum virulence.

Bacterial Proteins↗

PubMed2006

Bioinformatics-enabled identification of the HrpL regulon and type III secretion system effector proteins of Pseudomonas syringae pv. phaseolicola 1448A.

The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To identify genes encoding type III effectors and other potential virulence factors that are regulated by the HrpL alternative sigma factor, we used a hidden Markov model, weight matrix model, and type III targeting-associated patterns to search the genome of P. syringae pv. phaseolicola 1448A, which recently was sequenced to completion. We identified 44 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related T3SS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time polymerase chain reaction, and all were found to be upregulated by HrpL. Six of the candidate type III effectors were assayed for T3SS-dependent translocation into plant cells using the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function; however, they do have homologs in the model strain P. syringae pv. tomato DC3000 (PSPTO2105 and PSPTO0834, respectively) that are similarly upregulated by HrpL. Mutations were constructed in the DC3000 homologs and found to reduce bacterial growth in host Arabidopsis leaves. These results establish the utility of the bioinformatic or candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.

Adenylyl Cyclases↗