Toshio Kodama

Osaka University, Suika, Ōsaka, Japan

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Publications (46)176.32 Total impact

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    ABSTRACT: Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease that causes massive die-offs in farmed shrimps. Recent outbreaks of AHPND in Asia have been causing great losses for shrimp culture, and have become a serious socioeconomic problem. The causative agent of AHPND is Vibrio parahaemolyticus, which is typically known to cause food-borne gastroenteritis in humans. However, there have been few reports of the epidemiology of V. parahaemolyticus AHPND strains, and the genetic relationship among AHPND strains is unclear. Here, we report the genetic characterization of V. parahaemolyticus strains isolated from AHPND outbreaks in Thailand. We found eight isolates from AHPND-suspected shrimps and pond water that were positive for AHPND markers, AP1 and AP2. PCR analysis confirmed that none of these eight AP-positive AHPND strains possess the genes for the conventional virulence factors to humans, such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and type III secretion system 2. Phylogenetic analysis by the multilocus sequence typing showed that the AHPND strains are genetically diverse, suggesting that AHPND strains were not derived from a single genetic lineage. Our study represents the first report of molecular epidemiology of AHPND-causing V. parahaemolyticus strains using the multilocus sequence typing, and provides an insight into their evolutionary mechanisms.
    No preview · Article · Nov 2015 · FEMS Microbiology Letters
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    ABSTRACT: A novel bacterial type III secretion effector, VopV, from the enteric pathogen Vibrio parahaemolyticus has been identified as a key factor in pathogenicity due to its interaction with cytoskeletal actin. One of the repeat units in the long repetitive region of VopV, named VopVrep1, functions as an actin-binding module. Despite its importance in pathogenesis, the manner in which the effector binds to actin and the subsequent effects on actin dynamics remain unclear. Here, we report the molecular basis of the VopVrep1/actin interaction. VopVrep1 exists as an unstructured protein in solution but potently and specifically binds filamentous actin (F-actin) and not globular actin (G-actin). The F-actin/VopVrep1 complex was directly visualized at 9.6-Å resolution using electron cryomicroscopy (cryoEM) and helical image reconstitution. The density map revealed the binding site of VopVrep1 at the interface between two actin strands, which is close to the binding site of the bicyclic heptapeptide toxin phalloidin. Consistent with this observation, VopVrep1 alone prevented the depolymerization of F-actin. Overall, VopVrep1 demonstrated unique characteristics in comparison to known actin-binding proteins, but was relatively similar to phalloidin. The phalloidin-like behavior, targeting the interstrand region of actin filaments to stabilize the filament structure, likely contributes to the pathogenicity of V. parahaemolyticus.
    Preview · Article · Jun 2015 · Scientific Reports
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    ABSTRACT: Vibrio parahaemolyticus is an important pathogen that causes food-borne gastroenteritis in humans. The type III secretion system encoded on chromosome 2 (T3SS2) plays a critical role in the enterotoxic activity of V. parahaemolyticus. Previous studies have demonstrated that T3SS2 induces actin stress fibers in various epithelial cell lines during infection. This stress fiber formation is strongly related to pathogenicity, but the mechanisms that underlie T3SS2-dependent actin stress fiber formation and the main effector have not been elucidated. In this study, we identified VopO as a critical T3SS2 effector protein that activates the RhoA-ROCK pathway, which is an essential pathway for the induction of the T3SS2-dependent stress fiber formation. We also determined that GEF-H1, a RhoA guanine nucleotide exchange factor (GEF), directly binds VopO and is necessary for T3SS2-dependent stress fiber formation. The GEF-H1-binding activity of VopO via an alpha helix region correlated well with its stress fiber-inducing capacity. Furthermore, we showed that VopO is involved in the T3SS2-dependent disruption of the epithelial barrier. Thus, VopO hijacks the RhoA-ROCK pathway in a different manner compared with previously reported bacterial toxins and effectors that modulate the Rho GTPase signaling pathway.
    Preview · Article · Mar 2015 · PLoS Pathogens
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    ABSTRACT: Vibrio parahaemolyticus is a leading causative agent of seafood-borne gastroenteritis worldwide. Most clinical isolates from patients with diarrhea possess two sets of genes for the type III secretion system (T3SS) on each chromosome (T3SS1 and T3SS2). T3SS is a protein secretion system that delivers effector proteins directly into eukaryotic cells. The injected effectors modify the normal cell functions by altering or disrupting the normal cell signaling pathways. Of the two sets of T3SS genes present in V. parahaemolyticus, T3SS2 is essential for enterotoxicity in several animal models. Recent studies have elucidated the biological activities of several T3SS2 effectors and their roles in virulence. This review focuses on the regulation of T3SS2 gene expression and T3SS2 effectors that specifically target the actin cytoskeleton.
    Preview · Article · Dec 2014 · Cellular Microbiology
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    ABSTRACT: Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.
    Preview · Article · Mar 2014 · Infection and immunity
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    ABSTRACT: Vibrio parahaemolyticus is a Gram-negative marine bacterium that causes acute gastroenteritis in humans. The virulence of V. parahaemolyticus is dependent upon a type III secretion system (T3SS2). One effector for T3SS2, VopC, is a homolog of the catalytic domain of cytotoxic necrotizing factor (CNF), and was recently reported to be a Rho family GTPase activator and to be linked to internalization of V. parahaemolyticus by nonphagocytic cultured cells. Here, we provide direct evidence that VopC deamidates Rac1 and CDC42, but not RhoA, in vivo. Our results also suggest that VopC, through its activation of Rac1, contributes to formation of actin stress fibers in infected cells. Invasion of host cells, which occurs at a low frequency, does not seem linked to Rac1 activation, but instead appears to require CDC42. Finally, using an infant rabbit model of V. parahaemolyticus infection, we show that the virulence of V. parahaemolyticus is not dependent upon VopC-mediated invasion. Genetic inactivation of VopC did not impair intestinal colonization nor reduce signs of disease, including fluid accumulation, diarrhea, and tissue destruction. Thus, although VopC can promote host cell invasion, such internalization is not a critical step of the disease process, consistent with the traditional view of V. parahaemolyticus as an extracellular pathogen.
    No preview · Article · Dec 2013 · Cellular Microbiology
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    ABSTRACT: Resistance nodulation cell division (RND)-type efflux transporters play the main role in intrinsic resistance to various antimicrobial agents in many gram-negative bacteria. Here, we estimated 12 RND-type efflux transporter genes in Vibrio parahaemolyticus. Because VmeAB has already been characterized, we cloned the other 11 RND-type efflux transporter genes and characterized them in Escherichia coli KAM33 cells, a drug hypersusceptible strain. KAM33 expressing either VmeCD, VmeEF, or VmeYZ showed increased minimum inhibitory concentrations (MICs) for several antimicrobial agents. Additional four RND-type transporters were functional as efflux pumps only when co-expressed with VpoC, an outer membrane component in V. parahaemolyticus. Furthermore, VmeCD, VmeEF, and VmeYZ co-expressed with VpoC exhibited a broader substrate specificity and conferred higher resistance than that with TolC of E. coli. Deletion mutants of these transporter genes were constructed in V. parahaemolyticus. TM32 (ΔvmeAB and ΔvmeCD) had significantly decreased MICs for many antimicrobial agents and the number of viable cells after exposure to deoxycholate were markedly reduced. Strains in which 12 operons were all disrupted had very low MICs and much lower fluid accumulation in rabbit ileal loops. These results indicate that resistance nodulation cell division-type efflux transporters contribute not only to intrinsic resistance but also to exerting the virulence of V. parahaemolyticus.
    Full-text · Article · Jun 2013 · MicrobiologyOpen
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    ABSTRACT: Twelve Vibrio cholerae isolates with genes for a type III secretion system (T3SS) were detected among 110 environmental and 14 clinical isolates. T3SS-related genes were distributed among the various serogroups and pulsed-field gel electrophoresis of NotI-digested genomes showed genetic diversity in these strains. However, the restriction fragment length polymorphism profiles of the T3SS-related genes had similar patterns. Additionally, naturally competent T3SS-negative V. cholerae incorporated the ca. 47 kb gene cluster of T3SS, which had been integrated into a site on the chromosome by recombination. Therefore, it is suggested that horizontal gene transfer of T3SS-related genes occurs among V. cholerae in natural ecosystems.
    No preview · Article · May 2013 · Microbiology and Immunology
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    ABSTRACT: A total of 12 Vibrio cholerae isolates with genes for a type III secretion system (T3SS) were detected from 110 environmental and 14 clinical isolates. T3SS-related genes were distributed among the various serogroups, and these strains showed genetic diversity in the PFGE of NotI-digested genomes. However, the restriction fragment length polymorphism profiles of the T3SS-related genes showed similar patterns. Additionally, natural competent T3SS-negative V. cholerae incorporated the ca. 47kb gene cluster of T3SS, which integrated into a site of the chromosome by recombination. Therefore, it was suggested that the horizontal gene transfer of T3SS-related genes occurred among V. cholerae in the natural ecosystem.
    No preview · Article · Feb 2013 · Microbiology and Immunology
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    ABSTRACT: Author Summary V. parahaemolyticus is Gram-negative pathogen that causes a food poisoning in human. To date, a number of bacterial factors that play a role in V. parahaemolyticus virulence have been characterized, yet little is known about the host factors contributing to the disease process and susceptibility to these pathogens. IL-1β, in addition to TNF-α, is thought to be involved in inflammatory responses and disease development during infection with the pathogen, but the mechanisms of IL-1β production remain poorly defined. In this work we found that both the NLRP3 and NLRC4 inflammasomes are activated by thermostable direct hemolysins (TDHs) and type III secretion system 1 (T3SS1) in response to V. parahaemolyticus infection. The activated inflammasomes then triggers the activation of caspase-1, a cysteine protease that is essential for IL-1β processing and release. Furthermore, we identified T3SS1 secreted effector proteins, VopQ and VopS, which prevent mainly NLRC4 inflammasome activation. VopQ and VopS induce autophagy and the inactivation of Rho GTPases, including Cdc42, respectively, and these cellular events interfere with the assembly of specks, the platform of inflammasome activation. Collectively, T3SS1 effector-based suppression of inflammasome activation may provide important insights into bacterial strategies for evading inflammasome-mediated host immune responses.
    Full-text · Article · Jan 2013 · PLoS Pathogens
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    ABSTRACT: The type III secretion system (T3SS) of gram-negative bacteria involves dedicated protein translocation machinery that directly injects proteins into target cells. Pathogenic bacteria already benefit from this unique system. The successful functional cloning of this useful tool into non-pathogenic bacteria would help establish novel clinical and basic biotechnology strategies in areas such as vaccine administration, the development of screening systems for anti-T3SS drugs and the target-specific delivery of bioactive compounds. In this study, we successfully cloned the Vibrio parahaemolyticus T3SS1 genetic locus into a non-pathogenic Escherichia coli K-12 strain. Assays performed here revealed that the T3SS1 cloned into the E. coli K-12 strain has the ability to translocate V. parahaemolyticus T3SS1 secreted proteins. Importantly, we also observed this system to allow the E. coli K-12 strain to inject foreign protein, as well as the V. parahaemolyticus T3SS effector, into cultured cells. These results demonstrate a prospective useful tool with experimental and therapeutic applications.
    No preview · Article · Sep 2012 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H(+)-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death.
    Preview · Article · Jul 2012 · PLoS Pathogens
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    ABSTRACT: The pathogenesis of the diarrheal disease caused by Vibrio parahaemolyticus, a leading cause of seafood-associated enteritis worldwide, is dependent upon a type III secretion system, T3SS2. This apparatus enables the pathogen to inject bacterial proteins (effectors) into the cytosol of host cells and thereby modulate host processes. T3SS effector proteins transit into the host cell via a membrane pore (translocon) typically formed by 3 bacterial proteins. We have identified the third translocon protein for T3SS2: VopW, which was previously classified as an effector protein for a homologous T3SS in V. cholerae. VopW is a hydrophilic translocon protein; like other such proteins, it is not inserted into the host cell membrane but is required for insertion of the two hydrophobic translocators, VopB2 and VopD2, that constitute the membrane channel. VopW is not required for secretion of T3SS2 effectors into the bacterial culture medium; however, it is essential for transfer of these proteins into the host cell cytoplasm. Consequently, deletion of vopW abrogates the virulence of V. parahaemolyticus in several animal models of diarrheal disease. Unlike previously described hydrophilic translocators, VopW is itself translocated into the host cell cytoplasm, raising the possibility that it functions as both a translocator and an effector.
    Full-text · Article · May 2012 · Infection and immunity
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    ABSTRACT: Among three haemolysins identified thus far in Escherichia coli, alpha-haemolysin (HlyA) is encoded on the pathogenicity islands of extraintestinal pathogenic strains, while enterohaemolysin (EhxA) is encoded on the virulence plasmids of enterohaemorrhagic E. coli (EHEC) strains. In contrast, the gene for haemolysin E (HlyE) is located on the E. coli chromosome backbone and is therefore widely distributed among E. coli strains. However, because hlyE gene expression is repressed by the H-NS protein and because the gene has been disrupted in many strains, its haemolytic activity cannot be detected in wild-type strains by routine screening on blood agar plates. In this study, we found that the HlyE-derived haemolytic activity of enteropathogenic E. coli (EPEC) O55 : H7 can be detected after anaerobic cultivation on a washed blood agar plate (EHX plate) that is used to detect the production of EhxA. We also found that the haemolytic activity of EHEC O157 : H7 observed on EHX plates under aerobic and anaerobic growth conditions is derived from EhxA and HlyE, respectively; this differential expression of the two haemolysins occurs at the transcriptional level. Our analysis of 60 E. coli strains of various pathotypes and phylogenies for their repertoires of haemolysin genes, haemolytic phenotypes and hlyE gene sequences revealed that HlyE activity can generally be detected on EHX plates under anaerobic growth conditions if the gene is intact. Furthermore, our results indicate that hlyE gene inactivation occurred in three of the five E. coli lineages (phylogroups A, B1 and B2), which demonstrates phylogroup-specific gene disruption patterns.
    No preview · Article · Dec 2011 · Microbiology
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    ABSTRACT: The enteropathogen Vibrio parahaemolyticus possesses two sets of type III secretion systems, T3SS1 and T3SS2. Effector proteins secreted by these T3SSs are delivered into host cells, leading to cell death or diarrhea. However, it is not known how specific effectors are secreted through a specific T3SS when both T3SSs are expressed within bacteria. One molecule thought to determine secretion specificity is a T3SS-associated chaperone; however, no T3SS2-specific chaperone has been identified. Therefore, we screened T3SS2 chaperone candidates by a pull-down assay using T3SS2 effectors fused with glutathione-S-transferase. A secretion assay revealed that the newly identified cognate chaperone VocC for the T3SS2-specific effector VopC was required for the efficient secretion of the substrate through T3SS2. Further experiments determined the chaperone-binding domain and the amino-terminal secretion signal of the cognate effector. These findings, in addition to the previously identified T3SS1-specific chaperone, VecA, provide a strategy to clarify the specificity of effector secretion through T3SSs of V. parahaemolyticus.
    Preview · Article · Nov 2011 · FEMS Microbiology Letters
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    ABSTRACT: Vibrio vulnificus secretes a hemolysin/cytolysin (VVH) that induces cytolysis in target cells. A detergent resistant membrane domain (DRM) fraction of the cells after sucrose gradient centrifugation includes cholesterol-rich membrane microdomains which have been called "lipid rafts". It was reported that some pore-forming toxins require association with DRM and/or lipid rafts to exert their cytotoxicity. It has also been thought that cellular cholesterol is involved in VVH cytotoxicity because VVH cytotoxicity was inhibited by pre-incubation with cholesterol. However, both cellular localization and mode of action of VVH cytotoxicity remain unclear. In this study, we investigated the relationship between VVH localization on the cellular membrane and its cytotoxicity. Oligomers of VVH were detected from DRM fractions by sucrose gradient ultracentrifugation but all of these oligomers shifted from DRM fractions to non-DRM fractions after treatment with methyl-beta-cyclodextrin (MβCD), a cholesterol sequestering agent. On the other hand, immunofluorescence analysis showed that VVH did not co-localize with major lipid raft markers on cellular membrane of CHO cells. These data suggested that VVH localized at membrane regions which are relatively abundant in cholesterol but which are not identical with lipid rafts. To determine the linkage between localization and cytotoxicity of VVH, cytotoxicity was evaluated in MβCD-treated CHO cells. The cytotoxicity of VVH was not decreased by the MβCD treatment. In addition, the amount of VVH oligomer did not decrease in MβCD-treated CHO cells. Thus, we found that the amount of oligomer on cellular membrane is important for induction of cytotoxicity, whereas localization to lipid rafts on the cellular membrane was not essential to cytotoxicity.
    Preview · Article · Oct 2011 · PLoS ONE
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    ABSTRACT: Vibrio parahaemolyticus, a Gram-negative halophilic bacterium that causes acute gastroenteritis in humans, is characterized by two type III secretion systems (T3SS), namely T3SS1 and T3SS2. T3SS2 is indispensable for enterotoxicity but the effector(s) involved are unknown. Here, we identify VopV as a critical effector that is required to mediate V. parahaemolyticus T3SS2-dependent enterotoxicity. VopV was found to possess multiple F-actin-binding domains and the enterotoxicity caused by VopV correlated with its F-actin-binding activity. Furthermore, a T3SS2-related secretion system and a vopV homologous gene were also involved in the enterotoxicity of a non-O1/non-O139 V. cholerae strain. These results indicate that the F-actin-targeting effector VopV is involved in enterotoxic activity of T3SS2-possessing bacterial pathogens.
    No preview · Article · Oct 2011 · Cell host & microbe
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    ABSTRACT: AexU is a type three secretion system (TTSS) effector of Aeromonas hydrophila which has an in vitro ADP-ribosyltransferase (ART) and GTPase-activating protein (GAP) activities on Rac1, RhoA and Cdc42. Here we show that, AexU of Aeromonas veronii bv. sobria AeG1 strain disrupts actin cytoskeleton of HeLa cells during AeG1 infection, aexU transfection or direct application of AexU protein. Such cellular disruption was rescued by either inactivation of AexU-GAP activity by substitution of arginine residue 143 to alanine or expression of a constitutively active (CA) Rac1 but not CA RhoA or CA Cdc42. On the other hand, AexU was found co-localized with β4-integrin probably through its Arg-Gly-Asp (RGD) integrin binding motif (319-321) residues. Interestingly, direct application of GST-AexU-HA fusion protein caused significant cytotoxic effect on β4-integrin expressing HT-29 cells. In contrast, β4-integrin blockade with a specific antibody reduced such cytotoxicity. Consequently, AexU cytotoxic effect was exaggerated with a greater expression of β4-integrin in Caco-2 and HeLa cells, while it was incompetent on β4-integrin non-expressing CHO cells. As far as we know, this is a novel TTSS effector which specifically inactivates Rac1 to disrupt actin cytoskeleton and has an alternative cytotoxic pathway through β4-integrin mediation.
    Full-text · Article · Sep 2011 · Microbial Pathogenesis
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    ABSTRACT: The enteropathogen Vibrio parahaemolyticus possesses two sets of type III secretion systems, T3SS1 and T3SS2. Effector proteins secreted by these T3SSs are delivered into host cells, leading to cell death or diarrhea. However, it is not known how specific effectors are secreted through a specific T3SS when both T3SSs are expressed within bacteria. One molecule thought to determine secretion specificity is a T3SS‐associated chaperone; however, no T3SS2‐specific chaperone has been identified. Therefore, we screened T3SS2 chaperone candidates by a pull‐down assay using T3SS2 effectors fused with glutathione‐S‐transferase. A secretion assay revealed that the newly identified cognate chaperone VocC for the T3SS2‐specific effector VopC was required for the efficient secretion of the substrate through T3SS2. Further experiments determined the chaperone‐binding domain and the amino‐terminal secretion signal of the cognate effector. These findings, in addition to the previously identified T3SS1‐specific chaperone, VecA, provide a strategy to clarify the specificity of effector secretion through T3SSs of V. parahaemolyticus.
    No preview · Article · Jan 2011 · FEMS Microbiology Letters
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    ABSTRACT: Vibrio parahaemolyticus, a bacterial pathogen, causes human gastroenteritis. A type III secretion system (T3SS2) encoded in pathogenicity island (Vp-PAI) is the main contributor to enterotoxicity and expression of Vp-PAI encoded genes is regulated by two transcriptional regulators, VtrA and VtrB. However, a host-derived inducer for the Vp-PAI genes has not been identified. Here, we demonstrate that bile induces production of T3SS2-related proteins under osmotic conditions equivalent to those in the intestinal lumen. We also show that bile induces vtrA-mediated vtrB transcription. Transcriptome analysis of bile-responsive genes revealed that bile strongly induces expression of Vp-PAI genes in a vtrA-dependent manner. The inducing activity of bile was diminished by treatment with bile acid sequestrant cholestyramine. Finally, we demonstrate an in vivo protective effect of cholestyramine on enterotoxicity and show that similar protection is observed in infection with a different type of V. parahaemolyticus or with non-O1/non-O139 V. cholerae strains of vibrios carrying the same kind of T3SS. In summary, these results provide an insight into how bacteria, through the ingenious action of Vp-PAI genes, can take advantage of an otherwise hostile host environment. The results also reveal a new therapeutic potential for widely used bile acid sequestrants in enteric bacterial infections.
    Full-text · Article · Oct 2010 · PLoS ONE