Vibrio parahaemolyticus, one of the human-pathogenic vibrios, causes three major types of clinical illness: gastroenteritis, wound infections, and septicemia. Thermostable direct hemolysin (TDH) secreted by this bacterium has been considered a major virulence factor of gastroenteritis because it has biological activities, including cytotoxic and enterotoxic activities. Previous reports revealed that V. parahaemolyticus strain RIMD2210633, which contains tdh, has two sets of type III secretion system (T3SS) genes on chromosomes 1 and 2 (T3SS1 and T3SS2, respectively) and that T3SS1 is responsible for cytotoxicity and T3SS2 is involved in enterotoxicity, as well as in cytotoxic activity. However, the relative importance and contributions of TDH and the two T3SSs to V. parahaemolyticus pathogenicity are not well understood. In this study, we constructed mutant strains with nonfunctional T3SSs from the V. parahaemolyticus strain containing tdh, and then the pathogenicities of the wild-type and mutant strains were evaluated by assessing their cytotoxic activities against HeLa, Caco-2, and RAW 264 cells, their enterotoxic activities in rabbit ileal loops, and their lethality in a murine infection model. We demonstrated that T3SS1 was involved in cytotoxic activities against all cell lines used in this study, while T3SS2 and TDH had cytotoxic effects on a limited number of cell lines. T3SS2 was the major contributor to V. parahaemolyticus-induced enterotoxicity. Interestingly, we found that both T3SS1 and TDH played a significant role in lethal activity in a murine infection model. Our findings provide new indications that these virulence factors contribute to and orchestrate each distinct aspect of the pathogenicity of V. parahaemolyticus.Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that inhabits estuarine and coastal waters and can be isolated from seafood (6,7,9). It causes acute gastroenteritis in humans after they consume contaminated raw or undercooked seafood. Although this microorganism is better known for causing gastroenteritis, it also can cause wound infections and septicemia (5,23,36).Most clinical isolates of V. parahaemolyticus from patients with diarrhea show ␤-hemolysis on Wagatsuma agar (37). This phenomenon is known as the Kanagawa phenomenon (KP) and is considered a good marker to distinguish between pathogenic and nonpathogenic V. parahaemolyticus strains (25, 37). Thermostable direct hemolysin (TDH), which is responsible for the KP (28, 43), has multiple biological activities, including hemolysis, enterotoxicity, cytotoxicity, and cardiotoxicity (11-13, 26, 29, 32, 34, 38). For this reason, TDH has been considered a major virulence factor of V. parahaemolyticus.Whole-genome sequencing of a KP-positive V. parahaemolyticus strain revealed that this strain contains two sets of gene clusters for the type III secretion system (T3SS), T3SS1 and T3SS2, one on each of its two chromosomes (21). T3SS gene clusters have been detected in numerous Gram-negative animal and plant pathogens, wh...

Vibrio parahaemolyticus is a bacterial pathogen causative of food-borne gastroenteritis. Whole-genome sequencing of V. parahaemolyticus strain RIMD2210633, which exhibits Kanagawa phenomenon (KP), revealed the presence of two sets of the genes for the type III secretion system (T3SS) on chromosomes 1 and 2, T3SS1 and T3SS2, respectively. Although T3SS2 of the RIMD2210633 strain is thought to be involved in human pathogenicity, i.e., enterotoxicity, the genes for T3SS2 have not been found in trh-positive (KP-negative) V. parahaemolyticus strains, which are also pathogenic for humans. In the study described here, the DNA region of approximately 100 kb that surrounds the trh gene of a trh-positive V. parahaemolyticus strain, TH3996, was sequenced and its genetic organization determined. This revealed the presence of the genes for a novel T3SS in this region. Animal experiments using the deletion mutant strains of a gene (vscC2) for the novel T3SS apparatus indicated that the T3SS is essential for the enterotoxicity of the TH3996 strain. PCR analysis showed that all the trh-positive V. parahaemolyticus strains tested possess the novel T3SS-related genes. Phylogenetic analysis demonstrated that although the novel T3SS is closely related to T3SS2 of KP-positive V. parahaemolyticus, it belongs to a distinctly different lineage. Furthermore, the two types of T3SS2 lineage are also found among pathogenic Vibrio cholerae non-O1/non-O139 strains. Our findings demonstrate that these two distinct types are distributed not only within a species but also beyond the species level and provide a new insight into the pathogenicity and evolution of Vibrio species.Vibrio parahaemolyticus is a gram-negative halophilic marine and estuarine bacterium which is an important pathogen causative of food-borne gastroenteritis and traveler's diarrhea (1). Although most V. parahaemolyticus strains are nonpathogenic for humans, a limited population of these organisms causes human diseases. Almost all clinical V. parahaemolyticus isolates produce the thermostable direct hemolysin (TDH) and/or the TDH-related hemolysin (TRH), which are encoded by the tdh and trh genes, respectively (5, 21). The Kanagawa phenomenon (KP), a beta-type hemolysis on a special blood agar (Wagatsuma agar) (28), is known as a good marker of pathogenic strains (5, 21). V. parahaemolyticus strains which exhibit KP possess the two tdh genes tdhA (tdh2) and tdhS (tdh1) but not the trh gene (6,19,21). In contrast, KP-negative clinical V. parahaemolyticus strains possess the trh gene only or both the trh and tdh genes, while the majority of the nonpathogenic strains possess neither tdh nor trh.TDH and TRH, which have several biological activities in common (5,20,30,33), are considered to be the major virulence factors in clinical V. parahaemolyticus strains (5, 30).However, several studies have demonstrated that although the enterotoxicity was reduced in tdh-or trh-deleted mutant strains from that in the parent strains, the enterotoxic activity of these mutant strains partiall...

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.

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.

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.C lostridium perfringens, a spore-forming anaerobic rod, is a member of the normal intestinal flora in humans and animals and a component of soil and sewage microbiota (1-4). C. perfringens is the causative agent of various human diseases, including gas gangrene and food-borne gastroenteritis (5-10). The pathogenicity of C. perfringens is attributed to various toxins produced by the organism, including alpha, beta, epsilon, and iota toxins that classify C. perfringens isolates into five toxin types (A to E), theta toxin, NetB, and C. perfringens enterotoxin (CPE) (10-13).CPE, which is mainly produced by type A C. perfringens, is associated with human gastrointestinal (GI) illnesses, such as food-borne gastroenteritis, antibiotic-associated diarrhea, and sporadic diarrhea (14-16). CPE is a 35-kDa protein, and the cpe gene is located in the chromosome or on a plasmid (17)(18)(19)(20). After orally ingested CPE-positive C. perfringens reaches the GI tract, sporulating C. perfringens produces CPE, and the toxin causes clinical symptoms, such as diarrhea and abdominal cramping. In the clinical diagnosis of gastroente...

Vibrio parahaemolyticus is an important pathogen causing food-borne disease worldwide. An 80-kb pathogenicity island (Vp-PAI), which contains two tdh (thermostable direct hemolysin) genes and a set of genes for the type III secretion system (T3SS2), is closely related to the pathogenicity of this bacterium. However, the regulatory mechanisms of Vp-PAI's gene expression are poorly understood. Here we report that two novel ToxR-like transcriptional regulatory proteins (VtrA and VtrB) regulate the expression of the genes encoded within the Vp-PAI region, including those for TDH and T3SS2-related proteins. Expression of vtrB was under control of the VtrA, as vector-expressed vtrB was able to recover a functional protein secretory capacity for T3SS2, independent of VtrA. Moreover, these regulatory proteins were essential for T3SS2-dependent biological activities, such as in vitro cytotoxicity and in vivo enterotoxicity. Enterotoxic activities of vtrA and/or vtrB deletion strains derived from the wild-type strain were almost absent, showing fluid accumulation similar to non-infected control. Whole genome transcriptional profiling of vtrA or vtrB deletion strains revealed that the expression levels of over 60 genes were downregulated significantly in these deletion mutant strains and that such genes were almost exclusively located in the Vp-PAI region. These results strongly suggest that VtrA and VtrB are master regulators for virulence gene expression in the Vp-PAI and play critical roles in the pathogenicity of this bacterium.

Summary 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.

Vibrio parahaemolyticus is a leading cause of seafood‐borne bacterial gastroenteritis in humans. Since its discovery in 1950, this bacterium has been isolated in widespread outbreaks and in sporadic cases of gastroenteritis worldwide. Although the exotoxin, thermostable direct hemolysin, had been the focus of extensive research on the pathogenicity of V. parahaemolyticus, the whole‐genome sequencing of a clinical isolate, RIMD2210633 strain, was a breakthrough in this field. The possession of two sets of gene clusters for type III secretion systems (T3SS1 and T3SS2) was unveiled by that genome project. T3SS is a protein export apparatus that delivers bacterial proteins, called effectors, directly into the host's cytosol, to disrupt host cell function. The subsequent studies have established that T3SS2, which is encoded in an 80 kb pathogenicity island called V. parahaemolyticus pathogenicity island (Vp‐PAI), is closely related to enteropathogenicity. Recent functional analyses of Vp‐PAI‐encoded genes revealed the sophisticated mechanisms in V. parahaemolyticus for sensing the intestinal environment and host cell contact, and a dozen T3SS2‐exported proteins encoded in Vp‐PAI. In this review, we summarize recent advances in V. parahaemolyticus research regarding the control of the expression of Vp‐PAI‐encoded genes, structural components and the secretory regulation of T3SS2, and the biological activities of T3SS2‐exported effectors. Thus, Vp‐PAI‐encoded T3SS2 becomes an important key in the postgenomic era to shed light on the enteropathogenic mechanism of V. parahaemolyticus.