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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 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.

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SummaryVibrio parahaemolyticus is a leading causative agent of seafood-borne gastroenteritis worldwide. Most clinical isolates from patients with diarrhoea 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 signalling 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.

Vibrio parahaemolyticus is a Gram-negative pathogen that causes food-borne gastroenteritis. A major virulence determinant of the organism is a type III secretion system (T3SS2) encoded on a pathogenicity island, Vp-PAI. Vp-PAI gene expression is regulated by two transcriptional regulators, VtrA and VtrB, whose N-terminal regions share homology with an OmpR-family DNA-binding domain. VtrA activates the gene expression of VtrB, which in turn activates Vp-PAI gene expression; however, the mechanism of this transcriptional activation by VtrA is not well understood. In this study, we determined that VtrA is a membrane protein with a transmembrane (TM) domain, which was required for its transcriptional regulatory activity. Although the N-terminal region of VtrA alone is insufficient for its transcriptional regulatory activity, forced oligomerization using the leucine-zipper dimerization domain of yeast GCN4 conferred transcriptional regulatory activity and a greater affinity for the promoter region of vtrB. A ToxR-based assay demonstrated that VtrA oligomerizes in vivo. We also showed that bile, a host-derived activator of VtrA, induces the oligomerization of VtrA, which requires the C-terminal domain. The promoter region of vtrB contained repetitive T-rich DNA elements, which are important for vtrB transcriptional activation and are conserved among T3SS2-possessing Vibrio species. These findings propose that VtrA is active as oligomers, which may facilitate its N-terminus binding the target DNA, thus enhancing its transcriptional regulatory activity.

This study found, for the first time, that SEQ, a nonclassical SE, had remarkable stability to heat treatment and enzyme degradation and exhibited significant emetic activity, indicating that SEQ is a high-risk toxin in food poisoning.

We proposed a microfluidic device integrated with removable membranes for culturing a 3D tissue—a spheroid—directly on an ‘on-chip vascular bed’.