Gastroenteritis cases associated with non-toxigenic strains of Vibrio parahaemolyticus have been reported in many countries, suggesting the contribution of novel virulence factors. One candidate is zonula occludens toxin (Zot), which increases the intestinal permeability by other bacteria. Recently we identified prophages belonging to the Inoviridae family encoding putative Zot-like toxins in Chilean strains. Based on this information we performed sequence-based analyses of these toxins, followed by phylogenetic and structural analyses using computational tools. Our results showed that Zots found in Chilean V. parahaemolyticus strains are grouped into three different phylogenetic clusters, sharing two conserved motifs (Walker A and B) in their N-terminal region. These motifs are also conserved in Zots from the human pathogens Vibrio cholerae, Neisseria meningitidis and Campylobacter concisus. Although Zots of V. parahaemolyticus do not possess the FCIGRL sequence responsible for the effects produced by V. cholerae, they do possess a conserved secondary structure within their C-terminal region with Zots proteins able to disrupt the intestinal barrier, which is interesting since it has been suggested that the structure and not the Zot sequence would be responsible for the biological effects. This preliminary study provides the basis to study the function of Zots found in V. parahaemolyticus on the intestinal barrier and their possible role as a virulence factor.

Vibrio parahaemolyticus is an important human pathogen that is currently the leading cause of shellfish-borne gastroenteritis in the world. Particularly, the pandemic strain has the capacity to induce cytotoxicity and enterotoxicity through its Type 3 Secretion System (T3SS2) that leads to massive cell death. However, the specific mechanism by which T3SS2 induces cell death remain unclear and the contribution in mitochondrial stress is not fully understood. In this work, we evaluated the contribution of T3SS2 of V. parahaemolyticus to generate mitochondrial stress and cell death during infection in human intestinal HT-29 cells. To evaluate this, infection assays were carried out to evaluate mitochondrial transition pore opening, mitochondrial fragmentation, ATP quantification, and cell viability during infection. Our results showed that the Δvscn1 (T3SS2+) mutant strain contributes to generating the sustained opening of the mitochondrial transition pore. Furthermore, it generates perturbations in the ATP production in infected cells, leading to a significant decrease in cell viability and loss of membrane integrity. Our results suggest that T3SS2 from V. parahaemolyticus plays a role in generating mitochondrial stress and cell death in human intestinal HT-29 cells. It is important to highlight that this study represents the first report indicating the involvement of T3SS2 from V. parahaemolyticus in generating mitochondrial stress, its impact on the mitochondrial pore, and its effect on ATP production in human cells.