Vp1659 Is a Vibrio parahaemolyticus Type III Secretion System 1 Protein That Contributes to Translocation of Effector Proteins Needed To Induce Cytolysis, Autophagy, and Disruption of Actin Structure in HeLa Cells

Vibrio parahaemolyticus harbors two type III secretion systems (T3SSs; T3SS1 and T3SS2), of which T3SS1 is involved in host cell cytotoxicity. T3SS1 expression is positively regulated by ExsA, and it is negatively regulated by ExsD. We compared the secretion profiles of a wild-type strain (NY-4) ofV. parahaemolyticus with those of an ExsD deletion mutant (ΔexsD ) and with a strain of NY-4 that overexpresses T3SS1 (NY-4:pexsA ). From this comparison, we detected a previously uncharacterized protein, Vp1659, which shares some sequence homology with LcrV fromYersinia . We show thatvp1659 expression is positively regulated by ExsA and is negatively regulated by ExsD. Vp1659 is specifically secreted by T3SS1 ofV. parahaemolyticus , and Vp1659 is not required for the successful extracellular secretion of another T3SS1 protein, Vp1656. Mechanical fractionation showed that Vp1659 is translocated into HeLa cells in a T3SS1-dependent manner and that deletion of Vp1659 does not prevent VopS from being translocated into HeLa cells during infection. Deletion ofvp1659 significantly reduces cytotoxicity when HeLa cells are infected byV. parahaemolyticus , while complementation of the Δvp1659 strain restores cytotoxicity. Differential staining showed that Vp1659 is required to induce membrane permeability in HeLa cells. We also show evidence that Vp1659 is required for actin rearrangement and the induction of autophagy. On the basis of these data, we conclude that Vp1659 is a T3SS1-associated protein that is a component of the secretion apparatus and that it is necessary for the efficient translocation of effector proteins into epithelial cells.