Characterization of VPA0450, A Type III Secreted Effector Protein from Vibrio Parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium first isolated over 60 years ago after a major outbreak of food poisoning in Japan. It is now recognized as a significant cause of gastroenteritis associated with the consumption of raw or undercooked seafood. The recent emergence of pandemic strains has made the study of V. parahaemolyticus a priority in the field of bacterial pathogenesis. Virulence caused by V. parahaemolyticus has traditionally been attributed to the presence of one or more thermostable direct hemolysins. Genome sequencing of V. parahaemolyticus identified two distinct Type III Secretion Systems (T3SS). T3SS1, on chromosome 1, was shown to translocate four effectors, VopQ, VopR, VopS, and VPA0450, resulting in cytotoxicity of cultured host cells. VopQ has been shown to rapidly induce autophagy upon translocation into a host cell. VopS AMPylates Rho-family guanosine triphosphatases leading to the collapse of the actin cytoskeleton and host cell rounding prior to lysis. Herein we show that VPA0450 is a phosphatidylinositol phosphatase with homology to the inositol polyphosphate 5-phosphatase catalytic domain of the eukaryotic enzyme synaptojanin. VPA0450 was sufficient to induce membrane blebbing and the delocalization actin-binding proteins from the plasma membrane. VPA0450 contributes to cytotoxicity as strains deleted for vpa0450 induced cell lysis less efficiently than wild-type strains. VPA0450 compromised membrane integrity by hydrolyzing the D5 phosphate from phosphotidylinositide (4,5) bisphosphate, thereby disrupting adaptor protein binding sites required for proper membrane and cytoskeleton dynamics, likely contributing to cell death by facilitating lysis. Preliminary studies have shown the C-terminus of VPA0450 is necessary for localization of this effector to the plasma membrane, possibly by binding membranes and phosphoinositides. An improved system was developed for making chromosomal gene deletions in V. parahemaolyticus. New parent strains were created in which the positive regulators of each T3SS were deleted. Additional strains demonstrated that the cytotoxicity seen during infection with T3SS1 positive strains is attributed solely to T3SS1 effectors. Infection with a strain deleted for vopQ, vopS and vpa0450 uncovered the phenotype for VopR. Bioinformatic analysis of VopR identified effector homologs in other pathogens, homologous eukaryotic enzymes, and a catalytic triad.