Title: Characterization of the Type III Secretion System in non-O1, non-O139 strains of Vibrio cholerae
Description: V. cholerae is a Gram-negative bacterial pathogen that causes the diarrheal disease cholera. There are more than 200 known serogroups of V. cholerae, but all seven pandemics have been caused by two serogroups, O1 and O139. The major virulence mechanisms of O1 and O139 strains have been well characterized; the type IV pilus, TCP, and cholera toxin, CT, are required to cause disease. While V. cholerae O1 and O139 remain the major etiologic agents of cholera, non-O1, non-O139 strains are causing sporadic disease via undefined virulence mechanisms globally. A clinical non-O1, non-O139 V. cholerae strain, AM19226, was sequenced and a novel Type III Secretion System (T3SS) was identified. T3SS is a protein secretion system that injects effector proteins directly from the cytoplasm of the bacteria into the cytosol of the eukaryotic host cell. We demonstrated that the T3SS is functional and is required for intestinal colonization in AM19226. We also identified VopF, an effector that nucleates actin in vitro and induces protrusion formation in infected or transfected cells. Next, we defined the entire T3SS pathogenicity island in AM19226 and identified a divergent T3SS pathogenicity island in additional non-O1, non-O139 strains. Comparing the two V. cholerae T3SS pathogenicity islands, we identified a IT VopF homolog, VopN, which promotes stress fiber formation instead of protrusion formation in host cells. This difference in cellular structures may result from the distinct localization of the homologs. Using the infant mouse model, vopN can only partially complement a vopF deletion, which has a defect in colonizing the small intestine. Moreover, we determined that AM19226 uses T3SS to disrupt the cytoskeleton and loosen tight junctions in polarized T84 cells. The in vitro data was corroborated by the in vivo diarrheal response in infected adult rabbits.

Lastly, VopM, an essential colonization factor with a putative transmembrane domain, was identified as a type III effector. The translocation of VopM is dependent on a chaperone VceM. A putative outer membrane protein, VomA, is hypothesized to recognize and bind VopM. Accordingly, deletion of vceM or vomA rendered the strains avirulent. This dissertation has advanced our understanding of the virulence mechanisms in TCP-CT-, T3SS+, non-O1, non-O139 V. cholerae strains.