Function Characterization of RHO Inactivation by Vibrio Cholerae RTX Toxin

Vibrio cholerae is responsible for causing the life-threatening diarrheal disease cholera. The World Health Organization confirmed 236,896 cases and 6,311 deaths from cholera in 2006. V. cholerae uses a multitude of virulence factors to colonize and infect humans. <P> Although not directly responsible for eliciting the massive diarrheal response, the V. cholerae RTX toxin (MARTXVc) is involved in pathogenesis and contributes to disease. Incubation of various mammalian cell types with MARTXVc+ strains of V. cholerae causes cell rounding, cross-linking of actin monomers into higher order structures, and inactivation of the Rho GTPases. Recent evidence shows that MARTXVc is composed of at least three functionally distinct domains: the actin cross-linking domain (ACD) that is necessary and sufficient for cross-linking actin, the Rho-inactivation domain (RID) that is sufficient for inactivating Rho, and the cysteine protease domain (CPD) whose function is unknown. <P> The isolation of the MARTXVc domain responsible for inactivating Rho presents an exciting research opportunity. Preliminary evidence gathered by a graduate student in our laboratory indicates that the mechanism for Rho-inactivation by RID is unlike any used by known bacterial toxins. That RID may utilize a unique approach to inactivate Rho further emphasizes the importance of characterizing its mechanism of action because it may help to elucidate host-cell signaling pathways. <P> The focus of my application is to further characterize the mechanism of Rho inactivation by RID. My two specific aims are to: 1) Isolate regions and residues of the MARTXVc RID membrane localization domain, which are important for function. I will carry out a structure/function analysis to establish the portions of RID that are required for localization to the plasma membrane of HeLa cells. 2) Identify binding partners for MARTXVc RID within eukaryotic cells. I will perform co-immunoprecipitations on intoxicated cells to isolate proteins from HeLa cells that interact with RID. <P> PUBLIC HEALTH RELEVANCE: By characterizing the mechanism of action of RID (and MARTXVc), we will enhance our understanding of the strategies V. cholerae utilizes to cause disease. This knowledge may assist in the production of a vaccine or antibiotic that is able to protect those in afflicted regions from cholera. A domain found within RID is found in numerous toxins from many human and animal pathogens, thereby increasing the applicability of our findings to other diseases. The further characterization of components involved in the regulation of Rho GTPases from the proposed research will also benefit eukaryotic cell signaling research.