Genetic and Biochemical Study of V. Cholerae RTX Toxin

Abstract The life-threatening diarrheal disease cholera is caused by toxigenic strains of the Gram-negativeorganism Vibrio cholerae. In addition to the well-characterized ADP-ribosylating cholera toxin (CT), V.cholerae secretes a novel toxin that is the founding member of new family of Multifunctional, AutoprocossinRTX toxins (MARTX). The MARTX toxin of V. cholerae (MARTX-Vc) contributes to virulence in mice and maybe an important factor contributing to persistent colonization both in cholera patients and non-symptomaticcarriers.. MARTX-Vc is of intrinsic interest due to its novel biochemical properties and mode of action. At apredicted size of >480 kDa, the toxin is a multifunctional toxin. Thus far, we have described three activitiesassociated with this toxin. We have demonstrated that this toxin depolymerizes actin stress fibers andcovalently crosslinks actin into oligomers. We have demonstrated that this toxin causes rounding of cells dueto inactivation of RhoGTPases by an unknown mechanism. Finally, this toxin has a cysteine protease domainwith autoprocessing activity that may also have cellular targets resulting in cytotoxicity. Each of these activitieshas been mapped to a specific domain of the toxin. A fourth domain homologous to the alpha-beta hydrolasefamily of proteins is also a putative activity domain. In this grant proposal, we will pursue the multiple catalytic activities of the toxin by studying themechanism of action of the independent activity domains. A structure function analysis of the actincrosslinking domain will be performed and the regions essential for catalysis and actin binding will be identified.The target of the RhoGTPase inactivating domain will be identified and characterized. The requirements forautocleavage site recognition and inositol hexakisphosphate binding will be investigated and other potentialsites of cleavage identified. In all, this work will further our understanding of the mechanism of action of thistoxin and provide insight into its role in disease and will also provide information about related toxins that sharethe novel functional domains carried by this unique multifunctional toxin. Lay summary The life-threatening diarrheal disease cholera is caused the Gram-negative organism Vibrio cholerae.In addition to the well-characterized ADP-ribosylating cholera toxin (CT), V. cholerae secretes a novel toxinthat is the founding member of new family of the RTX toxins, the Multifunctional-Autoprocessing RTX toxins(MARTX). The MARTX toxin of V. cholerae contributes to virulence in mice and may be an important factorcontributing to persistent colonization both in cholera patients and non-symptomatic carriers. This proposalseeks to understand the cellular mechanism of action of this toxin that has three known enzymatic activities:actin crosslinking, Rho-inactivation, and autoprocessing. Completion of this research will impact not only ourunderstanding of cholera pathogenesis, but also the function of other uncharacterized toxins produced by otherhuman pathogens that share these unique enzymatic activities.