Identification of a His-Asp-Cys Catalytic Triad Essential for Function of the Rho Inactivation Domain (RID) of Vibrio cholerae MARTX Toxin

Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. For V. cholerae to colonize the intestinal epithelium, accessory toxins such as the multifunctional autoprocessing repeats-in-toxin (MARTX Vc ) toxin are required. MARTX toxins are composite toxins comprised of arrayed effector domains that carry out distinct functions inside the host cell. Among the three effector domains of MARTX Vc is the Rho inactivation domain (RID Vc ) known to cause cell rounding through inactivation of small RhoGTPases. Using alanine scanning mutagenesis in the activity subdomain of RID Vc , four residues, His-2782, Leu-2851, Asp-2854, and Cys-3022, were identified as impacting RID Vc function in depolymerization of the actin cytoskeleton and inactivation of RhoA. Tyr-2807 and Tyr-3015 were identified as important potentially for forming the active structure for substrate contact but are not involved in catalysis or post translational modifications. Finally, V. cholerae strains modified to carry a catalytically inactive RID Vc show that the rate and efficiency of MARTX Vc actin cross-linking activity does not depend on a functional RID Vc , demonstrating that these domains function independently in actin depolymerization. Overall, our results indicate a His-Asp-Cys catalytic triad is essential for function of the RID effector domain family shared by MARTX toxins produced by many Gram-negative bacteria. Background: The Vibrio cholerae MARTX toxin Rho inactivation domain (RID Vc ) currently has an unknown mechanism of action. Results: Residues His-2782, Leu-2851, Asp-2854, and Cys-3022 are shown to be essential for RID Vc activity. Conclusion: His-Asp-Cys form a catalytic triad necessary for enzymatic modification of the cellular target. Significance: RID effectors are Clan CE cysteine endopeptidases essential for Rho GTPase inactivation.