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 (MARTXVc) 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 MARTXVc is the Rho inactivation domain (RIDVc) known to cause cell rounding through inactivation of small RhoGTPases. Using alanine scanning mutagenesis in the activity subdomain of RIDVc, four residues, His-2782, Leu-2851, Asp-2854, and Cys-3022, were identified as impacting RIDVc 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 RIDVc show that the rate and efficiency of MARTXVc actin cross-linking activity does not depend on a functional RIDVc, 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.