Abstract
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.
Highlights
The Vibrio cholerae multifunctional autoprocessing repeats-in-toxin (MARTX) toxin Rho inactivation domain (RIDVc) currently has an unknown mechanism of action
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
Using V. cholerae carrying a MARTX toxin with an inactivated RIDVc, we show that RIDVc activity does not alter patterns or dynamics of actin cross-linking by ACDVc in intoxicated epithelial cells, suggesting that the increased availability of G-actin in the cell due to RIDVc activity does not enhance the activity of the ACDVc
Summary
The Vibrio cholerae MARTX toxin Rho inactivation domain (RIDVc) currently has an unknown mechanism of action. It is related to the glutamine synthetase family of enzymes and irreversibly connects G-actin monomers via isopeptide bonds between residues Lys-50 and Glu-270 in the presence of ATP and Mg2ϩ/ Mn2ϩ [11,12,13,14,15] Another effector domain, the ␣-hydrolase domain, has been identified by analysis of CPD processing sites [7, 16] and by sequence homology to ␣-hydrolase family members [17], its mechanism of action has not been assessed experimentally. In silico analysis of the AD predicts a circularly permuted papain-like thiol protease-fold suggesting RIDVc could function as a cysteine protease or acyltransferase [21] This putative catalytic activity is not likely directed against RhoA as the cell is fully restored upon removal of toxin-producing bacteria even when eukaryotic protein biosynthesis is blocked using cycloheximide [18]. Using V. cholerae carrying a MARTX toxin with an inactivated RIDVc, we show that RIDVc activity does not alter patterns or dynamics of actin cross-linking by ACDVc in intoxicated epithelial cells, suggesting that the increased availability of G-actin in the cell due to RIDVc activity does not enhance the activity of the ACDVc
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