Vibrio cholerae is the causative agent of the diarrheal disease cholera. Many virulence factors contribute to intestinal colonization and disease including the Multifunctional Autoprocessing RTX toxin (MARTX Vc ). The Rho-inactivation domain (RID) of MARTX Vc is responsible for inactivating the Rho-family of small GTPases, which leads to depolymerization of the actin cytoskeleton. Based on a deletion analysis of RID to determine the minimal functional domain, we have identified a subdomain at the N terminus of RID that is homologous to the membrane targeting C1 domain of Pasteurella multocida toxin. A GFP fusion to this subdomain from RID colocalized with a plasma membrane marker when transiently expressed within HeLa cells and can be found in the membrane fraction following subcellular fractionation. This C1-like subdomain is present in multiple families of bacterial toxins, including all of the clostridial glucosyltransferase toxins and various MARTX toxins. GFP-fusions to these homologous domains are also membrane associated, indicating that this is a conserved membrane localization domain (MLD). We have identified three residues (Y23, S68, R70) as necessary for proper localization of one but not all MLDs. In addition, we found that substitution of the RID MLD with the MLDs from two different effector domains from the Vibrio vulnificus MARTX toxin restored RID activity, indicating that there is functional overlap between these MLDs. This study describes the initial recognition of a family of conserved plasma membrane-targeting domains found in multiple large bacterial toxins.bacterial toxin | multifunctional autoprocessing RTX toxin | PMT | structural modeling | Vibrio cholerae B acterial pathogens use a wide array of secreted proteins to intoxicate mammalian cells and mediate disease. Many effectors target a specific organelle, necessitating strategies to traffic to certain subcellular locations after entry into the cytosol. For example, the cytolethal distending toxin active subunit, CdtB, uses a short N-terminal nuclear localization sequence attached to traffic to the nucleus (1). Similarly, many Type III secretion effectors contain distinct targeting signals: BteA uses a 130 aa N-terminal sequence to target lipid rafts (2); ExoS and YopE use internal 21 aa domains to associate with the host membrane (3); and ExoU uses a C-terminal sequence to localize to the membrane (4). For each of these effectors, subcellular localization is determined by a specific aa sequence separate from the catalytic site.The multifunctional-autoprocessing RTX toxins (MARTX) are large bacterial toxins composed of multiple effector domains released by autoprocessing upon translocation into the host cell cytosol (5). The best characterized member of this family is the MARTX of Vibrio cholerae (MARTX Vc ), which is predicted to be 4545 aa in length (6, 7). MARTX Vc carries three effector domains delivered by autoprocessing of which two function to disrupt the actin cytoskeleton. G-actin is covalently cross-linked into oligomers by th...
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