The Gram-negative pathogen Vibrio cholerae causes diarrheal disease through the export of enterotoxins. The V. cholerae RTX toxin was previously identified and characterized by its ability to round human laryngeal epithelial (HEp-2) cells. Further investigation determined that cell rounding is caused by the depolymerization of actin stress fibers, through the unique mechanism of covalent actin cross-linking. In this study, we identify a domain within the full-length RTX toxin that is capable of mediating the cross-linking reaction when transiently expressed within eukaryotic cells. A structure͞function analysis of the actin cross-linking domain (ACD) reveals that a 412-aa, or a 47.8-kDa, region is essential for cross-linking activity. When this domain is deleted from the full-length toxin gene, actin cross-linking, but not cell rounding, is eliminated, indicating that this toxin carries multiple dissociable activities. The ACD shares 59% amino acid identity with a hypothetical protein from V. cholerae, VC1416, and transient expression of the C-terminal domain of VC1416 also results in actin crosslinking in eukaryotic cells. The presence of this second ACD linked to an Rhs-like element suggests that V. cholerae acquired the domain by horizontal gene transfer and the ACD was inserted into the RTX toxin by gene duplication through the evolution of V. cholerae.Vibrio vulnificus ͉ VgrG ͉ covalent actin cross-linking

The type III secretion system (T3SS) is a highly conserved protein delivery system found in multiple Gram-negative pathogens, including Yersinia pseudotuberculosis. Most studies of Yersinia species type III intoxication of host cells have focused on the bacterial determinants that promote assembly and function of the secretion system. In this study, we performed a pooled RNA interference (RNAi) screen to identify mammalian host proteins required for the cytotoxic effects associated with the Yersinia translocated substrate YopE, a GTPase-activating protein (GAP) that inactivates the small Rho GTPases. Cell populations were positively selected for short hairpin RNAs (shRNAs) that interfere with YopE activity using a combination of fluorescence resonance energy transfer (FRET) and flow cytometry, and the degree of enrichment was determined by deep sequencing. Analysis of the candidates identified by the enrichment process revealed that many were important for the initial step of Y. pseudotuberculosis T3SS function, YopB/D pore formation. These candidates included shRNA that depleted downstream effectors of RhoA signaling, coated pit formation, and receptors involved in cell signaling, including the chemokine receptor CCR5 (chemokine [C-C motif] receptor 5). Depletion of CCR5 in 293T cells yielded a defect in YopB/D pore formation and effector translocation, while both phenotypes could be complemented by overexpression of CCR5 protein. Yop effector translocation was also decreased in isolated primary phagocytic cells from a Ccr5−/− knockout mouse. We postulate that CCR5 acts to promote translocation by modulating cytoskeletal activities necessary for proper assembly of the YopB/D translocation pore. Overall, this study presents a new approach to investigating the contribution of the host cell to T3SS in Y. pseudotuberculosis.