Disruption of Barrier Integrity by Salmonella typhimurium Requires Activation of Cdc42 and Rac1 in Epithelial Cells

Gastrointestinal infections in humans have been associated with a number of diseased condition, including stomach ulcers, gastroenteritis, Crohn's disease, and rheumatic arthritis. Such infections often cause altered intestinal permeability through perturbation of the tight junctions that hold epithelial cells together. The objective of the present studies was to detennine whether the enteric pathogens Salmonella, Yersinia, and Rotavirus can disrupt the integrity of the epithelial barrier, and, if so, how this is achieved. Another aim was to elucidate regulation of the epithelial batrier in relation to the structure of the cytoskeleton.To accomplish these goals, we assessed the mechanism of enhanced cytotoxicity of Yersinia YopE and the response to this protein by its target in the epithelial bartier, both of which require contact between the bacteria and the eukaryotic cells. YopK appeared to control Yop effector delivery by regulating the size of the translocation pore, and enhanced translocation was accompanied by decreased transepithelial resistance and disruption of barrier function. We also examined the interaction of Yersinia with polarized MDCK cells to detemrine the target of these bacteria. We found that wild-type Yersinia adhered apically to the tight junction areas, and, in adjacent cells, these contact points displayed s1 integrins and tight junction proteins that allowed localized invasin-mediated binding and translocation of cytotoxins. Studying signal transduction pathways involved in the disruption of barrier function by Salmonella typhimurium, we found that infection with the wild-type strain increased the level of activated. Rac1 and Cdc42 small G-proteins and caused them to accumulate apically in MDCK cells, and this was prevented by appropriate inhibitors. Activation of these proteins was a prerequisite of disruption of barrier integrity by S. typhimurium. We also considered specific effects of the rota virus non-structural protein NSP4 on the function of tight junctions. NSP4 has been desctibed as the first viral enterotoxin, and we found that incubation of noncontluent MDCK-1 cells with NSP4 prevented development of the permeability barrier, as well as lateral targeting of the tight junction-associated zonula occludence-1 protein.In conclusion, our results provide strong evidence that the studied pathogens perturb the epithelial barrier by binding to specific cell receptors to deliver cytotoxins (Yesinia); by interfering with cell signaling pathways (Salmonella); and by impairing normal formation of tight junctions (NSP4).