Breaching the epithelial barrier : Salmonella enterica serovar Typhimurium invasion and disruption of tight junctions

By invading epithelial cells and disrupting cell-cell junctions, Salmonella enterica serovar Typhimurium (S. Typhimurium) employs both transcellular and paracellular mechanisms in order to overcome the barrier function of epithelial cell monolayers. The research presented in this thesis has focused on defining and elucidating both the host and bacterial factors required for S. Typhimurium to breach the epithelial barrier. Internalization of S. Typhimurium absolutely requires the actin cytoskeleton, yet only a few of the cytoskeletal components involved in this process have been identified. The recruitment of actin-associated proteins to the site of invasion was investigated in order to identify host proteins that may play a role in S. Typhimurium invasion. The role of several key cytoskeletal proteins was assayed to determine whether they were required for S. Typhimurium invasion. The actin-severing protein, gelsolin, was found to inhibit the invasion process while ezrin did not play an important role during S. Typhimurium invasion. The contribution of the recruited Src homology (SH) 2 adaptors to invasion was further investigated. While not involved in bacterial internalization itself, the adaptors Nck and ShcA influenced adherence of S. Typhimurium to non-phagocytic cells. Using the murine model of S. Typhimurium-induced colitis we investigated whether S. Typhimurium disrupted epithelial tight junctions in vivo. S. Typhimurium infection of mice caused a significant increase in intestinal permeability and resulted in the redistribution of tight junction proteins ZO-1 and claudin-3 in the colon. Using Salmonella pathogenicity island-1 (SPI-1)-secreted effector mutants we found that SopB, SopE, SopE2, and SipA are the specific effectors responsible for disruption of tight junction structure and function in vitro. Tight junction disruption by S. Typhimurium was prevented by inhibiting host protein geranylgeranylation but was not dependent on host protein synthesis or secretion of host-derived products. These data suggest that SPI-1-secreted effectors utilize their ability to stimulate Rho family GTPases to disrupt tight junction structure and function. Because the ability of S. Typhimurium to penetrate the intestinal epithelium is key to its pathogenesis, these studies significantly enhance our understanding host-pathogen interaction and the molecular events leading to disease.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat