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Zurawski, Daniel V.

Cell and Molecular Biology Program

2004

Ph. D.

Salmonella enterica serovar Typhimurium (S. typhimurium) is a gram negative species of bacteria that is a model system to study Salmonella enterica serovar Typhi, the bacterial agent responsible for typhoid fever in humans. There are some 16 million cases reported every year, with 600,000 of these leading to death. More is being discovered about how Salmonella accomplishes this deadly feat. S. typhimurium invades enterocytes in the small intestine or is phagocytosed by cells monitoring the Peyer's patch, namely macrophages. Once engulfed, S. typhimurium resides inside a membrane-enclosed compartment called the Salmonella-containing vacuole (SCV). In order to survive the deleterious effects of phagosome and replicate, S. lyphimurium uses a Type III secretion system (TTSS) called Salmonella Pathogenicity Island 2 (SPI2) to secrete effector proteins into cells that modify normal trafficking in the host. Modified trafficking leads to avoidence of oxidative burst and iNOS delivery to the phagosome in macrophage. Secondly, tubular membrane extensions called Salmonella-induced filaments (Sifs) are formed and extend from the SCV. The formation of Sifs coincides directly with the onset of replication and is an essential virulence trait. SPI2 was shown to be required for Sifs after a screen was done for S. typhimurium mutants that were Sif-deficient A SPI2 gene, sseA, had an unknown function and no homology to known proteins. An sseA null mutant ( sseA) was defective for Sifs and for secretion of SseB, the putative SPI2 translocon sheath. It was also revealed that the total amount of SseB is significantly reduced in the sseA mutant. SseB accumulation and export were restored when SseA was provided in trans on a plasmid. Loss of SseB secretion was specific to SseB because SseC, just downstream, was still secreted in the sseA mutant. Co-purification studies demonstrated that SseA directly binds to SseB. Collectively, these results demonstrated that SseA functions as a TTSS chaperone for SseB. Subsequently, it was shown that SseA was also the chaperone for SseD, a putative translocon pore. Other mutants, originally isolated to be replication defective in epithelial cells, were further studied to see if they played a role in virulence. The mutations were mapped to MutH and MutS the DNA mismatch repair system, and SsaQ, a conserved inner membrane component of SPI2. Consistent with SPl2 function the SsaQ null mutant was shown to be defective for SseB secretion and Sif formation. MutH and MutS mutations showed that these proteins were not required for S. typhimurium virulence in mice. The interactions of SseA with its partners, SseB and SseD, were characterized further. A yeast two-hybrid screen indicated that SseA binding requires a C-terminal domain within both partners. The SseA-binding region within SseB was found to encompass a predicted coiled-coil. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization. This work has shown the importance of SPI2 in the disease process of S. typhimurium, and that SseA is a TTSS chaperone that is required for proper SseB and SseD secretion and function. Mapping the binding sites allowed for SseA classification.