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Suvarnapunya, Akamol E.

Microbiology and Molecular Genetics

2004

PhD

Salmonella enterica is a facultative intracellular pathogen that causes diseases as mild as gastroenteritis and as life-threatening as typhoid fever in many different host organisms, including humans. The ability of S enterica to survive and replicate within various types of host cells directly correlates to the establishment and maintenance of systemic disease. This paradigm was established by previous characterizations of serovar Typhimurium mutants that were defective for replication in macrophages, as well as mutants that were defective for replication in epithelial cells (Rep- mutants). To more completely understand how S typhimurium is able to survive and replicate within host cells, a genetic and molecular characterization of the Rep- mutants was undertaken. It was determined that the MudJ insertions in the three Rep- mutants had occurred in mutS and mutH, encoding enzymes of the DNA mismatch repair (MMR) system, and ssaQ, encoding a putative apparatus protein of the Salmonella Pathogenicity Island 2 (SPI2) Type III secretion system. While the SPI2 mutation was linked to the Rep- phenotype, it was discovered that the MMR mutations were unlinked. The hypermutation resulting from the MMR gene disruptions was likely to have given rise to secondary mutations that reduced intraepithelial survival. Indeed, an unknown secondary mutation in the mutS mutant abrogated SPI2 function. Thus, while SPI2 has since been well-established as a critical S typhimurium determinant of both intraepithelial and intramacrophage survival, the importance of DNA repair remained an open question. This question is even more significant when considering the formidable challenges that Salmonella faces within macrophages. In order to survive and replicate within macrophages, Salmonella must be able to inhibit or at least manage macrophage induced oxidative stress, which is a primary host antibacterial mechanism. Therefore, the contribution of the intrinsically-redundant DNA base-excision repair (BER) system to intramacrophage survival was analyzed, since the BER system is specifically dedicated to oxidative damage repair. Several BER deletion mutants, particularly double mutants of bifunctional glycosylases ( nth/nei) or AP endonucleases ( xth/nfo), were defective for both intramacrophage survival and murine virulence, demonstrating for the first time that S typhimurium DNA is a critical target of macrophage oxidants. Concurrently, research by others suggested that SPI2 enables intramacrophage survival by inhibiting the intracellular targeting of oxidant generators. To clarify this still-emerging and controversial role of SPI2 in oxidant defense, the effects of SPI2 mutations on a mutant unable to initiate BER ( fpg/nth/nei) were assessed under macrophage oxidative stress. SPI2 was found to directly protect S typhimurium DNA from macrophage-induced mutation. Statistical comparisons of the intramacrophage survival of double SPI2/BER mutants to single mutants suggested that SPI2 and BER function synergistically to inhibit oxidant lethality. The SseF and SseG SPI2 effectors were implicated, whereas SifA was not. This shows for the first time that SPI2 enables intramacrophage survival by directly protecting a critical target of macrophage oxidants. The research presented here enhances our understanding of Salmonella pathogenesis by showing the importance of oxidative DNA damage to the macrophage Salmonella interaction, and may also be more broadly applicable to other intracellular pathogens.