PROJECT SUMMARY/ABSTRACT Enteric fever, caused by the Salmonella serovars S. Typhi and S. Paratyphi, accounts for nearly 15 million infections and 136,000 deaths each year. Our laboratory has developed a lethal small animal model for enteric fever using humanized mice engrafted with a functional human hematopoietic system. The ability of S. Typhi and S. Paratyphi A to cause lethal infections in humanized mice engrafted with human hematopoietic cells suggests that human mononuclear phagocytes are required for the pathogenesis of enteric fever. A genome-wide analysis of S. Typhi virulence determinants in humanized mice has confirmed some suspected essential virulence determinants but has also revealed unexpected differences between S. Typhi and non-typhoidal Salmonella serovars. Furthermore, we have discovered that S. Typhi persists in cultured human macrophages by preventing cell death, due to the absence of multiple SPI-2 type III secretion system effectors that play a central role in non-typhoidal Salmonella pathogenesis. We have recently found significant differences in the responses of enteric fever and non-typhoidal Salmonella to iron deprivation, observed that the gut microbiota antagonizes S. Typhi intestinal colonization, and shown important parallels between S. Paratyphi A and S. Typhi in their interactions with human macrophages and humanized mice. Our central hypothesis is that the virulence of enteric fever Salmonella serovars depends on the evasion of innate immunity and persistence in mononuclear phagocytes. The research plan will examine three specific aims: 1. Avoidance of Macrophage Cell Death by Enteric Fever Salmonella Serovars. Genetic and biochemical approaches will elucidate the molecular mechanisms by which S. Typhi promotes macrophage survival and assess the relevance of macrophage survival to S. Typhi virulence in humanized mice. 2. Interactions of Salmonella Typhi with the Gut Microbiota. The contributions of extracytoplasmic stress responses, microbiota antagonism and avoidance of macrophage cytotoxicity to the ability of S. Typhi to persistently colonize the intestinal tract will be investigated. 3. Salmonella Paratyphi A Virulence. A systematic analysis of S. Paratyphi virulence determinants and its mechanisms of iron acquisition and macrophage persistence will be performed. The proposed studies will advance our understanding of enteric fever pathogenesis and can lead to novel strategies for its prevention and treatment.