The appearance of multidrug-resistant Salmonella isolates and the HIV epidemic have contributed to the resurgence of salmonellosis, an infection that annually afflicts more than 1 billion people worldwide. Multiple clinical and experimental lines of evidence point to the NADPH oxidase as a critical host defense mechanism in resistance to acute Salmonella infections. Salmonella, an enteric pathogen adapted to the intracellular environment of phagocytes, resides in remodeled phagosomes that selectively block contact with lysosomes and endocytic vesicles harboring the NADPH oxidase. A recently discovered locus at centisome 30 of the Salmonella chromosome encodes a type III secretory system known as Salmonella pathogenicity island 2 (SPI2) that disrupts maturation of the Salmonella phagosome. The primary goal of my laboratory is to understand the mechanisms by which this intracellular pathogen remodels its phagosome and evades the antimicrobial armamentarium of professional phagocytes. In the present proposal, we plan to test the hypothesis that SPI2 effectors decrease TNFRp55-stimulated ganglioside synthesis, thus blocking the migration of NADPH oxidase-harboring vesicles to the vicinity of the Salmonella phagosome. We specifically plan: 1) To identify SPI2 effector proteins that block trafficking of the NADPH oxidase. Attenuation of SPI2 mutants in macrophages and mice, coupled to techniques in molecular and cell biology, biochemistry and microscopy will be used to identify effector proteins that block NADPH oxidase trafficking. 2) To identify points in the TNFRp55-stimulated sphingomyelin pathway which are inhibited by SPI2 effector proteins. Lipid biochemistry, enzymology and bacterial genetics will be used to identify points in the sphingomyelin pathway inhibited by SPI2 effectors. And 3) To determine the kinetics of secretion and intracellular location of SPI2 effectors that inhibit the trafficking of the NADPH oxidase. Cell biology, immunology and microbial genetics will be used to study the early intracellular expression of SPI2 effectors and their distribution relative to the NADPH oxidase, TNFRp55 and the Salmonella phagosome. These studies will not only shed light on the cell biology of the NADPH oxidase but will also identify potential molecular targets common to intracellular pathogens such as Salmonella, Mycobacterium, and Legionella that are capable of thwarting the normal maturation of the nascent phagosome.
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