The ability of Salmonella to survive within professional phagocytes is paramount to its pathogenicity, as strains incapable of survival in mononuclear phagocytes are severely attenuated in mice. Survival of Salmonella within phagocytes is tightly associated with its ability to halt maturation of the phagosome along the endocytic pathway. We have recently discovered that Salmonella pathogenicity island 2 (SPI2)-encoded effector proteins remodel the phagosome by interfering with the TNFalpha/TNFRp55 signaling cascade that directs delivery ofNADPH oxidase-containing vesicles to the vicinity of the Salmonella phagosome. I plan to test the hypothesis that, by cleaving glucosylceramide, glucosylceramidase interferes with TNFRp55 signaling and blocks migration of NADPH oxidase-harboring vesicles to the vicinity of the Salmonella phagosome.
The specific aims are: 1) To determine the role of glucosylceramidase in the pathogenesis of Salmonella infections. Glucosylceramidase mutants will be constructed to examine the effect on both survival of Salmonella in macrophages and inhibition NADPH oxidase trafficking. 2) To examine the effect of Salmonella-encoded glucosylceramidase on macrophage lipid metabolism. Wildtype and glucosylceramidase-deficient Salmonella will be used to infect macrophages to assess global changes in the sphingomyelin pathway. Purified glucosylceramidase will be tested for lipid metabolizing activity, and immunocytochemistry will be used to visualize the effect of glycosylceramidase on trafficking of NADPH oxidase-containing vesicles. 3) To determine the role of glucosylceramidase on avoiding IFN( activated trafficking of NADPH oxidase. In this section, I will elucidate the mechanisms by which IFN gamma stimulates anti-Salmonella activity dependent upon the NADPH oxidase. The ability of IFNgamma to stimulate glucosylceramide synthesis will be examined as well. These studies will not only enhance our understanding of Salmonella pathogenesis but will also identify potential targets for the development of new therapeutic strategies against intracellular pathogens.
