Evasion of NADPH oxidase by Salmonella lipases

描述（由申请人提供）：沙门氏菌在专业吞噬细胞内存活的能力对其致病性至关重要，因为无法在单核吞噬细胞中存活的菌株在小鼠中严重减弱。沙门氏菌在吞噬细胞内的存活与其沿内吞途径阻止吞噬体成熟的能力密切相关。我们最近发现沙门氏菌致病性岛2 （SPI2）编码的效应蛋白通过干扰TNFalpha/TNFRp55信号级联来重塑吞噬体，该信号级联指导含有nadph氧化酶的囊泡递送到沙门氏菌吞噬体附近。我计划验证这样的假设，即通过切割葡萄糖神经酰胺，葡萄糖神经酰胺酶干扰TNFRp55信号，并阻止含有NADPH氧化酶的囊泡向沙门氏菌吞噬体附近的迁移。具体目标是：

DESCRIPTION (provided by applicant): 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.