Evasion of NADPH oxidase by Salmonella lipases

沙门氏菌脂肪酶逃避 NADPH 氧化酶

• 批准号: 6915100
• 负责人: Bruce D MCCOLLISTER
• 金额: $ 11.45万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6915100
• 关键词: NAD(P)H dehydrogenase; SDS polyacrylamide gel electrophoresis; Salmonella infections; bacterial genetics; bacterial proteins; bactericidal immunity; gene expression; gene mutation; genetically modified animals; glucosylceramidase; host organism interaction; immunocytochemistry; interferon gamma; intracellular; intracellular transport; laboratory mouse; lipase; lipid metabolism; macrophage; phagocytosis; polymerase chain reaction; protein transport; southern blotting

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.

描述(由申请人提供)：沙门氏菌在专业吞噬细胞中存活的能力对其致病性至关重要，因为在小鼠中无法在单核吞噬细胞中存活的菌株会严重减弱。沙门氏菌在吞噬细胞内的存活与其阻止吞噬小体沿内吞途径成熟的能力密切相关。我们最近发现，沙门氏菌致病岛2(SPI2)编码的效应蛋白通过干扰TNFpha/TNFRp55信号级联来重塑吞噬小体，该信号级联将含有NADPH氧化酶的囊泡运送到沙门氏菌吞噬小体附近。我计划测试这一假设，即通过裂解葡萄糖神经酰胺，葡萄糖神经酰胺酶干扰TNFRp55信号转导，并阻止携带NADPH氧化酶的小泡迁移到沙门氏菌吞噬小体附近。具体目标是： 1)探讨葡萄糖神经酰胺酶在沙门氏菌感染发病机制中的作用。 将构建葡萄糖神经酰胺酶突变体，以检测其对沙门氏菌在巨噬细胞中的存活和抑制NADPH氧化酶转运的影响。 2)检测沙门氏菌编码的葡糖神经酰胺酶对巨噬细胞脂代谢的影响。野生型和葡萄糖神经酰胺酶缺乏的沙门氏菌将被用来感染巨噬细胞，以评估鞘磷脂途径的全球变化。纯化的葡萄糖基神经酰胺酶将被用来测试脂质代谢活性，并将用免疫细胞化学来观察糖基神经酰胺酶对含NADPH氧化酶的囊泡运输的影响。 3)研究葡萄糖神经酰胺酶在阻止NADPH氧化酶活化转运中的作用。在这一部分，我将阐明干扰素-γ依赖于NADPH氧化酶刺激抗沙门氏菌活性的机制。此外，还将检测IFNGamma刺激葡萄糖神经酰胺合成的能力。这些研究不仅将加强我们对沙门氏菌发病机制的了解，还将为开发针对细胞内病原体的新治疗策略确定潜在的靶点。