Evasion of NADPH oxidase by Salmonella lipases

沙门氏菌脂肪酶逃避 NADPH 氧化酶

• 批准号: 6721064
• 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/6721064
• 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生存的能力对于其致病性至关重要，因为单核吞噬细胞中无法生存的菌株在小鼠中严重减弱。沙门氏菌在吞噬细胞中的存活与其停止沿着内吞途径的吞噬体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tnfalpha/tnfrp55信号级联反重塑吞噬体，该信号级联指导将含量氧化酶的囊泡递送至鲑鱼吞噬孢子的囊泡。我计划检验以下假设：葡萄糖基酰胺酶通过切割葡萄糖基酰胺，会干扰TNFRP55信号传导，并阻止NADPH氧化酶 - 磨牙囊泡迁移到沙门氏菌吞噬体的占地。具体目的是： 1）确定葡萄糖基酰胺酶在沙门氏菌感染的发病机理中的作用。 将构建葡萄糖基酰胺酶突变体，以检查巨噬细胞中沙门氏菌存活和抑制NADPH氧化酶运输的影响。 2）检查沙门氏菌编码的葡萄糖基酰胺酶对巨噬细胞脂质代谢的影响。野生型和缺陷型沙门氏菌将用于感染巨噬细胞以评估鞘磷脂途径的全球变化。将测试纯化的葡萄糖基酰胺酶的脂质代谢活性，并将使用免疫细胞化学来可视化糖基质酶对含NADPH氧化酶含量囊泡的运输的影响。 3）确定葡萄糖基酰胺酶在避免IFN（激活的NADPH氧化酶的运输）中的作用。在本节中，我将阐明IFN \ Gamma刺激抗盐对NADPH氧化酶刺激抗盐酸氧化酶的机制。仅IFNGamma刺激葡萄糖层的疾病疗法的能力促进了我们的良好的研究，我们的研究均应促进我们的研究。发病机理，但还将确定针对细胞内病原体开发新的治疗策略的潜在靶标。