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，因为无法在单核吞噬细胞中存活的菌株在小鼠中严重减毒。吞噬细胞内沙门氏菌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 TNFα/TNFRp55 信号级联来重塑吞噬体，该级联指导将含有 NADPH 氧化酶的囊泡递送到沙门氏菌吞噬体附近。我计划测试以下假设：通过裂解葡萄糖神经酰胺，葡萄糖神经酰胺酶会干扰 TNFRp55 信号传导并阻止含有 NADPH 氧化酶的囊泡迁移到沙门氏菌吞噬体附近。具体目标是： 1) 确定葡萄糖神经酰胺酶在沙门氏菌感染发病机制中的作用。 将构建葡糖神经酰胺酶突变体来检查对巨噬细胞中沙门氏菌存活和抑制 NADPH 氧化酶运输的影响。 2) 研究沙门氏菌编码的葡萄糖神经酰胺酶对巨噬细胞脂质代谢的影响。野生型和葡萄糖神经酰胺酶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整体变化。将测试纯化的葡萄糖神经酰胺酶的脂质代谢活性，并使用免疫细胞化学来可视化糖神经酰胺酶对含有 NADPH 氧化酶的囊泡运输的影响。 3) 确定葡萄糖神经酰胺酶在避免 IFN（激活 NADPH 氧化酶运输）方面的作用。在本节中，我将阐明 IFN\gamma 依赖 NADPH 氧化酶刺激抗沙门氏菌活性的机制。IFNgamma 刺激葡萄糖神经酰胺的能力这些研究不仅将增强我们对沙门氏菌发病机制的了解，还将确定开发新药物的潜在靶标。针对细胞内病原体的治疗策略。