Salmonella evasion of NADPH oxidase-dependent killing

沙门氏菌逃避 NADPH 氧化酶依赖性杀灭

• 批准号: 6805158
• 负责人: Andres Vazquez-Torres
• 金额: $ 34万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6805158
• 关键词: NAD(P)H dehydrogenase; SDS polyacrylamide gel electrophoresis; Salmonella; Salmonella infections; bacterial genetics; bacterial proteins; chromatography; drug resistance; enteric bacteria; free radical oxygen; gangliosides; gene expression; genetically modified animals; intracellular; laboratory mouse; phagocytes; phagocytosis; polymerase chain reaction; sphingomyelins; vesicle /vacuole; western blottings

DESCRIPTION (provided by applicant): The appearance of multidrug-resistant Salmonella isolates and the HIV epidemic have contributed to the resurgence of salmonellosis, an infection that annually afflicts more than 1 billion people worldwide. Multiple clinical and experimental lines of evidence point to the NADPH oxidase as a critical host defense mechanism in resistance to acute Salmonella infections. Salmonella, an enteric pathogen adapted to the intracellular environment of phagocytes, resides in remodeled phagosomes that selectively block contact with lysosomes and endocytic vesicles harboring the NADPH oxidase. A recently discovered locus at centisome 30 of the Salmonella chromosome encodes a type III secretory system known as Salmonella pathogenicity island 2 (SPI2) that disrupts maturation of the Salmonella phagosome. The primary goal of my laboratory is to understand the mechanisms by which this intracellular pathogen remodels its phagosome and evades the antimicrobial armamentarium of professional phagocytes. In the present proposal, we plan to test the hypothesis that SPI2 effectors decrease TNFRp55-stimulated ganglioside synthesis, thus blocking the migration of NADPH oxidase-harboring vesicles to the vicinity of the Salmonella phagosome. We specifically plan: 1) To identify SPI2 effector proteins that block trafficking of the NADPH oxidase. Attenuation of SPI2 mutants in macrophages and mice, coupled to techniques in molecular and cell biology, biochemistry and microscopy will be used to identify effector proteins that block NADPH oxidase trafficking. 2) To identify points in the TNFRp55-stimulated sphingomyelin pathway which are inhibited by SPI2 effector proteins. Lipid biochemistry, enzymology and bacterial genetics will be used to identify points in the sphingomyelin pathway inhibited by SPI2 effectors. And 3) To determine the kinetics of secretion and intracellular location of SPI2 effectors that inhibit the trafficking of the NADPH oxidase. Cell biology, immunology and microbial genetics will be used to study the early intracellular expression of SPI2 effectors and their distribution relative to the NADPH oxidase, TNFRp55 and the Salmonella phagosome. These studies will not only shed light on the cell biology of the NADPH oxidase but will also identify potential molecular targets common to intracellular pathogens such as Salmonella, Mycobacterium, and Legionella that are capable of thwarting the normal maturation of the nascent phagosome.

描述（由申请人提供）：多重耐药沙门氏菌分离株的出现和艾滋病毒的流行导致了沙门氏菌病的死灰复燃，这种感染每年困扰着全世界超过 10 亿人。多项临床和实验证据表明 NADPH 氧化酶是抵抗急性沙门氏菌感染的关键宿主防御机制。沙门氏菌是一种适应吞噬细胞内环境的肠道病原体，存在于重塑的吞噬体中，选择性地阻止与含有 NADPH 氧化酶的溶酶体和内吞囊泡的接触。最近在沙门氏菌染色体第 30 号染色体上发现的一个基因座编码了一种称为沙门氏菌致病性岛 2 (SPI2) 的 III 型分泌系统，该系统会破坏沙门氏菌吞噬体的成熟。我实验室的主要目标是了解这种细胞内病原体重塑其吞噬体并逃避专业吞噬细胞的抗菌武器的机制。在本提案中，我们计划测试以下假设：SPI2 效应器减少 TNFRp55 刺激的神经节苷脂合成，从而阻止含有 NADPH 氧化酶的囊泡迁移到沙门氏菌吞噬体附近。我们具体计划： 1) 鉴定阻止 NADPH 氧化酶运输的 SPI2 效应蛋白。巨噬细胞和小鼠中 SPI2 突变体的减弱，与分子和细胞生物学、生物化学和显微镜技术相结合，将用于鉴定阻止 NADPH 氧化酶运输的效应蛋白。 2) 确定 TNFRp55 刺激的鞘磷脂途径中被 SPI2 效应蛋白抑制的点。脂质生物化学、酶学和细菌遗传学将用于识别鞘磷脂途径中受 SPI2 效应子抑制的点。 3) 确定抑制 NADPH 氧化酶运输的 SPI2 效应子的分泌动力学和细胞内位置。细胞生物学、免疫学和微生物遗传学将用于研究 SPI2 效应子的早期细胞内表达及其相对于 NADPH 氧化酶、TNFRp55 和沙门氏菌吞噬体的分布。这些研究不仅将揭示 NADPH 氧化酶的细胞生物学，还将确定细胞内病原体（如沙门氏菌、分枝杆菌和军团菌）常见的潜在分子靶点，这些病原体能够阻碍新生吞噬体的正常成熟。