Characterizing the targets of phagocytic superoxide in Salmonella

沙门氏菌吞噬超氧化物靶标的表征

• 批准号: 9083232
• 负责人: JAMES M. SLAUCH
• 金额: $ 37.52万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-15 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9083232
• 关键词: 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one; Address; Animals; Bacteria; Bacterial DNA; Biochemical; Biochemistry; Biological Assay; Blood Circulation; Cause of Death; Cells; Cessation of life; Chemicals; Co-Immunoprecipitations; Complex; Cytoplasm; Data; Devices; Disease; Gene Expression; Genes; Genetic; Genetic screening method; Goals; Immune response; Immune system; In Vitro; Infection; Laboratories; Lead; Leukocytes; Membrane Proteins; Methods; Methylphenazonium Methosulfate; Modeling; Mus; Mutagenesis; Mutation; Natural Immunity; Nitrogen; Organ; Oxidative Stress; Oxygen; Pathogenesis; Phagocytes; Phagosomes; Phenotype; Physiological; Physiology; Prevention; Process; Production; Publishing; Qualifying; Reactive Oxygen Species; Regulation; Research; Research Personnel; Resistance; Respiratory Burst; Role; Salmonella; Salmonella enterica; Salmonella infections; Salmonella typhimurium; Signal Transduction; Stress; Superoxide Dismutase; Superoxides; System; Techniques; Time; Variant; antimicrobial; bacterial genetics; base; cell injury; foodborne; foodborne pathogen; gene product; improved; in vitro Assay; in vivo; innovation; insight; killings; macrophage; mutant; novel; oxidative damage; pathogen; pathogenic bacteria; periplasm; prevent; public health relevance; response

 DESCRIPTION (provided by applicant): Macrophages produce reactive oxygen species (ROS) and other antimicrobial substances to kill bacteria. Salmonella Typhimurium is a major food-borne pathogen capable of surviving within macrophages. SodCI, the periplasmic superoxide dismutase, directly and specifically protects Salmonella against superoxide, the primary phagocytic ROS. However, the mechanism by which phagocytic superoxide damages bacteria is unknown. Dogma states that bacterial DNA is a primary target of oxidative damage in the macrophage, consistent with known effects of ROS produced endogenously in the bacterial cytoplasm. But we have shown that, contrary to dogma, the targets of the phagocytic oxidative burst are not the bacterial DNA or other cytoplasmic molecules. Rather, phagocytic superoxide damages an extracytoplasmic bacterial target. Our goal is to understand the physiological basis of bacterial sensitivity to phagocytic superoxide and the mechanisms by which pathogenic bacteria protect against this innate immune response. Novel genetic and biochemical approaches are used to identify and study the bacterial targets of phagocytic superoxide. A variation on a high-throughput transposon mutagenesis technique that we call "differential TnSeq" was used to identify genes that show synthetic, epistatic, or suppressive genetic interaction with sodCI. The ytfL gene was identified as being epistatic to sodCI. Genes with the same "phenotypic profile" as ytfL are significantly enriched in genes that also show genetic interactions with sodCI. These data lead us to hypothesize that YtfL and the products of these related genes comprise a complex or system that is sensitive to exogenous superoxide, and that this system is normally protected by SodCI. The identified genes will be further characterized using in vivo and in vitro assays. To facilitate these studies, we have developed a novel in vitro device that, for the first time, allows the production of superoxide in the laboratoy at levels that mimic those produced in the phagocyte. These innovative approaches, along with the co-investigators' combined expertise in bacterial genetics, pathogenesis, physiology, and biochemistry of oxidative stress, make us uniquely qualified to address this fundamental aspect of innate immunity. Completion of the specific aims will increase our ability to prevent and treat, not only Salmonella infection, but also diseases caused by other pathogens.

 描述（由申请人提供）：大环内酯产生活性氧（ROS）和其他抗菌物质来杀死细菌。鼠伤寒沙门氏菌是一种能够在巨噬细胞内存活的主要食源性病原体。SodCI，周质超氧化物歧化酶，直接和特异性地保护沙门氏菌免受超氧化物，主要的吞噬细胞ROS。然而，吞噬超氧化物破坏细菌的机制尚不清楚。Dogma指出，细菌DNA是巨噬细胞中氧化损伤的主要靶标，与细菌细胞质中内源性产生的ROS的已知作用一致。但我们已经表明，与教条相反，吞噬细胞氧化爆发的目标不是细菌DNA或其他细胞质分子。相反，吞噬超氧化物破坏细胞质外细菌靶标。我们的目标是了解细菌对吞噬性超氧化物的敏感性的生理基础，以及病原菌保护这种先天免疫反应的机制。新的遗传和生物化学方法被用于识别和研究吞噬超氧化物的细菌靶标。我们称之为“差异TnSeq”的高通量转座子诱变技术的变体用于鉴定显示与sodCI的合成、上位或抑制性遗传相互作用的基因。ytfL基因被鉴定为对sodCI具有上位性。与ytfL具有相同“表型谱”的基因显著富集在也显示与sodCI的遗传相互作用的基因中。这些数据使我们假设YtfL和这些相关基因的产物包含对外源性超氧化物敏感的复合物或系统，并且该系统通常受到SodCl的保护。将使用体内和体外测定进一步表征鉴定的基因。为了促进这些研究，我们开发了一种新的体外装置，首次允许在实验室中产生超氧化物，其水平模拟吞噬细胞中产生的超氧化物。这些创新的方法，沿着共同研究者在细菌遗传学、发病机理、生理学和氧化应激生物化学方面的综合专业知识，使我们有独特的资格来解决先天免疫的这个基本方面。具体目标的实现将提高我们预防和治疗的能力， 不仅是沙门氏菌感染，还有其他病原体引起的疾病。