Characterizing the targets of phagocytic superoxide in Salmonella

沙门氏菌吞噬超氧化物靶标的表征

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：巨噬细胞产生活性氧（ROS）和其他抗菌物质以杀死细菌。鼠伤寒沙门氏菌是一种能够在巨噬细胞中生存的主要食物病原体。 Sodci是周质超氧化物歧化酶，直接和特异性地保护沙门氏菌免受主要吞噬性ROS的超氧化物。但是，吞噬超氧化物损害细菌的机制尚不清楚。教条指出，细菌DNA是巨噬细胞中氧化损伤的主要靶标，与细菌细胞质内源产生的ROS的已知作用一致。但是我们已经表明，与教条形成鲜明对比的是，吞噬氧化物爆发的靶标不是细菌DNA或其他细胞质分子。相反，吞噬性超氧化物会损害外质细菌靶标。我们的目标是了解细菌对吞噬超氧化物的生理基础，以及致病细菌保护这种先天免疫响应的机制。新型的遗传和生化方法用于识别和研究吞噬超氧化吞噬的药理学靶标。我们称为“差异TNSEQ”的高通量转座子诱变技术的变化用于识别显示出合成，认识或抑制性遗传与SODCI的基因。 YTFL基因被确定为对Sodci的认识。具有与YTFL相同的“表型谱”的基因显着富集在与SODCI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括对外源性超氧化物敏感的复杂或系统，并且该系统通常受SODCI的保护。使用体内和体外测定法将进一步表征所鉴定的基因。为了促进这些研究，我们开发了一种新型的体外装置，这首先允许在实验室中以模仿吞噬细胞产生的水平生产超氧化物。这些创新的方法，以及共同投资者在细菌遗传学，发病机理，生理学和氧化应激的生物化学方面的综合专业知识，使我们具有独特的资格来解决这种先天免疫的基本方面。完成特定目标的完成将增加我们预防和治疗的能力不仅沙门氏菌感染，还由其他病原体引起的疾病。