SALMONELLA RESISTANCE TO CATIONIC PROTEINS IN PMN

沙门氏菌对 PMN 中阳离子蛋白的抗性

• 批准号: 3140383
• 负责人: John K Spitznagel
• 金额: $ 15.26万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3140383
• 关键词: Salmonella infections; Salmonella typhimurium; bacterial genetics; bacterial polysaccharides; bactericidal immunity; binding proteins; host organism interaction; human tissue; laboratory mouse; lipid structure; lipopolysaccharides; membrane activity; membrane lipids; membrane permeability; molecular cloning; mutant; neutrophil; phagocytosis; radiotracer; site directed mutagenesis; tissue /cell culture; virulence

Salmonella cause over 40,000 reported infections and 500 deaths annually at a cost of $50M. The most dangerous one escape the early neutrophil (PMN) reaction in the lamina propria and disseminate. Are some Salmonella typhimurium more resistant to oxygen-independent killing by PMN cationic antimicrobial proteins (CAP) and therefore more likely to disseminate? Recently, genetically determined changes in lipid A have been implicated in such resistance. Aim 1: To study the resistance of Salmonella to CAP with standard molecular genetics, to genetically and physically characterize the gene(s) that control it, and to identify the mechanisms; Aim 2: To study binding of CAP by the mutant Salmonella, the permeabilizing action of CAP, and the disorganizing action of CAP on LPS and the outer membrane; Aim 3: To examine LPS structure of mutant alleles; and Aim 4: to compare side by side in the same PMN monolayers the resistance of Salmonella mutants and the parent strain O2-independent antimicrobial action. Spontaneous and Tn10 insertional mutants resistant to both polymyxin B and CAP or resistant only to CAP will be used. Techniques: insertional mutagenesis, generalized transduction enzymes, agarose gel electrophoresis, and probing or Southern transfer with a Tn10 probe. Gene(s) will be cloned with established techniques or radioiodinated, biologically active cationic proteins will be studied. Studies on permeabilization of strains and LPS release from strains will be done with established methods. The structure of lipid A will be studied with fast-atom bombardment electron mass spectrometry, laser desorption, and nuclear magnetic resonance spectroscopy. (The mutations (in the wild-type background), together with wild-type Salmonella, will be compared side by side in the same monolayer of human PMN to test resistance to killing in anaerobic conditions.) We use drug resistance or metabolic markers to separate and count mutant and parent strains surviving side by side in PMN. Bacterial survival rates are calculated from inputs estimated from metabolically incorporated 3H-uracil and 35S- methionine.

沙门氏菌导致超过40，000例报告的感染和500例死亡 每年花费5000万美元。 最危险的一个逃脱 固有层中的早期中性粒细胞（PMN）反应， 传播。 一些鼠伤寒沙门氏菌是否对 PMN阳离子抗菌蛋白的非氧依赖性杀伤作用 (CAP)因此更容易传播 最近， 基因决定的脂质A的变化与 这样的抵抗。 目的1：研究沙门氏菌对大肠杆菌的耐药性， CAP具有标准分子遗传学，从基因和身体上 表征控制它的基因，并鉴定 目的2：研究突变体与CAP的结合作用 沙门氏菌，CAP的透化作用，以及 CAP对LPS和外膜的作用;目的3：检测LPS 突变等位基因的结构;以及目标4：并排比较 在相同的PMN单层中，沙门氏菌突变体的抗性和 亲株具有不依赖O2的抑菌作用。 自发 和对多粘菌素B和CAP均具有抗性的Tn 10插入突变体 或仅对CAP有抗性。 技术：插入 诱变，广义转导酶，琼脂糖凝胶 电泳和用Tn 10探针或Southern转移 探针 基因将使用已建立的技术克隆，或 放射性碘标记的生物活性阳离子蛋白质将被 研究了 菌株的透化作用和LPS释放的研究 将使用已建立的方法进行分离。 结构 将用快原子轰击电子质量 光谱法、激光解吸和核磁共振 谱 (The突变（在野生型背景中）， 与野生型沙门氏菌一起， 在相同的人PMN单层中，以测试对杀伤的抗性 在厌氧条件下）。 我们利用抗药性或代谢 分离和计数存活的突变株和亲本菌株的标记物 并排在PMN中。 细菌存活率计算如下： 从代谢结合的3 H-尿嘧啶和35 S- 蛋氨酸。