SALMONELLA RESISTANCE TO CATIONIC PROTEINS IN PMN

沙门氏菌导致40,000多例报告感染和500人死亡 每年耗资5000万美元。最危险的逃脱 早期中性粒细胞(PMN)反应在固有层和 传播。一些鼠伤寒沙门氏菌对 PMN阳离子抗菌蛋白的氧非依赖性杀灭作用 (上限)，因此更有可能传播？最近， 基因决定的类脂A的变化与 如此抗拒。目的1：了解沙门氏菌对沙门氏菌的耐药性。 带有标准分子遗传学的帽子，从基因和身体上 确定控制它的基因(S)的特征，并鉴定 机制；目的2：研究突变体与CAP的结合 沙门氏菌、CAP的通透作用和解体作用 CAP对内毒素及其外膜的作用；目的3：检测内毒素 突变等位基因的结构；和目标4：并排比较 在相同的PMN单层中，沙门氏菌突变株和 亲本菌株O2不依赖抑菌作用。自发 和Tn10插入突变体对多粘菌素B和CAP都具有抗性 或者只对CAP耐药。技巧：插入式 诱变、通用转导酶、琼脂糖凝胶 Tn10的电泳法、探针法或Southern转移法 探测器。基因(S)将用现有的技术或 放射性碘标记的生物活性阳离子蛋白将是 学习。细菌通透性与内毒素释放的研究 从菌株中分离将用既定的方法进行。该结构 将用快原子轰击电子质量研究脂类A的 光谱分析、激光解吸和核磁共振 光谱学。(突变(在野生型背景中)， 与野生型沙门氏菌一起，将被并列比较 在同一单层人中性粒细胞中测试抗杀性 在厌氧条件下。)我们使用耐药性或新陈代谢 分离和计数存活的突变菌株和亲本菌株的标记 在PMN中并肩而行。细菌存活率的计算公式为 从代谢结合的~3H-尿嘧啶和~(35)S估计的输入- 蛋氨酸。

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.