MECAHNISMS OF ACTION OF C PERFRINGENS EXTEROTOXIN

产气荚膜梭菌肠毒素(CPE)已被确定为导致几种最常见的食源性和非食源性胃肠道(GI)疾病症状的致病因子。该项目的长期目标是全面了解CPE相关胃肠道疾病的发病机制，包括确定CPE的作用机制，并确定预防或治疗这些疾病的策略。为了实现这一目标，在下一个赠款期间将追求以下具体目标：1)通过Northern分析和“抗受体”抗体研究，评估Claudins作为CPE受体对人类肠道细胞的重要性；如果Claudins被确认为重要的CPE受体，将通过对一系列Claudin随机突变体结合CPE和传递细胞毒作用的表型来探索CPE的相互作用；2)通过免疫印迹和免疫沉淀分析鉴定含CPE的小和大复合体的真核蛋白质成分；然后将通过抗体抑制研究来剖析每个真核复合蛋白对CPE作用的重要性，3)对高分辨率的MAP CPE功能区进行定点突变，包括最近发现的受体结合区和毒素的大复合体形成区；用这些CPE突变体产生的结果将在CPE的三维结构的背景下被解释，以及4)通过CPE质粒在非食源性疾病分离株中的物理定位来剖析CPE阳性菌株的分子发病机制，确定CPE质粒是否可以转移到其他产气荚膜梭菌中，评估食物中毒菌株的染色体CPE是否是可动员的转座子，以及确定两个组分调节系统和/或指数生长期阻遏因子HPR是否在调节CPE的孢子形成相关表达中发挥作用。

Clostridium perfringens enterotoxin (CPE) has now been conclusively identified as the virulence factor responsible for symptoms associated with several of the most common foodborne and nonfoodborne gastrointestinal (GI) illnesses of bacterial origin. The long term objective of this project is to fully understand the pathogenesis of CPE-associated GI diseases, including identification of the mechanism of action of CPE, and to identify strategies to prevent or treat these illnesses. To progress towards this goal, the following specific aims will be pursued in the next grant period: 1) evaluating the importance of claudins as CPE receptors for human intestinal cells through Northern analyses and "anti-receptor" antibody studies; if claudins are confirmed as important CPE receptors, claudin: CPE interactions will be explored by phenotyping a series of claudin random mutants for their ability to bind CPE and convey cytotoxicity, 2) identifying the eucaryotic protein constituents of CPE-containing small and large complexes by immunoblot and immunoprecipitation analyses; the importance of each eucaryotic complex protein for CPE action will then be dissected through antibody inhibition studies, 3) using site-directed mutagenesis to high-resolution map CPE functional regions, including the recently identified receptor-binding and large complex-forming regions of the toxin; results generated with these CPE mutants will then be interpreted within the context of the 3-D structure of CPE, and 4) dissecting the molecular pathogenesis of cpe-positive isolates by physical mapping of the cpe plasmid in nonfoodborne disease isolates, determining whether the cpe plasmid can be transferred to other C. perfringens isolates, evaluating whether the chromosomal cpe of food poisoning isolates is dn a mobilizable transposon, and determining whether two component regulatory systems and/or the exponential growth phase repressor Hpr play a role in regulating the sporulation-associated expression of CPE.