MECAHNISMS OF ACTION OF C PERFRINGENS EXTEROTOXIN

产气荚膜梭菌肠毒素（CPE）现已被最终确定为与几种最常见的食源性和非食源性胃肠道（GI）细菌源性疾病相关症状的毒力因子。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，下一个资助期将实现以下具体目标：1)通过Northern分析和“抗受体”抗体研究评估claudin作为人类肠道细胞CPE受体的重要性；如果证实claudin是重要的CPE受体，claudin: CPE相互作用将通过对一系列claudin随机突变体进行表型分析，以了解其结合CPE和传递细胞毒性的能力。2)通过免疫印迹和免疫沉淀分析鉴定含有CPE的大小复合物的真核蛋白成分；每个真核生物复合物蛋白对CPE作用的重要性将通过抗体抑制研究进行剖析，3)使用定点诱变技术来高分辨率绘制CPE功能区域，包括最近发现的受体结合区和毒素的大型复合物形成区；4)通过对非食源性疾病分离株的CPE质粒进行物理定位，剖析CPE阳性分离株的分子发病机制，确定CPE质粒是否可以转移到其他产气荚膜荚膜杆菌分离株中，评估食物中毒分离株的染色体CPE是否不是可移动的转座子。并确定双组分调控系统和/或指数生长阶段抑制因子Hpr是否在调控CPE的产孢相关表达中起作用。

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.