Mechanisms of Action of C. perfringens Enterotoxin

描述(由申请人提供)：产气荚膜梭菌肠毒素(CPE)导致A型产气荚膜梭菌食物中毒的胃肠道(Gl)症状，A型产气荚膜梭菌是美国第三大常见食源性疾病，以及某些非食源性人类Gl疾病。该项目的长期目标是通过重点研究CPE的独特作用机制来了解这些疾病的发病机制，从而确定预防或控制CPE相关的G1疾病的策略。为了实现这一目标，下一个资助期将追求以下目标：1)通过Western blotting从CPE复合体中电洗脱的蛋白质来进一步评估CPE与Claudin受体的相互作用，使用Claudin-1或-4转染体来区分CPE与来自相同或相反的Claudin纤维的Claudin相互作用，使用定点突变来识别结合CPE的特定Claudin区域/氨基酸，并通过免疫组织化学/sds-PAGE评价CPE是否在体内与claudin相互作用，2)通过对CPE复合体中蛋白质的质量分析来继续分析CPE作用中的结合后步骤，通过SDS-PAGE确定每个复合体中存在多少CPE蛋白，通过免疫荧光显微镜检测CPE内化到宿主细胞，并进行Western印迹/ELISA法评估体内和体外CPE作用的脂筏、激活的钙蛋白酶或炎症的贡献，3)利用定点突变/荧光光谱技术进一步研究CPE的结构功能关系，以鉴定参与受体结合和CPE插入膜的特定CPE氨基酸，并通过X射线衍射确定CPE蛋白的三维结构，以及4)通过PCR进一步分析CPE阳性的A型产气荚膜杆菌的分子发病机制，通过构建等基因的β2毒素突变体，研究CPE质粒的多样性，并评价β2毒素在CPE相关的非食源性G1病发病机制中的作用。

DESCRIPTION (provided by the applicant): Clostridium perfringens enterotoxin (CPE) is responsible for the gastrointestinal (Gl) symptoms of C. perfringens type A food poisoning, the 3rd most common foodborne illness in the USA, as well as certain nonfoodborne human Gl diseases. The long-term objective of this project is to identify strategies to prevent or control CPE-associated Gl disease by understanding the pathogenesis of these illness via a focus on CPE's unique mechanism of action. To progress towards this goal, the next grant period will pursue the following aims: 1) further evaluating CPE interactions with claudin receptors by Western blotting electroeluted proteins from CPE complexes, using claudin-1 or -4 transfectants to distinguish whether CPE interacts with claudins from the same or apposing claudin fibrils, using site-directed mutagenesis to identify specific claudin regions/amino acids that bind CPE, and evaluating by immunohistochemistry/SDS-PAGE whether CPE interacts with claudins in vivo, 2) continued analysis of post-binding steps in CPE action by mass spectroscopy analyses of proteins in CPE complexes, determining via SDS-PAGE how many CPE proteins are present in each complex, testing for CPE internalization into host cells by immunofluorescent microscopy, and conducting Western blot/ELISA evaluations of the contribution of lipid rafts, activated calpain, or inflammation to CPE action in vivo and in vitro, 3) additional study of the CPE structure function relationship using site-directed mutagenesis/fluorescent spectroscopy techniques to identify specific CPE amino acids involved in receptor binding and CPE insertion into membranes, and x-ray diffraction to determine the 3-D structure of the CPE protein, and 4) further analyzing the molecular pathogenesis of CPE-positive C. perfringens type A isolates by using PCR, microarrays and Southern blots to study the diversity of the cpe plasmid and by evaluating the contribution of beta2 toxin to the pathogenesis of CPE-associated nonfoodborne Gl disease by constructing isogenic beta2 toxin mutants.