Mechanisms of Action of C. Perfringens Enterotoxin

产气荚膜梭菌肠毒素（CPE）引起的胃肠道（GI）症状是第二常见的 细菌性食源性疾病在美国和5-15%的疟疾相关腹泻病例。预防/控制 CPE相关的胃肠道疾病，并进一步开发CPE作为抗癌剂，该项目旨在 了解CPE在GI疾病中的独特作用机制。为了实现这一目标，MERIT 扩展将。目的A，通过确定CPE与claudin受体的结构，进一步评估CPE与claudin受体的相互作用。 紧密连接蛋白-4和与紧密连接蛋白-4结合的CPE，绘制CPE结合所必需的紧密连接蛋白-4残基，评估是否紧密连接蛋白与CPE结合， 低CPE结合亲和力仍然可以传递CPE细胞毒性，并研究了封闭蛋白受体诱饵作为 针对CPE介导的GI疾病的治疗;目的B，继续分析CPE作用中的后结合步骤， CPE复合物的质谱分析，使用claudin- 表达转染子，进行扫描透射电子显微镜以评估复合物质量， 同源性，研究线粒体蛋白在CPE诱导的细胞死亡中的作用，评估CPE复合物 在体内形成，并确定CPE是否诱导肠道炎症反应，可能有助于GI 目的C，通过完成CPE的结构， 进行定点突变以确认CPE结构分析所提示的关键功能区域， 通过诱变评估推定的CPE跨膜结构域对CPE作用的贡献， 生物物理方法（荧光光谱和SCAM），并测试CPE氨基酸45-52 目的D进一步分析CPE阳性的分子发病机制 通过检测cpe质粒是否接合转移到正常植物群C中，产气荚膜杆菌菌株 小鼠肠道;比较（通过测序）A型分离株与C型和D型分离株的cpe基因座结构 分离物;通过构建SigF和SigG突变体评估CPE表达的分子调控;并测试 CPE阳性A型分离株产生的其他毒素是否通过构建 等基因cpe、pfoA、pic和/或cpb 2敲除突变体，并在兔回肠袢中测试它们的毒力。 相关性（见说明）： 本项目的目的是确定产气荚膜梭菌肠毒素（CPE）的作用， 导致美国第二大常见食源性疾病的症状， 腹泻相关性腹泻。这些见解将产生特定的疗法来预防这些疾病 并允许进一步开发CPE作为潜在的癌症治疗剂。

Clostridium perftingens enterotoxin (CPE) causes the gastrointestinal (GI) symptoms ofthe 2nd most common bacterial foodborne disease in the USA and 5-15% of antibiotic-associated diarrhea cases. To prevent/control CPE-associated GI disease, and further development ofCPE as an anti-cancer agent, this project seeks to understand CPE's unique mechanism of action during GI disease. To progress towards this goal, the MERIT extension will. Aim A, further evaluate CPE interactions with claudin receptors by determining the structure of claudin-4 and CPE bound to claudin-4, map claudin-4 residues essential for CPE binding, evaluate if claudins with low CPE binding affinity can still convey CPE cytotoxicity and investigate the use of claudin receptor decoys as therapeutics against CPE-mediated GI disease; Aim B, continue analysis of post-binding steps in CPE action by mass spectrometry analysis of CPE complexes, evaluate the process of CPE complex formation using claudin- expresing transfectants, perform scanning transmission electron microscopy to evaluate complex mass and homogeneity, investigate the role of mitochondrial proteins in CPE-induced cell death, evaluate CPE complex formation in vivo, and determine if CPE induces intestinal inflammatory responses that might contribute to GI disease; Aim C, conduct additional study of CPE structure/function analyses by finishing the structure of CPE, perform site-directed mutagenesis to confirm key functional regions suggested by analysis ofthe CPE structure, evaluate the contribution of a putative CPE membrane-spanning domain to CPE action by mutagenesis and biophysical approaches (fluorescence spectroscopy and SCAM) and test whether CPE amino acids 45-52 represent an oligomerization latch domain; Aim D, further analyze the molecular pathogenesis of CPE-positive type A isolates by testing if the cpe plasmid conjugatively transfers to normal flora C. perfringens strains in the mouse intestines; compare (by sequencing) the cpe locus organization in type A isolates vs. type C and D isolates; evaluate the molecular regulation of CPE expression by constructing SigF and SigG mutants; and test whether other toxins produced by CPE-positive type A isolates contribute to GI pathogenesis by constructing isogenic cpe, pfoA, pic and/or cpb2 knockout mutants and testing their virulence in rabbit ileal loops. RELEVANCE (See Instructions): The purpose of this project is to determine the action of Clostridium perfringens enterotoxin (CPE), which is responsible for the symptoms of the 2nd most common foodborne illness in the USA and many cases of antibiotic-associated diarrhea. These insights will generate specific therapeutics to prevent these illnesses and allow further development of CPE as a potential cancer therapeutic.