Mechanisms of Action of C. Perfringens Enterotoxin

产气荚膜梭菌肠毒素(CPE)引起的胃肠道(GI)症状居第二位 在美国，细菌性食源性疾病和与抗生素相关的腹泻病例中有5%-15%。预防/控制 CPE相关的胃肠道疾病，以及CPE作为抗癌药物的进一步发展，该项目旨在 了解CPE在胃肠道疾病中的独特作用机制。要朝着这个目标前进，优点是 分机会的。目的A，通过测定CPE与Claudin受体的结构进一步评价CPE与Claudin受体的相互作用 Claudin-4和CPE与Claudin-4结合，MAP Claudin-4残基是CPE结合所必需的，评估Claudins与 低的CPE结合亲和力仍然可以传递CPE的细胞毒性，并研究Claudin受体诱骗作为 治疗CPE介导的胃肠道疾病；目的B，继续分析CPE结合后的步骤，通过 CPE络合物的质谱分析，评价CPE络合物的形成过程 表达转染体，进行扫描透射电子显微镜观察复合体质量和 同质性，研究线粒体蛋白在CPE诱导的细胞死亡中的作用，评估CPE复合体 在体内形成，并确定CPE是否诱导可能导致GI的肠道炎症反应 目的C，通过完成CPE的结构，对CPE的结构/功能分析进行进一步的研究， 进行定点突变以确认CPE结构分析所建议的关键功能区域， 评价一个假定的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 通过测试cpe质粒是否接合地转移到正常产气荚膜弧菌菌株来分离A型 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基因座组织 通过构建Sigf和Sigg突变体来评估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cpb2基因敲除突变体，并在兔回肠上测试其毒力。 相关性(请参阅说明)： 本项目的目的是确定产气荚膜梭菌肠毒素(CPE)的作用，该毒素是 对美国第二种最常见的食源性疾病的症状和许多病例负责 抗生素相关性腹泻。这些见解将产生预防这些疾病的特定疗法。 并允许将CPE进一步发展为潜在的癌症治疗方法。

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.