Structural mechanisms of Clostridium difficile pathogenesis

描述（由申请方提供）：艰难梭菌是一种革兰氏阳性、孢子形成厌氧菌，可感染结肠，引起一系列人类疾病，包括腹泻、伪膜性结肠炎和毒性巨结肠。与C.艰难梭菌相关疾病是实质性的和增加，使C。这是一个重大的公共卫生问题。C.艰难的发病机制是TcdA和TcdB，这两种大型同源毒素能够修饰真核宿主细胞内的多个靶点。毒素含有与宿主受体结合、跨膜递送、自动加工和宿主Rho蛋白的酶失活相关的四个功能结构域。与受体的相互作用、递送动力学、自动加工的容易性和对细胞底物的特异性受到宿主细胞内不同毒素同种型之间的序列变异和不同环境条件的影响。拟议的研究计划的中心目标是阐明毒素功能的结构和分子机制。我们提出了一种混合的方法，结合冷冻电子显微镜（cryo-EM），X射线晶体学，生物化学和基于细胞的功能研究。在目的1中，我们将阐明的TcdA全毒素的结构，使用冷冻EM和探针的多个构象阶段，访问的毒素暴露于宿主环境的线索（低pH值，肌醇-6-磷酸和还原剂）。在目标2中，我们将确定TcdA递送结构域的X射线晶体结构，并确定与膜孔形成相关的序列。在目标3中，我们将比较TcdA与TcdB的葡糖基转移酶性质。对这两种毒素的保守和不同特征的机制理解将为治疗性抑制剂设计提供必要的平台。 公共卫生相关性：艰难梭菌是一种产毒素细菌，是医院获得性腹泻和腹泻相关性腹泻的常见原因。与C.艰难梭菌相关疾病是实质性的和增加，使C。这是一个重大的公共卫生问题。拟议项目的目标是确定两种主要毒素TcdA和TcdB破坏宿主细胞功能的结构和分子机制。

DESCRIPTION (provided by applicant): Clostridium difficile is a gram-positive, spore-forming anaerobe that infects the colon, causing a range of human disease including diarrhea, pseudomembranous colitis, and toxic megacolon. The incidence, severity, and costs associated with C. difficile associated disease are substantial and increasing, making C. difficile a significant public health concern. The principle virulence factors in C. difficile pathogenesis are TcdA and TcdB, two large homologous toxins capable of modifying multiple targets within eukaryotic host cells. The toxins contain four functional domains that are associated with host-receptor binding, delivery across a membrane, autoprocessing, and the enzymatic inactivation of host Rho-proteins. The interaction with receptors, the kinetics of delivery, the ease of autoprocessing, and the specificity for cellular substrates are affected by sequence variation among different toxin isoforms and distinct environmental conditions within the host cell. The central objective of the proposed research program is to elucidate structural and molecular mechanisms of toxin function. We propose a hybrid approach that combines cryo-electron microscopy (cryo-EM), X-ray crystallography, biochemical and cell-based functional studies. In Aim 1, we will elucidate a structure of the TcdA holotoxin using cryo-EM and probe the multiple conformational stages that are accessed as the toxin is exposed to host environmental cues (low pH, inostol-6-phosphate and reductant). In Aim 2, we will determine the X-ray crystal structure of the TcdA delivery domain and identify the sequences that are associated with membrane pore formation. In Aim 3, we will compare the glucosyltransferase properties of TcdA to those of TcdB. A mechanistic understanding of the conserved and divergent features of these two toxins will provide a necessary platform for therapeutic inhibitor design. PUBLIC HEALTH RELEVANCE: Clostridium difficile is a toxin-producing bacterium that is a frequent cause of hospital-acquired and antibiotic-associated diarrhea. The incidence, severity, and costs associated with C. difficile associated disease are substantial and increasing, making C. difficile a significant public health concern. The goal of the proposed project is to identify the structural and molecular mechanisms by which the two primary toxins, TcdA and TcdB, disrupt host cell function.