Structural mechanisms of Clostridium difficile pathogenesis

描述(申请人提供)：艰难梭菌是一种革兰氏阳性、芽胞形成的厌氧菌，可感染结肠，导致一系列人类疾病，包括腹泻、伪膜性结肠炎和毒性巨结肠。与艰难梭菌相关的疾病的发病率、严重性和成本都是巨大的，而且正在增加，这使得艰难梭菌成为一个重要的公共卫生问题。艰难梭菌致病的主要毒力因子是TcdA和TcdB，这是两种能够在真核宿主细胞内修饰多靶点的大型同源毒素。这些毒素包含四个功能结构域，分别与宿主受体结合、跨膜传递、自动处理和宿主Rho蛋白的酶失活有关。不同毒素异构体之间的序列差异和宿主细胞内不同的环境条件影响着与受体的相互作用、递送动力学、自动处理的简易性和细胞底物的特异性。建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。我们提出了一种结合冷冻电子显微镜(Cryo-EM)、X射线结晶学、生化和基于细胞的功能研究的混合方法。在目标1中，我们将利用冷冻EM来阐明TcdA全毒素的结构，并探索当毒素暴露于宿主环境线索(低pH、6-肌醇-6-磷酸和还原剂)时所经历的多个构象阶段。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和TcdB的葡萄糖转移酶特性。从机制上理解这两种毒素的保守和差异特征将为治疗抑制剂的设计提供必要的平台。

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.