Structural mechanisms of Clostridium difficile pathogenesis

描述（由申请人提供）：艰难梭菌是一种革兰氏阳性、孢子形成的厌氧菌，可感染结肠，引起一系列人类疾病，包括腹泻、假膜性结肠炎和中毒性巨结肠。难辨梭菌相关疾病的发病率、严重程度和费用都很高，而且还在增加，使难辨梭菌成为一个重大的公共卫生问题。艰难梭菌致病机制的主要毒力因子是TcdA和TcdB，这两种能够修饰真核宿主细胞内多个靶点的大同源毒素。该毒素包含四个功能域，它们与宿主受体结合、跨膜递送、自动加工和宿主rho蛋白的酶失活有关。与受体的相互作用、传递动力学、自动处理的便利性以及对细胞底物的特异性受到宿主细胞内不同毒素同工型和不同环境条件之间序列变化的影响。提出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射线晶体学，生化和细胞功能研究的混合方法。在目标1中，我们将使用冷冻电镜阐明TcdA全毒素的结构，并探测毒素暴露于宿主环境线索（低pH值、醇缩醇-6-磷酸和还原剂）时进入的多个构象阶段。在Aim 2中，我们将确定TcdA传递域的x射线晶体结构，并确定与膜孔形成相关的序列。在Aim 3中，我们将比较TcdA和TcdB的葡萄糖基转移酶特性。对这两种毒素的保守性和差异性特征的机制理解将为治疗性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.