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。艰难梭菌感染(CDI)的发病率、严重性和成本正在增加，使艰难梭菌成为一个重大的公共卫生问题。艰难梭菌致病的主要毒力因子是TcdA和TcdB，这是两种能够进入和修饰真核宿主细胞内多个靶点的同源毒素。这项提议是为了跟进前一个资金周期中的两项重要发现，即TcdA的晶体结构和TcdB的细胞受体。在目标1中，我们将使用TcdA递送结构域的结构作为了解宿主内膜内毒素孔形成的框架。电子显微镜、交联剂和荧光强度测量的混合方法将产生毒素孔的结构图。在目标2中，我们将使用电子显微镜、诱变、定量结合研究和X射线结晶学相结合的方法来定义TcDB与其受体PVRL3的相互作用。在目标3中，我们将评估TcDB-PVRL3相互作用在小鼠中毒和感染模型中的相关性。这些目标的结果将提供对允许毒素进入宿主细胞的事件的分子理解，并为治疗干预提供框架。

 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 infection (CDI) are 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 entering and modifying multiple targets within eukaryotic host cells. This proposal is designed to follow up two important discoveries made in the previous funding cycle, a crystal structure of TcdA and the cellular receptor for TcdB. In Aim 1, we will use the structure of the TcdA delivery domain as a framework for understanding toxin pore formation within the host endosomal membrane. A hybrid approach of electron microscopy, crosslinking, and fluorescence intensity measurements will generate a structural view of the toxin pore. In Aim 2, we will define the interaction of TcdB with its receptor, PVRL3, using a hybrid of electron microscopy, mutagenesis, quantitative binding studies, and X-ray crystallography. In Aim 3, we will evaluate the relevance of the TcdB-PVRL3 interaction in mouse models of intoxication and infection. Results from these aims will provide a molecular understanding of the events that allow toxins access to the host cell and a framework for therapeutic intervention.