Structural Mechanisms of Clostridioides difficile pathogenesis

摘要 艰难梭状芽胞杆菌是一种革兰氏阳性、芽胞形成的厌氧菌，它感染结肠，导致一系列 人类疾病，包括腹泻、伪膜性结肠炎和中毒性巨结肠。美国中心 疾病控制中心报告，2017年，估计有223,900例艰难梭菌感染(CDI)。 在美国住院的患者估计有12,800人死亡。社区的发病率- 获得性CDI也很常见，使艰难梭菌成为一个重大的公共卫生问题。细菌产生一种毒素， Tcdb，它是大多数CDI症状的原因。拟议项目的目标是定义 TcdB与宿主细胞表面受体蛋白结合的结构基础和生理后果。在……里面 目标1，我们将使用组合定义TcdB-受体相互作用中的结构和关键残基 结构生物学、诱变和数量结合方法。我们将使用此信息来生成 TcdB受体结合位点有明确突变的艰难梭菌。在目标2中，我们将评估细胞 在人类外植体中毒模型的背景下毒素的趋向性，利用显著的 高分辨率光学显微镜成像技术进展。这些研究将与 一种新的TcdB中毒细胞模型和CDI小鼠模型的机制研究。总而言之， 这些研究将确定与已定义的Tcdb-受体相互作用相关的生理后果，并 预计将为推进新的治疗和CDI预防战略提供一个机械框架。

Summary Clostridioides 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 United States Centers for Disease Control reports that, in 2017, there were 223,900 estimated cases of C. difficile infection (CDI) in hospitalized patients in the United States with an estimated 12,800 deaths. The incidence of community- acquired CDI is also common making C. difficile a significant public health concern. The bacterium makes a toxin, TcdB, which is responsible for the majority of CDI symptoms. The goal of the proposed project is to define the structural basis and physiologic consequences of TcdB binding to receptor proteins on the host cell surface. In Aim 1, we will define the structures and key residues involved in TcdB-receptor interactions using a combination of structural biology, mutagenesis, and quantitative binding approaches. We will use this information to generate C. difficile strains with defined mutations in TcdB receptor binding sites. In Aim 2, we will evaluate the cellular tropism of the toxin in the context of a human explant intoxication model, taking advantage of significant technological advances in high resolution light microscopy imaging. These studies will be paired with mechanistic studies in a novel cellular model of TcdB intoxication as well as the mouse model of CDI. Collectively, these studies will define the physiologic consequences associated with defined TcdB-receptor interactions and are expected to provide a mechanistic framework for advancing novel therapeutic and CDI prevention strategies.