Structural Mechanisms of Clostridioides difficile pathogenesis

总结 艰难梭菌是一种革兰氏阳性、孢子形成的厌氧菌，感染结肠，引起一系列的结肠炎。 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 疾病控制报告称，2017年，估计有223，900例C。艰难梭菌感染（CDI） 美国的住院患者估计有12，800人死亡。社区的发生率- 获得性CDI也很常见，使C。这是一个重大的公共卫生问题。细菌会产生毒素 TcdB，这是大多数CDI症状的原因。拟议项目的目标是确定 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 目的1，我们将确定结构和关键残基参与TcdB受体相互作用使用的组合 结构生物学、诱变和定量结合方法。我们将使用这些信息来生成 C.在TcdB受体结合位点中具有确定突变的艰难梭菌菌株。在目标2中，我们将评估细胞 在人外植体中毒模型的背景下，利用显著的 高分辨率光学显微镜成像的技术进步。这些研究将与 在TcdB中毒的新细胞模型以及CDI的小鼠模型中的机制研究。总的来说， 这些研究将确定与确定的TcdB-受体相互作用相关的生理后果， 有望为推进新型治疗和CDI预防策略提供机制框架。

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.