Genetic Mechanisms of Sporulation Induction in C. difficile

项目摘要/摘要 艰难梭状芽胞杆菌(前称梭状芽孢杆菌)是引起严重腹泻的主要医院病原菌 一种传染性很强且难以治疗的疾病。艰难梭菌的形成和传播很容易 从受感染的宿主中排出具有传染性的孢子。艰难梭菌的孢子形态对大多数消毒剂都有抵抗力。 对细菌在宿主肠道外的生存至关重要。胃肠道是唯一 已知的自然环境支持艰难梭菌孢子的形成，但我们对孢子是如何形成的知之甚少 形成是由细菌启动的。这个项目的长期目标是发现分子 控制艰难梭菌产孢量启动的机制。根据我们的数据，我们假设有几个 早期产孢蛋白在独立的途径中发挥作用，通过控制 激活主转录调控因子Spo0A。本应用程序的具体目标是定义 作用于Spo0A并描绘控制Spo0A的分子途径的直接调控机制 产孢子的启动。利用我们以前在艰难梭菌分子遗传学、基因调控、 和革兰氏阳性肠道的发病机制，我们将通过两个详细的实验达到目标 明确的目标。在目标1中，我们将扩展我们当前的研究，以发现Spo0A的直接交互合作伙伴 一种遗传和生化方法的结合。同时，目标2扩展了上位性分析以构建 Spo0A调控通路，然后对起始级联中的蛋白质进行功能分析。这 研究具有创新性，因为它结合了生物分子和遗传方法来解决根本问题 关于这一重要而复杂的生物过程的问题。这些目标的完成预计将暴露出 孢子萌发过程中的潜在脆弱性。这项工作是制定理性战略的重要一步 通过防止宿主内形成有感染性的孢子来阻止艰难梭菌的传播。

Project Summary/Abstract Clostridioides difficile (formerly Clostridium) is a major nosocomial pathogen that causes severe diarrheal disease that is highly infectious and difficult to treat. C. difficile is easily transmitted due to the formation and expulsion of contagious spores from infected hosts. The spore form of C. difficile is resistant to most disinfectants and is critical for the survival of the bacterium outside of the host intestine. The gastrointestinal tract is the only natural environment known to support C. difficile spore formation, but we understand little about how spore formation is initiated by the bacterium. The long-term goal of this project is to uncover the molecular mechanisms that control the initiation of C. difficile sporulation. Based on our data, we hypothesize that several early sporulation proteins function in independent pathways to regulate the initiation of sporulation by controlling activation of the master transcriptional regulator, Spo0A. The specific objectives of this application are to define the direct regulatory mechanisms that act upon Spo0A and to delineate the molecular pathways that control sporulation initiation. Capitalizing on our previous experiences in C. difficile molecular genetics, gene regulation, and Gram-positive intestinal pathogenesis, we will meet the objectives through the experiments detailed in two specific aims. In Aim 1, we will extend our current studies to uncover direct interacting partners of Spo0A using a combination of genetic and biochemical approaches. In parallel, Aim 2 expands epistatic analyses to construct Spo0A regulatory pathways, followed by functional analyses of the proteins within the initiation cascade. This research is innovative because it combines biomolecular and genetic approaches to resolve a fundamental question about this important and complex biological process. Completion of these aims is expected to expose potential vulnerabilities in spore initiation. This work is an essential step in the development of rational strategies to impede C. difficile transmission by preventing the formation of infectious spores in the host.