Spore Assembly in Clostridium Difficile

 描述（由申请人提供）：孢子是重要的医院病原体艰难梭菌传播疾病的主要机制，但对这种专性厌氧菌如何形成耐药孢子形式知之甚少。而阻止孢子组装将打破C.艰难的感染，目前没有治疗针对这一发展过程。发展这种疗法的一个主要障碍是缺乏对C。艰难梭菌形成孢子。我们的长期目标是了解C。艰难梭菌组装保护性蛋白质孢子外壳，这是功能性孢子形成所必需的。我们最近确定了第一个孢子形态发生蛋白在梭菌SpoIVA和SipL，并表明它们直接相互作用。在本申请中，我们的目的是阐明SpoIVA和SipL协调调节孢子形成的分子机制。我们将使用互补的遗传学，生物化学和细胞学方法来确定孢子组装过程中SpoIVA-SipL复合物形成的功能意义，确定新的SpoIVA和/或SipL相互作用的合作伙伴，调节孢子组装，并定位SpoIVA和SipL孢子形成过程中。通过询问SpoIVA和SipL相互作用和招募外壳蛋白到孢子形成过程中的前孢子的机制，拟议的研究将阐明孢子形成的关键调控因子的功能，确定梭菌中新的形态发生蛋白，并开发新的成像方法用于在厌氧条件下定位活细胞中的蛋白质。由于SipL仅在梭菌中发现，这些研究将扩大我们对梭菌中孢子组装如何调节的有限理解。建议工作的完成将为合理设计能够识别C的屏幕提供必要的机械见解。艰难梭菌孢子形成抑制剂，也可能确定新的潜在治疗靶点。因此，这些研究可能会导致新的治疗方法，可以防止疾病传播的C。艰难梭菌和其它梭菌孢子形成病原体。

 DESCRIPTION (provided by applicant): Spores are major mechanism by which the important nosocomial pathogen Clostridium difficile transmits disease, yet little is known about how this obligate anaerobe forms the resistant spore form. While preventing spore assembly would break the destructive cycle of infection and re-infection that characterizes C. difficile infections, no therapies currently target this developmental process. A major obstacle to developing such therapies is the absence of a basic understanding of how C. difficile forms a spore. Our long-term goal is to understand how C. difficile assembles the protective proteinaceous spore coat, which is necessary for functional spore formation. We recently identified the first spore morphogenetic proteins in the Clostridia as SpoIVA and SipL and showed that they directly interact. In this application, we aim to elucidate the molecular mechanisms by which SpoIVA and SipL coordinately regulate spore formation. We will use complementary genetic, biochemical, and cytological methods to determine the functional significance of SpoIVA-SipL complex formation during spore assembly, identify new SpoIVA and/or SipL interacting partners that regulate spore assembly, and localize SpoIVA and SipL during spore formation. By interrogating the mechanism by which SpoIVA and SipL interact and recruit coat proteins to the forespore during sporulation, the proposed studies will elucidate the functions of key regulators of spore formation, identify new morphogenetic proteins in the Clostridia, and develop new imaging methods for localizing proteins in live cells under anaerobic conditions. Since SipL is found exclusively in the Clostridia, these studies will expand our limited understanding of how spore assembly is regulated in the Clostridia. Completion of the proposed work will provide the mechanistic insight necessary for rationally designing screens that can identify C. difficile sporulation inhibitors and may also identify new potential therapeutic targets. As a result, these studies may lead to novel therapeutics that can prevent disease transmission by C. difficile and other clostridial spore-forming pathogens.