Spore Assembly in Clostridium Difficile

 描述(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抗性孢子形态知之甚少。虽然阻止孢子聚集将打破艰难梭菌感染和再感染的破坏性循环，但目前还没有针对这一发育过程的治疗方法。开发这种疗法的一个主要障碍是缺乏对艰难梭菌如何形成孢子的基本了解。我们的长期目标是了解艰难梭菌如何组装保护性的蛋白质孢子皮，这是功能性孢子形成所必需的。我们最近在梭状芽胞杆菌中发现了第一个孢子形态发生蛋白SpoIVA和SipL，并表明它们直接相互作用。在这一应用中，我们旨在阐明SpoIVA和SipL协同调节孢子形成的分子机制。我们将利用互补的遗传、生化和细胞学方法来确定SpoIVA-SipL复合体在孢子组装过程中形成的功能意义，寻找新的调控孢子组装的SpoIVA和/或SipL相互作用伙伴，并在孢子形成过程中定位SpoIVA和SipL。通过研究SpoIVA和SipL在孢子形成过程中相互作用并将外壳蛋白招募到前孔的机制，本研究将阐明孢子形成的关键调节因子的功能，鉴定梭状芽胞杆菌中新的形态发生蛋白，并开发新的成像方法来定位厌氧条件下活细胞中的蛋白质。由于SipL仅在梭状芽胞杆菌中发现，这些研究将扩大我们对芽胞组装如何在梭状芽胞杆菌中调节的有限理解。拟议工作的完成将提供合理设计筛选所需的机械洞察力，这些筛选可以识别艰难梭菌孢子形成抑制剂，还可能识别新的潜在治疗靶点。因此，这些研究可能会导致新的治疗方法，可以防止艰难梭菌和其他形成梭状芽胞的病原体传播疾病。

 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.