Proteolytic Regulation of Spore Germination in Clostridium difficile

描述（由申请人提供）：孢子萌发对于主要医院病原体艰难梭菌启动和传播感染至关重要，但对调节这一复杂发育过程的分子机制知之甚少。由于知识上的差距阻碍了预防C。我们的长期目标是阐明C.艰难孢子萌发成营养细胞。萌发过程中的一个关键步骤是酶促去除孢子皮层，这是一层肽聚糖保护层，可使孢子保持休眠状态。在梭菌中，皮质降解依赖于SleC皮质水解酶被Csp家族蛋白酶蛋白水解激活。虽然只有单一的Csp蛋白酶足以诱导产气荚膜梭菌中的皮质水解，但我们已经表明，在C.艰难梭菌需要CspC和CspBA融合蛋白酶两者以在萌发剂添加时激活SleC。有趣的是，CspC和CspBA的CspA结构域都是假蛋白酶，我们和其他人已经证明它们调节皮质水解;事实上，CspC最近被鉴定为一种新的萌发受体。这些发现提出了一些重要的问题：假蛋白酶如何调节CspB蛋白酶的活性？调节蛋白水解如何激活SleC？我们的目标是在这个建议是确定CspC，CspBA和SleC协调控制皮质水解的分子机制。使用遗传、生物化学和结构方法，我们将鉴定CspB激活所需的CspC和CspA假蛋白酶内的区域。靶向诱变和晶体学研究的SLEC将被用来阐明调节蛋白水解激活SLEC的分子基础。最后，CspC，CspBA和SleC之间的相互作用将使用互补细菌双杂交，免疫沉淀和亲和纯化方法进行鉴定。总的来说，拟议的研究将增加我们对假酶如何控制酶活性以及C。艰难梭菌孢子对胆盐萌发物有感知和反应。这些研究将为开发降低C.艰难梭菌病的传播和复发

DESCRIPTION (provided by applicant): Spore germination is essential for the major nosocomial pathogen Clostridium difficile to initiate and transmit infection, yet little is known about the molecular mechanisms regulating this complex developmental process. Since this gap in knowledge has prevented the development of therapies that can prevent dissemination of C. difficile, our long-term goal is to elucidate the molecular basis by which C. difficile spores germinate into vegetative cells. A critical step during germination is the enzymatic removal of the spore cortex, a protective layer of peptidoglycan that maintains spores in a dormant state. In the Clostridia, cortex degradation depends on the SleC cortex hydrolase being proteolytically activated by Csp family proteases. While only a single Csp protease is sufficient to induce cortex hydrolysis in Clostridium perfringens, we have shown that in C. difficile both CspC and the CspBA fusion protease are required to activate SleC upon germinant addition. Intriguingly, CspC and the CspA domain of CspBA are both pseudoproteases that we and others have shown regulate cortex hydrolysis; indeed, CspC was recently identified as a novel germinant receptor. These findings raise a number of important questions: how do pseudoproteases regulate the activity of the CspB protease? How does regulated proteolysis activate SleC? Our objective in this proposal is to determine the molecular mechanisms by which CspC, CspBA, and SleC coordinately control cortex hydrolysis. Using genetic, biochemical and structural methods, we will identify regions within the CspC and CspA pseudoproteases required for CspB activation. Targeted mutagenesis and crystallographic studies of SleC will be used to elucidate the molecular basis by which regulated proteolysis activates SleC. Lastly, interactions between CspC, CspBA, and SleC will be identified using complementary bacterial two-hybrid, immunoprecipitation, and affinity purification approaches. Collectively, the proposed studies will increase our understanding of how pseudoenzymes can control enzyme activity and how C. difficile spores sense and respond to bile salt germinants. These studies will lay the foundation for developing therapeutics that can reduce C. difficile disease transmission and recurrence.