Proteolytic Regulation of Spore Germination in Clostridium difficile

描述（申请人提供）：孢子萌发是医院内主要病原体艰难梭菌发起和传播感染的必要条件，但调控这一复杂发育过程的分子机制尚不清楚。由于这种知识上的差距阻碍了治疗方法的发展，可以防止艰难梭菌的传播，我们的长期目标是阐明艰难梭菌孢子萌发成营养细胞的分子基础。发芽过程中的一个关键步骤是酶去除孢子皮层，这是一层肽聚糖的保护层，使孢子保持休眠状态。在梭状芽孢杆菌中，皮质降解依赖于被Csp家族蛋白酶水解激活的SleC皮质水解酶。虽然在产气荚膜梭菌中，只有一个Csp蛋白酶足以诱导皮层水解，但我们已经证明，在艰难梭菌中，CspC和CspBA融合蛋白酶都需要在萌芽添加时激活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层水解；事实上，CspC最近被鉴定为一种新的生发受体。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？受调节的蛋白水解如何激活SleC？我们的目标是确定CspC， CspBA和SleC协调控制皮质水解的分子机制。利用遗传、生化和结构方法，我们将确定CspC和CspA假蛋白酶中CspB激活所需的区域。SleC的靶向诱变和晶体学研究将用于阐明调节蛋白水解激活SleC的分子基础。最后，CspC、CspBA和SleC之间的相互作用将通过互补细菌双杂交、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艰难梭菌孢子如何感知和响应胆盐萌发的理解。这些研究将为开发能够减少艰难梭菌疾病传播和复发的治疗方法奠定基础。

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.