Proteolytic Regulation of Spore Germination in Clostridium difficile

描述(由申请人提供)：孢子萌发是医院内主要病原体艰难梭菌启动和传播感染所必需的，但对调节这一复杂发育过程的分子机制知之甚少。由于这种认识上的差距阻碍了可以防止艰难梭菌传播的治疗方法的开发，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。在萌发过程中的一个关键步骤是用酶去除孢子皮质，孢子皮质是维持孢子休眠状态的肽聚糖的保护层。在梭状芽胞杆菌中，皮层的降解依赖于SLEC皮层水解酶被CSP家族的蛋白水解酶激活。虽然只有一种CSP酶足以在产气荚膜梭菌中诱导皮质水解，但我们已经证明，在艰难梭菌中，CSPC和CspBA融合酶都需要在萌发添加时激活SLEC。有趣的是，CspBA的CSPC和CSPA结构域都是我们和其他人已经发现的调节皮质水解酶的假蛋白；事实上，CSPC最近被发现是一种新的萌发受体。这些发现提出了一些重要的问题：假蛋白水解酶如何调节CSPB蛋白水解酶的活性？受调控的蛋白分解如何激活SLEC？我们在这项建议中的目标是确定CSPC、CspBA和SLEC协调控制皮质水解酶的分子机制。利用遗传、生化和结构方法，我们将确定CSPB激活所需的CSPC和CSPA假蛋白酶中的区域。SLEC的定向突变和结晶学研究将被用来阐明受调控的蛋白分解激活SLEC的分子基础。最后，将使用互补细菌双杂交、免疫沉淀和亲和纯化方法鉴定CSPC、CspBA和SLEC之间的相互作用。总之，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发芽菌的理解。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.