Structural and Functional Analysis of the CspB Protease Required for Clostridium Spore Germination
Structural and Functional Analysis of the CspB Protease Required for Clostridium Spore Germination
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DOI：
10.1371/journal.ppat.1003165
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发表时间：
2013-02-01
期刊：
影响因子：
6.7
通讯作者：
Shen, Aimee
中科院分区：
文献类型：
--
作者：
Adams, Chloe M.;Eckenroth, Brian E.;Shen, Aimee
孢子是医院病原体艰难梭菌的主要传播形式，艰难梭菌是全球医疗保健相关腹泻的主要原因。成功传播C.艰难梭菌需要其哈代、抗性孢子在胃肠道中萌发成营养细胞。这个过程中的一个关键步骤是孢子皮层的降解，孢子皮层是孢子核心周围的一层厚厚的肽聚糖。在梭菌属中，皮质降解依赖于皮质水解酶SleC的蛋白水解激活。以前的研究表明，Csps是萌发过程中SleC裂解所必需的;然而，它们的作用机制仍然很难表征。在这项研究中，我们证明了CspB是一种枯草杆菌蛋白酶样丝氨酸蛋白酶，其活性是有效的SleC切割和C。艰难孢子萌发。通过将Csp家族成员CspB的第一晶体结构求解为1.6埃，我们确定了CspB内的关键结构域。与所有先前解决的原核枯草杆菌酶的结构相比，CspB前结构域保持紧密结合的野生型枯草杆菌酶结构域和空间上封闭的催化活性位点。结合生物化学和遗传学分析，结构揭示了Csp蛋白酶含有独特的对体外和在C.很难总的来说，我们的研究提供了对CspB活性和功能的第一个分子见解。这些研究可能为开发抑制剂提供信息，这些抑制剂可以防止梭菌孢子萌发，从而防止疾病传播。
Spores are the major transmissive form of the nosocomial pathogen Clostridium difficile, a leading cause of healthcare-associated diarrhea worldwide. Successful transmission of C. difficile requires that its hardy, resistant spores germinate into vegetative cells in the gastrointestinal tract. A critical step during this process is the degradation of the spore cortex, a thick layer of peptidoglycan surrounding the spore core. In Clostridium sp., cortex degradation depends on the proteolytic activation of the cortex hydrolase, SleC. Previous studies have implicated Csps as being necessary for SleC cleavage during germination; however, their mechanism of action has remained poorly characterized. In this study, we demonstrate that CspB is a subtilisin-like serine protease whose activity is essential for efficient SleC cleavage and C. difficile spore germination. By solving the first crystal structure of a Csp family member, CspB, to 1.6 angstrom, we identify key structural domains within CspB. In contrast with all previously solved structures of prokaryotic subtilases, the CspB prodomain remains tightly bound to the wildtype subtilase domain and sterically occludes a catalytically competent active site. The structure, combined with biochemical and genetic analyses, reveals that Csp proteases contain a unique jellyroll domain insertion critical for stabilizing the protease in vitro and in C. difficile. Collectively, our study provides the first molecular insight into CspB activity and function. These studies may inform the development of inhibitors that can prevent clostridial spore germination and thus disease transmission.