ECF Sigma Factors and the Cell Envelope Stress Response of Clostridium difficile

项目摘要 艰难梭菌是引起医院获得性感染性腹泻的最常见原因，它是 仅在美国，每年就有25万人感染，1.4万人死亡。尽管有临床上的影响 艰难梭菌对艰难梭菌定植宿主所需的因素知之甚少 逃避宿主的免疫防御。有几条证据表明先天免疫的组成部分 系统在控制艰难梭菌感染中起着重要作用。我们的长期目标是做得更好 了解艰难梭菌在感染期间如何抵抗先天免疫防御。细菌细胞 包膜是细胞存活所必需的，也是先天免疫系统许多成分的靶标。 溶菌酶是天然免疫系统的重要组成部分。我们发现艰难梭菌是高度 对溶菌酶具有抗药性。我们已经鉴定出艰难梭菌的胞外功能σ因子σV 由CSFV编码，由溶菌酶特异性诱导，对C. 艰难梭菌是艰难梭菌感染动物模型中的一个重要毒力因子。ECFσ因素 代表了一类重要的响应细胞包膜压力的信号转导系统。 σV在抗σ因子rsiV的蛋白水解性破坏后被激活。我们的数据显示 当RsiV直接与溶菌酶结合时，RsiV对蛋白酶变得敏感。X-射线晶体- RsiV-溶菌酶复合体的结构不仅揭示了与溶菌酶结合的区域，而且还揭示了 此外，RsiV可能作为溶菌酶抑制物发挥作用。这一发现提出了几个重要的问题： RsiV与溶菌酶的结合如何控制σV的激活？RsiV如何避开Site-1 在没有溶菌酶的情况下进行卵裂？哪些基因在介导溶菌酶抗性中起重要作用 艰难梭菌？在这里，我们建议1)使用RsiV-溶菌酶结构来确定RsiV的特征 和与溶菌酶相互作用所需的CD1560；2)确定信号肽和信号的作用 3)σV依赖基因在溶菌酶中的作用 艰难梭菌的耐药性和毒力。

Project Summary Clostridium difficile, the most common cause of hospital-acquired infectious diarrhea, is responsible for 250,000 infections and 14,000 deaths each year in the United States alone. Despite the clinical impact of C. difficile relatively little is known about the factors of C. difficile required to colonize the host and evade host immune defenses. Several lines of evidence suggest components of the innate immune system play an important role in controlling C. difficile infections. Our long-term goal is to better understand how C. difficile resists the innate immune defenses during an infection. The bacterial cell envelope is essential for cell viability and is the target of many components of the innate immune system. Lysozyme is an important component of the innate immune system. We found that C. difficile is highly resistant to lysozyme. We have identified a C. difficile Extra-Cytoplasmic Function (ECF) σ factor σV encoded by csfV, which is specifically induced by lysozyme and is critical to lysozyme resistance in C. difficile and is an important virulence factor in an animal model of C. difficile infection. ECF σ factors represent an important class of signal transduction systems which respond to cell envelope stresses. σV is activated upon the sequential proteolytic destruction of the anti-σ factor RsiV. Our data indicate that RsiV becomes sensitive to proteases upon binding directly to lysozyme. The X-ray crystal co- structure of the RsiV-lysozyme complex revealed not only the regions involved in binding lysozyme but also that RsiV likely functions as a lysozyme inhibitor. This finding raises several important questions: How is does RsiV binding to lysozyme binding control activation of σV? How does RsiV avoid site-1 cleavage in the absence of lysozyme? What genes are important for mediating lysozyme resistance in C. difficile? Here we propose to 1) Use the RsiV-lysozyme structure to determine the features of RsiV and CD1560 required for interaction with lysozyme; 2) Determine the role of signal peptidase and signal peptides in processing of RsiV; and 3) Define the contribution of σV-dependent genes in lysozyme resistance and virulence in C. difficile.