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

项目摘要 艰难梭菌是医院获得性感染性腹泻的最常见原因，是导致 仅在美国每年就有25万人感染，14,000人死亡。尽管有临床影响 梭difficile的因子知之甚少。艰难的需要殖民主机和 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C.艰难感染。我们的长期目标是更好地 了解C。艰难梭菌在感染期间抵抗先天免疫防御。细菌细胞 包膜对于细胞活力是必需的，并且是先天性免疫系统的许多组分的靶标。 溶菌酶是先天免疫系统的重要组成部分。我们发现C. difficile高度 抗溶菌酶。我们已经确定了一个C。艰难梭菌胞质外功能（ECF）σ因子σV CSFV基因编码的一种蛋白，是溶菌酶特异性诱导的，对C. 艰难梭菌，是一个重要的毒力因子，在动物模型的C。艰难感染ECF σ因子 代表一类重要的响应细胞包膜应激的信号转导系统。 σV在抗σ因子RsiV的连续蛋白水解破坏后被激活。我们的数据表明 RsiV在与溶菌酶直接结合后对蛋白酶变得敏感。X射线晶体 RsiV-溶菌酶复合物的结构不仅揭示了参与结合溶菌酶的区域， RsiV也可能起溶菌酶抑制剂的作用。这一发现提出了几个重要问题： RsiV与溶菌酶的结合如何控制σV的活化？RsiV如何避开site-1 在没有溶菌酶的情况下分裂？哪些基因对介导溶菌酶抗性很重要？ C. difficile？本文提出1）利用RsiV-溶菌酶结构来确定RsiV的特征 和CD 1560与溶菌酶相互作用所需; 2）确定信号肽酶和信号肽的作用 肽在RsiV加工中的作用; 3）确定溶菌酶中σ V依赖性基因的贡献 抗性和毒力。很难

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.