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.