PrsW Is Required for Colonization, Resistance to Antimicrobial Peptides, and Expression of Extracytoplasmic Function σ Factors in Clostridium difficile

Clostridium difficile is an anaerobic, Gram-positive, spore-forming, opportunistic pathogen that is the most common cause of hospital-acquired infectious diarrhea. In numerous pathogens, stress response mechanisms are required for survival within the host. Extracytoplasmic function (ECF) sigma factors are a major family of signal transduction systems, which sense and respond to extracellular stresses. We have identified three C. difficile ECF sigma factors. These ECF sigma factors, CsfT, CsfU, and CsfV, induce their own expressions and are negatively regulated by their cognate anti-sigma factors, RsiT, RsiU, and RsiV, respectively. The levels of expression of these ECF sigma factors increase following exposure to the antimicrobial peptides bacitracin and/or lysozyme. The expressions of many ECF sigma factors are controlled by site 1 and site 2 proteases, which cleave anti-sigma factors. Using a retargeted group II intron, we generated a C. difficile mutation in prsW, a putative site 1 protease. The C. difficile prsW mutant exhibited decreased levels of expression of CsfT and CsfU but not of CsfV. When expressed in a heterologous host, C. difficile PrsW was able to induce the degradation of RsiT but not of RsiU. When the prsW mutant was tested in competition assays against its isogenic parent in the hamster model of C. difficile infection, we found that the prsW mutant was 30-fold less virulent than the wild type. The prsW mutant was also significantly more sensitive to bacitracin and lysozyme than the wild type in in vitro competition assays. Taken together, these data suggest that PrsW likely regulates the activation of the ECF sigma factor CsfT in C. difficile and controls the resistance of C. difficile to antimicrobial peptides that are important for survival in the host.