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

Abstract

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) factors are a major family of signal transduction systems, which sense and respond to extracellular stresses. We have identified three C. difficile ECF factors. These ECF factors, CsfT, CsfU, and CsfV, induce their own expressions and are negatively regulated by their cognate anti-factors, RsiT, RsiU, and RsiV, respectively. The levels of expression of these ECF factors increase following exposure to the antimicrobial peptides bacitracin and/or lysozyme. The expressions of many ECF factors are controlled by site 1 and site 2 proteases, which cleave antifactors. 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 factor CsfT in C. difficile and controls the resistance of C. difficile to antimicrobial peptides that are important for survival in the host.Clostridium difficile is an anaerobic, Gram-positive, sporeforming, opportunistic pathogen and is the most common cause of hospital-acquired infectious diarrhea (3,29,32). C. difficile infections can range from mild diarrhea to life-threatening pseudomembranous colitis. People who develop C. difficile-associated disease are most commonly patients on antibiotics and/or who reside in hospitals or long-term care facilities. In recent years both the incidence and severity of C. difficile infections have increased in susceptible populations as well groups not traditionally considered to be at risk (33,38,39,43).In most patients, C. difficile infection is triggered by the disruption of the normal intestinal flora. C. difficile colonizes the intestinal tract of 12 to 20% of the adult population without causing disease (42). The disruption of the normal intestinal flora, most commonly due to treatment with antibiotics such as fluoroquinolones or clindamycin (13, 29), allows C. difficile to grow to high levels. How the normal flora blocks C. difficile growth is unclear. The normal flora may compete with C. difficile for nutrients and for binding sites in the intestinal tract or produce antimicrobial compounds that inhibit C. difficile growth (26, 45).The major known virulence determinants of C. diff...

Bacillus subtilis σ V Confers Lysozyme Resistance by Activation of Two Cell Wall Modification Pathways, Peptidoglycan O-Acetylation and d -Alanylation of Teichoic Acids

“…OatA provides lysozyme resistance in pathogenic Staphylococcus species (5, 7) and Lactococcus lactis (62). Furthermore, possible orthologs of V were found to be induced by lysozyme exposure in Enterococcus faecalis (42) and Clostridium difficile (35). Together, these results suggest that B. subtilis sigV, and therefore the V regulon, might be induced by lysozyme and thereby provide lysozyme resistance.…”

New Role for Human α-Defensin 5 in the Fight against Hypervirulent Clostridium difficile Strains

“…This was not an obvious finding: cationic AMPs share a mechanism of action, and C. difficile has evolved numerous strategies to evade their attack (28). C. difficile has been described to be resistant to bacterially derived AMPs, like bacitracin, nisin, gallidermin, vancomycin, and polymyxin B, but also to host-derived AMPs, like lysozyme (46,50). Furthermore, resistance to mammalian SMAP-29 and LL-37 was reported for epidemic-associated PCR ribotype 027 isolates (33).…”

“…The percentage of surviving bacteria was calculated as 100 ϫ (1 Ϫ r/c), where c is the number of CFU in untreated controls (SB6 medium alone) and r is the number of CFU in treated samples. The concentration of defensin that caused 50% bacterial growth inhibition (IC 50 ) was calculated from a plot of percent inhibition versus the logarithm of defensin concentration.…”

The Activity of V, an Extracytoplasmic Function Factor of Bacillus subtilis, Is Controlled by Regulated Proteolysis of the Anti- Factor RsiV

“…Homologs of the V system have been identified in Enterococcus faecalis and Clostridium difficile, where they are also induced in response to lysozyme (34)(35)(36). In E. faecalis, it has been demonstrated that V is required for resistance to lysozyme and is important for virulence in mouse models of systemic and urinary tract infection (35).…”