The intestinal pathogen Clostridium difficile is known to grow only within the intestines of mammals, yet little is known about how the bacterium subsists in this environment. In the intestine, C. difficile must contend with innate defenses within the host, such as cationic antimicrobial peptides (CAMPs) produced by the host and the indigenous microbiota. In this study, we investigated the mechanism of activation and regulation of the CprABC transporter system, which provides resistance to multiple CAMPs and shows homology to the immunity systems of bacterial antimicrobial peptide producers. The CprABC system proved to be controlled by a noncontiguous two-component system consisting of the CprK sensor kinase and an orphan response regulator (CD3320; CprR). The CprK-CprR regulators were shown to activate cprABCK transcription in a manner similar to that by lantibiotic regulatory systems. Unlike lantibiotic producer regulation, regulation by CprK-CprR was activated by multiple lantibiotics produced by diverse Gram-positive bacteria. We identified a motif within these lantibiotics that is likely required for activation of cpr. Based on the similarities between the Cpr system and lantibiotic systems, we propose that the CprABC transporter and its regulators are relatives of lantibiotic systems that evolved to recognize multiple substrates to defend against toxins made by the intestinal microbiota.

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Nylon-3 polymers (poly-β-peptides) have been investigated as synthetic mimics of host-defense peptides in recent years. These polymers are attractive because they are much easier to synthesize than are the peptides themselves, and the polymers resist proteolysis. Here we describe in vitro analysis of selected nylon-3 copolymers against Clostridium difficile, an important nosocomial pathogen that causes highly infectious diarrheal disease. The best polymers match the human host-defense peptide LL-37 in blocking vegetative cell growth and inhibiting spore outgrowth. The polymers and LL-37 were effective against both the epidemic 027 ribotype and the 012 ribotype. In contrast, neither vancomycin nor nisin inhibited outgrowth for the 012 ribotype. The best polymer was less hemolytic than LL-37. Overall, these findings suggest that nylon-3 copolymers may be useful for combatting C. difficle.

Clostridium difficile (also known as Peptoclostridium difficile) is a major nosocomial pathogen and a leading cause of antibioticassociated diarrhea throughout the world. Colonization of the intestinal tract is necessary for C. difficile to cause disease. Hostproduced antimicrobial proteins (AMPs), such as lysozyme, are present in the intestinal tract and can deter colonization by many bacterial pathogens, and yet C. difficile is able to survive in the colon in the presence of these AMPs. Our prior studies established that the Dlt pathway, which increases the surface charge of the bacterium by addition of D-alanine to teichoic acids, is important for C. difficile resistance to a variety of AMPs. We sought to determine what genetic mechanisms regulate expression of the Dlt pathway. In this study, we show that a dlt null mutant is severely attenuated for growth in lysozyme and that expression of the dltDABC operon is induced in response to lysozyme. Moreover, we found that a mutant lacking the extracytoplasmic function (ECF) sigma factor V does not induce dlt expression in response to lysozyme, indicating that V is required for regulation of lysozyme-dependent D-alanylation of the cell wall. Using reporter gene fusions and 5= RACE (rapid amplification of cDNA ends) analysis, we identified promoter elements necessary for lysozyme-dependent and lysozyme-independent dlt expression. In addition, we observed that both a sigV mutant and a dlt mutant are more virulent in a hamster model of infection. These findings demonstrate that cell wall D-alanylation in C. difficile is induced by lysozyme in a V -dependent manner and that this pathway impacts virulence in vivo.C lostridium difficile (Peptoclostridium difficile) causes nearly half a million infections in the United States each year, representing a significant public health threat (1). In order to cause infection, C. difficile must colonize the colon. As an important interface between the host and microbiota, the colon is an environment rich in host innate immune molecules and bacteriumderived antimicrobials made by the indigenous microbiota (2-6). These innate immune molecules and bacterially produced antimicrobials include a variety of cationic antimicrobial peptides (CAMPs), such as lysozyme, defensins, and bacteriocins (2,4,[7][8][9]. Understanding how C. difficile is able to resist killing in this antimicrobial-laden environment could better our understanding of the factors that contribute to the progression of C. difficile infections.A common mechanism of resistance to CAMPs in many bacteria is the alteration of the cell surface charge (10-12). One mechanism for increasing the surface charge is through the addition of D-alanine (D-Ala) to teichoic acids in the cell wall (10,12,13). The addition of D-Ala is mediated by four proteins, DltA, DltB, DltC, and DltD, encoded by the dlt operon (13). The Dlt pathway confers lysozyme resistance to Bacillus subtilis and Enterococcus faecalis (14,15). Previously, we demonstrated that the D-alanylation of the cell wall via ...

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