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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 ...

Clostridium (Clostridioides) difficile is a gastrointestinal pathogen that colonizes the intestinal tract of mammals and can cause severe diarrheal disease. Although C. difficile growth is confined to the intestinal tract, our understanding of the specific metabolites and host factors that are important for the growth of the bacterium is limited. In other enteric pathogens, the membrane-derived metabolite, ethanolamine (EA), is utilized as a nutrient source and can function as a signal to initiate the production of virulence factors. In this study, we investigated the effects of ethanolamine and the role of the predicted ethanolamine gene cluster (CD1907-CD1925) on C. difficile growth. Using targeted mutagenesis, we disrupted genes within the eut cluster and assessed their roles in ethanolamine utilization, and the impact of eut disruption on the outcome of infection in a hamster model of disease. Our results indicate that the eut gene cluster is required for the growth of C. difficile on ethanolamine as a primary nutrient source. Further, the inability to utilize ethanolamine resulted in greater virulence and a shorter time to morbidity in the animal model. Overall, these data suggest that ethanolamine is an important nutrient source within the host and that, in contrast to other intestinal pathogens, the metabolism of ethanolamine by C. difficile can delay the onset of disease.

Clostridium difficile is an anaerobic, Gram-positive pathogen that causes severe gastrointestinal disease in humans and other mammals. C. difficile is notoriously difficult to work with and, until recently, few tools were available for genetic manipulation and molecular analyses. Despite the recent advances in the field, there is no simple or cost-effective technique for measuring gene transcription in C. difficile other than direct transcriptional analyses (e.g., quantitative real-time PCR and RNA-seq), which are time-consuming, expensive and difficult to scale-up. We describe the development of an in vivo reporter assay that can provide qualitative and quantitative measurements of C. difficile gene expression. Using the Enterococcus faecalis alkaline phosphatase gene, phoZ, we measured expression of C. difficile genes using a colorimetric alkaline phosphatase assay. We show that inducible alkaline phosphatase activity correlates directly with native gene expression. The ability to analyze gene expression using a standard reporter is an important and critically needed tool to study gene regulation and design genetic screens for C. difficile and other anaerobic clostridia.