The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σ ^V in Response to Lysozyme

Abstract

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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“…Hamster studies were conducted as previously described with minor modifications (Woods et al ., ). Male and female Syrian golden hamsters ( Mesocricetus auratus ) weighing between 70 and 110 g were purchased from Charles River Laboratories.…”

“…Hamsters treated with clindamycin, but not inoculated with C. difficile spores, served as negative controls. Spores were prepared as previously described Edwards and McBride, 2016;Woods et al, 2016). Following inoculation, hamsters were monitored for signs of disease, such as diarrhoea or wet tail, weight loss and lethargy.…”

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“…DNase-I treated RNA from the rstA :: erm mutant (MC391) was obtained as described above. 5’ RACE was performed using the 5’/3’ RACE Kit, Second Generation (Roche), following the manufacturer’s instructions as previously reported (32). Briefly, first strand cDNA synthesis was performed using the rstA- specific primer oMC982, followed by purification with the High Pure PCR Product Purification Kit (Roche).…”

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“…Alkaline phosphatase (AP) assays were performed as previously described (46,54), with minor modifications. Cultures of C. difficile containing phoZ reporter constructs were grown overnight in BHIS medium supplemented with 0.1% taurocholate, 0.5% fructose, and 2 g thiamphenicol ml Ϫ1 .…”