Though mutating srtB has no effect on establishing infection, SaSrtB is required for persistence of the bacterium in mice [17]. Clostridium difficile, an anaerobic Gram-positive, spore-forming bacillus, is the leading cause of hospital-acquired infectious diarrhea in North America and Europe. Infection with C. difficile can result in a range of

clinical presentations, from mild self-limiting diarrhea to the life-threatening BB-94 in vivo pseudomembranous colitis (PMC), known collectively as C. difficile infection (CDI) [19]. MLST studies have identified that the C. difficile population structure forms at least five distinct Selleckchem JQEZ5 lineages that are all associated with CDI [20–22]. Complications of severe CDI can lead to toxic megacolon, Tozasertib chemical structure bowel perforation, sepsis and death in up to 25% of cases [23]. Broad-spectrum antibiotic usage is the greatest risk factor for development of CDI due to the consequent disruption of the intestinal microflora. Treatment of CDI with metronidazole and vancomycin

can exacerbate the problem by continuing to disrupt the intestinal microflora. This leaves the patient susceptible to relapse or re-infection. Approximately one third of patients experience CDI relapse following treatment, and those who relapse have a greater risk of succumbing to the infection [23]. A current imperative is the development of therapies that selectively target C. difficile, whilst leaving the intestinal microflora intact. The C. difficile reference Florfenicol strain 630 encodes a single predicted sortase, CD630_27180, which has high amino-acid similarity with SrtB of S. aureus and B. anthracis [24]. A second sortase encoded within the genome is interrupted by a stop codon prior to the catalytic cysteine and is considered a pseudogene.

Thus, in contrast to other Gram-positive bacteria, C. difficile appears to have only a single functional sortase. As such, a compound that inhibits the activity of C. difficile sortase could target the pathogen without disrupting the numerous Gram-negative bacteria that make up the intestinal flora. In this study, we demonstrate that the predicted sortase encoded by CD630_27180 recognizes and cleaves an (S/P)PXTG motif between the threonine and glycine residues. The cleavage of this motif is dependent on the conserved cysteine residue at position 209 in the predicted active site of the sortase. We have also identified seven putative sortase substrates, all of which contain the (S/P)PXTG motif. These substrates are conserved among the five C. difficile lineages and include potential adhesins, a 5’ nucleotidase, and cell wall hydrolases. Furthermore, we identified a number of small-molecule inhibitors by means of an in silico screen that inhibit the activity of the C. difficile SrtB. Results Conservation of the catalytically active residues of sortase The genome sequence of C.