Clostridium difficilehas a single sortase, SrtB, that can be inhibited by small-molecule inhibitors

Abstract

BackgroundBacterial sortases are transpeptidases that covalently anchor surface proteins to the peptidoglycan of the Gram-positive cell wall. Sortase protein anchoring is mediated by a conserved cell wall sorting signal on the anchored protein, comprising of a C-terminal recognition sequence containing an “LPXTG-like” motif, followed by a hydrophobic domain and a positively charged tail.ResultsWe report that Clostridium difficile strain 630 encodes a single sortase (SrtB). A FRET-based assay was used to confirm that recombinant SrtB catalyzes the cleavage of fluorescently labelled peptides containing (S/P)PXTG motifs. Strain 630 encodes seven predicted cell wall proteins with the (S/P)PXTG sorting motif, four of which are conserved across all five C. difficile lineages and include potential adhesins and cell wall hydrolases. Replacement of the predicted catalytic cysteine residue at position 209 with alanine abolishes SrtB activity, as does addition of the cysteine protease inhibitor MTSET to the reaction. Mass spectrometry reveals the cleavage site to be between the threonine and glycine residues of the (S/P)PXTG peptide. Small-molecule inhibitors identified through an in silico screen inhibit SrtB enzymatic activity to a greater degree than MTSET.ConclusionsThese results demonstrate for the first time that C. difficile encodes a single sortase enzyme, which cleaves motifs containing (S/P)PXTG in-vitro. The activity of the sortase can be inhibited by mutation of a cysteine residue in the predicted active site and by small-molecule inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0219-1) contains supplementary material, which is available to authorized users.

Clostridium difficile secreted Pro‐Pro endopeptidase PPEP‐1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831

“…In Gram-positive bacteria, virulence factors involved in adhesion to extracellular matrix proteins are often targeted to the peptidoglycan layer through a sortase mediated reaction. Previous studies have shown that the C. difficile sortase recognizes an (S/P)PXTG motif [19,32,33]. The PPEP-1 substrates CD2831 and CD3246, but not CbpA, contain the (S/P)PXTG motif and a recent study showed that CD2831 is indeed targeted to the cell wall of C. difficile [29].…”

Structural Insights into Substrate Recognition by Clostridium difficile Sortase

“…When the peptide is cleaved, the un-quenched fluorophore gives an enhanced fluorescent signal. To assess whether the previously described sortase inhibitors can inhibit the catalytic activity of Cd-SrtB ΔN26 , MTSET, AAEK1, and curcumin (Maresso et al, 2007; Hu et al, 2013; Donahue et al, 2014) (Supplementary Figure 2) were examined using the fluorescence resonance energy transfer (FRET)-based assay. The concentration-dependent inhibitory effects of MTSET (Figure 1B), AAEK1 (Figure 1C), and curcumin (Figure 1D) on the cleavage activity of the recombinant Cd-SrtB ΔN26 were observed as the fluorescence signals were reduced when the inhibitors were added to the reactions comprised of Cd-SrtB ΔN26 and fluorogenic peptides.…”

“…Our results show that the P4 residue of PPKTG in Cd-SrtB ΔN26 –PPKTG complex interacts with Asp166 for about 80% of the simulations time (10 ns) and forms hydrogen bonds with Asp166; while the P4 residues in NPKTG and NVQTG do not specifically interact with any residue in Cd-SrtB ΔN26 (Figure 4 and Supplementary Figure 5). Moreover, we assumed that a peptide would be subjected to a conformation that allows Cd-SrtB to achieve a better catalytic efficiency if the distance (DIS Cys-Thr ) between the sulfhydryl group of cysteine residue in Cd-SrtB and the carboxyl carbon of threonine residue in peptide is relatively short (Donahue et al, 2014; Chambers et al, 2015). We therefore examined distance distributions for four peptides of interest throughout 10 ns simulations.…”

“…In the genome of toxigenic C. difficile strain 630, only one functional sortase, the SrtB gene, is present (Donahue et al, 2014). C. difficile is a Gram-positive, anaerobic, and spore-forming bacterium that can colonize the gut if the normal intestinal microbiota is disrupted (Kelly and LaMont, 1998).…”

“…There have been many structural and functional studies on sortases from various Gram-positive pathogens (Spirig et al, 2011; Cascioferro et al, 2014; Bradshaw et al, 2015), and studies on C. difficile sortases were reported recently (Donahue et al, 2014; van Leeuwen et al, 2014; Chambers et al, 2015). Sa-SrtA has been extensively studied, and the catalytic mechanism underlying how Sa-SrtA anchors the surface protein to cell wall has been reported (Mazmanian et al, 1999; Perry et al, 2002).…”