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.
Highlights
Bacterial sortases are transpeptidases that covalently anchor surface proteins to the peptidoglycan of the Gram-positive cell wall
To determine whether Sortase B (SrtB) is conserved between C. difficile strains, representatives for each of the five distinct clades were chosen for analysis based on the availability of a fully annotated sequence: C. difficile strains 630 for Clade 1, R20291 and CD196 (RT027) for Clade 2 [29], M68 and CF5 (RT017) for Clade 3 [20], CD305 (RT023) for Clade 4, and M120 (RT078) for Clade 5 [20]
We developed a Fluorescence resonance energy transfer (FRET)-based assay to demonstrate that SrtB of C. difficile recognizes and cleaves the (S/P)PXTG motif between the threonine and glycine residues, and that cleavage is dependent on a single cysteine residue at position 209
Summary
Bacterial 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. Sortases are membrane-bound cysteine transpeptidases that anchor surface proteins to the peptidoglycan cell wall in Gram-positive bacteria. The sorting process is mediated by a conserved C-terminal cell wall sorting signal on the anchored protein, comprised of a C-terminal recognition sequence (often LPXTG, where X is any amino acid), followed closely by a hydrophobic transmembrane domain and a positively charged tail [1]. SaSrtA, which recognizes an LPXTG motif, is responsible for anchoring a variety of surface proteins involved in adherence and immune response evasion, and is essential for virulence in animal models [8,9]. Though mutating srtB has no effect on establishing infection, SaSrtB is required for persistence of the bacterium in mice [17]
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