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Open Access Research article

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

Elizabeth H Donahue1, Lisa F Dawson1, Esmeralda Valiente1, Stuart Firth-Clark2, Meriel R Major2, Eddy Littler2, Trevor R Perrior2 and Brendan W Wren1*

Author Affiliations

1 Pathogen Molecular Biology Department, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK

2 Domainex Ltd, 162 Cambridge Science Park, Milton Road, Cambridge CB4 0GH, UK

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BMC Microbiology 2014, 14:219  doi:10.1186/s12866-014-0219-1

Published: 31 August 2014

Abstract

Background

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.

Results

We 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.

Conclusions

These 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.

Keywords:
Clostridium difficile; Sortase; Cysteine protease; Fluorescence resonance energy transfer (FRET); Enzyme kinetics; Enzyme inhibitors