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

BMC Microbiology

Brendan W Wren²  Trevor R Perrior¹  Eddy Littler¹  Meriel R Major¹  Stuart Firth-Clark¹  Esmeralda Valiente²  Lisa F Dawson²  Elizabeth H Donahue² 

[1] Domainex Ltd, 162 Cambridge Science Park, Milton Road, Cambridge CB4 0GH, UK;Pathogen Molecular Biology Department, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK

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

Others  :  1140472
DOI  :  10.1186/s12866-014-0219-1

 received in 2014-06-06, accepted in 2014-08-12,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Donahue et al.; licensee BioMed Central Ltd.

【 图 表 】

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