Investigating the roles of the cell wall anchoring sortase enzyme and sorted proteins in Clostridium difficile

Donahue, EH; (2015) Investigating the roles of the cell wall anchoring sortase enzyme and sorted proteins in Clostridium difficile. PhD thesis, London School of Hygiene & Tropical Medicine. DOI:

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Clostridium difficile is a Gram-positive, anaerobic bacterium that is the most frequent cause of antibiotic-associated colitis and healthcare-acquired diarrhoea worldwide. In many Gram-positive bacteria, a membrane bound sortase enzyme covalently anchors surface proteins to the cell wall, a process that is essential for virulence. Sortase protein anchoring is mediated by a conserved cell wall sorting signal on the anchored protein, containing the “LPXTG-like” motif. Sequence analysis confirmed that C. difficile strain 630 encodes a single sortase, CD2718, but little is known about its function. In this study, we identify 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. A FRET-based assay was developed to confirm that recombinant CD2718 catalyses the cleavage of fluorescently labelled peptides containing (S/P)PXTG motifs in vitro. Mass spectrometry reveals the cleavage site to be between the threonine and glycine residues of the (S/P)PXTG peptide. Replacement of the predicted catalytic cysteine residue at position 209 with alanine abolishes CD2718 activity, as does addition of the cysteine protease inhibitor MTSET to the reaction. The activity of CD2718 can also be inhibited by several small-molecule inhibitors identified through an in silico screen. CD2718-mediated cleavage of a recombinant fusion protein containing the full length predicted sortase substrate CD0183 was also observed. These results demonstrate for the first time that C. difficile encodes a single sortase enzyme that recognises (S/P)PXTG sequences. The activity of CD2718 can be inhibited by rationally designed small-molecule inhibitors, and may be an appropriate target for downstream anti-infective therapies against C. difficile infection.

Item Type: Thesis
Thesis Type: Doctoral
Thesis Name: PhD
Contributors: Wren, BW (Thesis advisor);
Faculty and Department: Faculty of Infectious and Tropical Diseases > Dept of Pathogen Molecular Biology
Copyright Holders: Elizabeth Donahue


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