AbstractThe opportunistic pathogen Clostridium difficile is the most common cause of antibiotic-associated diarrhoea, with severity of disease ranging from mild diarrhoea to fulminant pseudomembranous colitis and death. It poses a major burden on healthcare providers, costing millions of pounds each year due to ward closures, isolation measures and prolonged illness. The current antibiotic therapy for C. difficile infection is effective, but high rates of relapse lead to ongoing misery for patients and spiralling costs for healthcare providers. Novel therapeutics for C. difficile are therefore desperately sought, and the antibiotic-induced nature of the disease has led to interest in development of non-antibiotic therapies.Sortase enzymes are responsible for covalent anchoring of specific proteins to the peptidoglycan of the cell wall of gram-positive bacteria. Following the discovery that the Sortase A enzyme of Staphylococcus aureus is essential for pathogenesis, sortase inhibitors are under investigation as novel therapeutics. Being ubiquitous in gram-positive bacteria, it is likely that other gram-positive pathogens require sortase enzymes for their pathogenesis and may be targets for development of sortase inhibitors.This work describes a characterisation of the sortase enzyme of C. difficile. To provide evidence for a role of the sortase in the cell wall biogenesis, a C. difficile sortase knockout strain was constructed by intron mutagenesis. Characterisation of this mutant led to the discovery that the putative adhesin CD0386 is anchored to the peptidoglycan of C. difficile by the sortase SrtB. To provide structural insight into the catalytic mechanism of the C. difficile sortase, an active site mutant was crystallised and its structure solved to 2.55Å by X-ray diffraction. The wall-linked protein CD0386 was also crystallised and subject to successful test diffraction. Analyses of SrtB reaction products by chromatography and mass spectroscopy indicate that the enzyme cleaves an SPKTG peptide motif and catalyses a transpeptidation reaction with a component of the C. difficile peptidoglycan.
|Date of Award||28 Jan 2014|
|Sponsors||Public Health England|
|Supervisor||K-Ravi Acharya (Supervisor) & C C Shone (Supervisor)|
Investigation of the <em>Clostridium difficile</em> Sortase by Gene Knockout, X-ray Crystallography and Biochemical Characterisation
Chambers, C. (Author). 28 Jan 2014
Student thesis: Doctoral Thesis › PhD