The Pathogenesis of Clostridium difficile infection

Abstract

Clostridium difficile is a major problem as the aetiological agent of antibiotic associated diarrhoea. The mechanism by which the bacterium colonises the gut is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The aims of this study were to further define roles for selected surface proteins using a knockout approach, to evaluate the feasibility of surface protein based immunotherapeutics and to obtain structural information using X-ray crystallography. Mutants of cell wall-binding domain (PFam04122) containing proteins CD1036, CD2735, CD2784, Cwp66, CD2791, Cwp84, CD2795 and the flagella cap (FliD) were created. Mutants were characterised with regard to growth, sporulation, toxin production, adhesion in vitro, and, for the Cwp84 mutant, using the in vivo hamster model. The surface-located cysteine protease, Cwp84, was found to play a key role in maturation of the C. difficile S-layer, yet the Cwp84 mutant still caused disease with a similar pathology to the wildtype. Culture supernatant levels of toxin A were increased in CD2735, Cwp66, CD2791, CD2795 and particularly in Cwp84 and FliD 24 hr cultures, while CD2735, Cwp66, CD2791, CD2795 mutants also showed reduced adherence to Caco-2 cells compared to the wild-type. Passively administered immunotherapy, generated to low pH surface protein extracts of the C. difficile R20291 strain, did not protect hamsters from challenge with the cognate strain. Structural studies were undertaken on the surface proteins CD2791, Cwp66 and CD2767. Crystallisation conditions were identified for a recombinant N-terminal domain of CD2767 and an X-ray data set collected to 2 Å, although the structure was not solved by molecular replacement. Together these results further our knowledge of C. difficile surface proteins, although further work is required to identify which surface proteins play key roles in vivo during infection.

Date of Award	1 May 2011
Original language	English
Awarding Institution	University of Bath
Supervisor	Ravi Acharya (Supervisor) & C C Shone (Supervisor)