Clostridium difficile is the leading cause of hospital acquired diarrhoea. With extended
hospitalisation, a high mortality rate and the risk of recurrence, C. difficile infection presents
a burden to both patients and the healthcare system. Symptoms of disease are primarily
mediated by the two major toxins released by C. difficile and are the focus of current
vaccine studies. Little is known of surface proteins in C. difficile, their role in colonisation
and their potential as antigens to reduce severity of disease. The surface of C. difficile is
composed of a peptidoglycan cell wall and an external S-layer. In many Gram positive
bacteria a membrane bound enzyme, sortase, covalently attaches specific proteins to the
cell wall.
In this study, seven potential sortase substrates were identified and shown to be expressed
in C. difficile 630, and at least four were shown to be localised to the cell wall. The substrate
CD0183 was shown to need its LPxTG like domain for correct sorting onto the cell wall by
sortase, which was shown to be a probable cysteine protease. An efficient cell wall
extraction protocol was developed alongside two novel assays for studying the sortase
mechanism which could provide future insights into this mechanism of protein sorting. The
highly conserved S-layer protein Cwp2 is co-transcribed with CD2790 and cwp66 and its
promoter is important for the expression of cwp66, especially during exponential growth. Slayer
proteins were shown to potentially reduce colonisation and disease in a multicomponent
vaccine, with Cwp2 in particular identified as an antigen which can prevent
diarrhoeal symptoms of disease in hamsters. Thus S-layer proteins are potentially important
components of a vaccine against C. difficile infection.