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.