Clostridium difficile infection (CDI) is the most common cause of nosocomial diarrhoea worldwide. Current treatment options have varied levels of success and there is no licensed vaccine. Most vaccines investigated to date use the C. difficile toxins, as these induce a protective immune response. However, this is only against the symptoms of CDI, therefore there is a need for antigens capable of preventing colonisation and transmission. The results of a C. difficile specific pan-protein array, screened against blood samples from CDI patients and healthy controls, was analysed and a shortlist of proteins identified where the IgG antibody response was significantly higher in the control group. Three of these were expressed and purified in E. coli, and tested against patient samples in an ELISA. For two of the three proteins, there was a higher IgG response in the healthy control group (albeit not significant) confirming the array results. Inactivation of three immunogenic proteins from the array in C. difficile R20291 revealed the putative permease CDR20291_0342, was not involved in efflux of the antimicrobials tested. Inactivation of the putative pilin protein, CDR20291_3343, did not influence surface motility, but motility was almost abolished in a flagella mutant. The putative cobalt transporter CDR20291_0330, was proposed to contribute to a putative vitamin B12 synthesis pathway, which is required for ethanolamine utilisation. Neither R20291Δ0330 or R20291 utilised ethanolamine in the conditions tested but it was found that inactivation of one component of the two-component regulatory system in C. difficile 630Δerm abolished ethanolamine utilisation. Finally, mechanisms of synthesising C. difficile glycoconjugates using bioconjugation were investigated, using a carrier protein and portion of the R20291 flagella glycan. A short acceptor peptide was designed and found to be glycosylated at all sites with a glycan from Campylobacter jejuni but it was not possible to detect glycosylation with the flagella glycan from C. difficile.