Identification of the receptor binding proteins for Clostridium difficile Bacteriophages

Clostridium difficile causes severe infectious diarrhoea referred to as C. difficile infection (CDI) and due to the organism being naturally resistant to antibiotics, alternative treatments for CDI are urgently required. Phages could provide an alternative source of antimicrobials for this pathogen due to their specificity, minimal disruption of microbiota and ability to self-amplify at the site of infection. However, the therapeutic development of phages will significantly benefit from a full understanding of the C. difficile phage infection process. To date no studies have identified the phage receptors binding proteins (RBPs) or the corresponding receptors on the bacterial surface that phages bind or adsorb, to establish infection. In other words, how does the first physical contact between phage receptor binding proteins located in the distal part of the phage tail and the surface of the bacterium occur? This project aims to identify the receptor binding proteins for two phages of C. difficile; phiCDHS1 (siphovirus), which infects CD105LC1 and CDR20291 that belong to the Ribotype 027 hypervirulent strains. In addition, phiCDMH1 (myovirus) that infects CD105HE1 ribotype 076. The approaches employed to identify the RBPs for these phages, were to over-express the four predicted phage tail fiber proteins Gp18, Gp19, Gp21 and Gp22 from CDHS1 phage and Gp29 and Gp30 from CDMH1. Which presumably, one or two of them is involved in the phage host binding. After significant optimisation, the expressed proteins were purified and polyclonal antibodies were generated against them. The antibodies were then used to neutralize phage infection, and were immunogold labelled to visualise the location of the proteins using TEM. The proteins were also crystallised in order to identify their structure. It was found that the anti-Gp22 protein was able to block phiCDHS1 infection, indicating that Gp22 is the protein responsible for C. difficile recognition. In addition, the anti-Gp29 protein was able to inhibit phiCDMH1 infection, which indicates that the Gp29 is the RBP for this phage. This is the first observation for C. difficile bacteriophages. This finding provides a novel insight into C. difficile bacteriophage biology and mechanisms of interaction with their hosts.