Phage-bacterial recognition is species-specific, determined by interactions between phage receptor-binding proteins (RBPs) and corresponding bacterial receptors. RBPs are diverse, and we present data demonstrating the identification and characterisation of a novel C. difficile phage RBP. Putative RBP was identified for CDHS-1, and overexpressed, purified, and polyclonal antibodies were raised and used in phage neutralization assays. Anti-gp22 neutralised CDHS-1, indicating it is the RBP. Immunogold-labelling and transmission electron microscopy confirmed this, enabling visualization of the protein locations. A detailed structural understanding was obtained from determining the three-dimensional structure of gp22 by X-ray crystallography. gp22 is a new RBP class consisting of an N-terminal L-shaped α-helical superhelix domain and a C-terminal Mg2+-binding domain. The protein is a stable homodimer in solution mediated via reciprocal contacts between an α-helical hairpin located within the superhelix domain and additional asymmetrical contacts between the ends of the short arm of each L-shaped protomer. The dimer resembles U-shape with a crossbar formed from the hairpin of each partner. C. difficile binding is Mg2+-dependent. CDHS-1 could not infect a C. difficile S-layer mutant suggesting the bacterial receptors are within the S-layer. These findings provide novel insights into phage biology and extend our knowledge of RBPs.
Identification of the receptor-binding protein of Clostridium difficile phage CDHS-1 reveals a new class of receptor-binding domains.