Elucidating the Mechanism of Clostridioides difficile Phage Infection and Identification of the Phage Receptor

Clostridioides difficile is a leading cause of healthcare-associated diarrhoea, exacerbated by rising antibiotic resistance and recurrent infections. Bacteriophage therapy offers a promising, highly specific alternative. However, its effective implementation requires a deeper molecular understanding of phage-host interactions, particularly receptor recognition and binding mechanisms. This study focuses on CDHS1, a C. difficile phage previously shown to exhibit enhanced lytic activity in the presence of magnesium ions (Mg²⁺), implicating divalent cations in phage adsorption and infectivity.Building on prior structural work that crystallised the CDHS1 receptor-binding protein (RBP), Gp22, in a magnesium-rich solution, this research investigates the role of Mg²⁺ in stabilising Gp22 and facilitating interaction with its putative receptor, the surface layer protein SlpA. Site-directed mutagenesis of three predicted Mg²⁺-binding residues (Asp540, Gln602, Glu605) was performed to generate binding-deficient mutants. Functional and biochemical characterisation revealed that Mg²⁺ enhances Gp22 stability and host-binding efficiency, supporting its structural and functional significance.To probe the phage and host interaction, SlpA was extracted from C. difficile and tested against Gp22 using pull-down assays and native PAGE electrophoresis. Despite evidence supporting a role for SlpA in phage recognition, in vitro binding was not observed, possibly due to loss of native conformation or absence of additional cofactors. Further, the effect of Mg²⁺ was tested in both chelating and supplemented conditions, yet did not yield detectable interactions under these assay formats.These findings suggest that while Mg²⁺ is essential for RBP stability and phage adsorption, stable receptor binding may require membrane-associated environments or complex host structures. This work provides novel insight into magnesium’s role in phage biology and highlights the need for advanced approaches, including computational modelling and in vivo assays, to fully elucidate the receptor recognition mechanisms in C. difficile phage therapy.