A tale of two phage tails: Engineering the host range of bacteriophages infecting Clostridioides difficile

Abstract Clostridioides difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea across the globe. Although treatable with a restricted number of antibiotics, the emergence of resistant variants and high relapse rates necessitate alternative countermeasures. Phage therapy represents an attractive option. However, its implementation is handicapped by the narrow host specificity of the C. difficile bacteriophages isolated to date. One strategy to rationally expand phage host range would be to make appropriate modifications to the phage receptor binding protein (RBP). Here, we identify the tail fibre as the RBP of two Myoviridae phages, ΦCD1801 and ΦCD2301, which were previously isolated and propagated using the C. difficile strains CD1801 (RT078) and CD2301 (RT014), respectively. Contrary to studies into reprogramming the host ranges of phage of other bacterial other species, exchanging the tail fibre genes ( tcf/tfp ) alone between the two phage was insufficient to change host specificity. Rather, alterations to host range were dependent their exchange together with a putative chaperone encoded by hyp , localised adjacent to the tail fibre gene. Capitalising on this discovery, CRISPR/Cas9 was used to change the host range of one phage to that of the other by swapping the respective tcf/tfp and hyp genes. Significantly, one of the resulting mutants, surpassed both parental phages in terms of host range and efficiency of infection. This is the first time that genome engineering has successfully expanded the host range of a C. difficile phage, a prerequisite for implementing phage for the treatment of CDI. Importance Alternatives to antibiotics for treating Clostridioides difficile infection (CDI) are urgently required. Phage therapy presents an attractive option as it has the potential to clear the infection with minimal microbiome disruption and eliminate the possibility of recurrence. However, the C. difficile bacteriophages isolated to date have highly restricted host ranges. Moreover, rational strategies to alter specificity have till now been precluded as the identity of the phage receptor binding proteins involved was largely unknown. Here, we demonstrated that tail fibre proteins and an associated putative chaperone determine the host range of two Myoviridae phage. This enabled the alteration of specificity through CRISPR-mediated genome editing and the creation of a phage derivative with a host range and infection efficiency exceeding that of the parental phages. This is the first time that the host range of a C. difficile phage has been successfully expanded through rational genome engineering.