Adsorption data.

Phage therapy is a promising alternative to antibiotics for treating Clostridioides difficile infections. Despite its fundamental importance, the initial and most critical step of C. difficile phage infection cycle - the precise recognition of a host receptor - remains poorly understood. The surface-layer protein SlpA was previously identified as a general phage receptor in C. difficile, but the molecular determinants of phage-S-layer interactions remain unclear. We investigated the structural features of SlpA required for phage recognition by engineering and expressing modified isoforms in FM2.5 and R20291 ΔslpA strains. By deleting specific SlpA domains and creating chimeric constructs from different isoforms, we assessed the capacity of seven phages to adsorb and infect the complemented strains. Our results demonstrate that both the LMW and HMW fragments of the S-layer protein contribute to phage specificity in an isoform-dependent manner. In addition, the LMW D2 domain is frequently required, but not always essential for productive infection. Several phages were also able to infect some of the complemented strains despite poor or delayed adsorption, highlighting differences in receptor binding. Interestingly, some phages adsorbed efficiently but failed to infect, showing that the capacity to bind to a host is not always predictive of a successful infection. Our findings reveal the complexity of phage-host interactions in C. difficile and provide new insights into the structural features of the S-layer protein that govern phage binding. These findings will be instrumental in guiding the future design of phage cocktails to target a broad spectrum of C. difficile clinical isolates.