Structural Remodeling of Phage φPNJ-6 Hoc Promotes Adhesion to the Intestinal Epithelium

Although the mechanisms underlying bacteriophage-host interactions have been extensively elucidated, the "nonlytic" interactions between bacteriophage and the host intestinal microenvironment remain an emerging field. Our previous work demonstrated that the Escherichia coli T4-like bacteriophage φPNJ-6 adheres to the mucin MUC2 via the capsid protein Hoc. However, whether a rigid lock-and-key model fully explains this interaction remains unclear. In this study, we employed CRISPR-Cas12a-mediated precise deletion to remove the key Hoc residues involved in MUC2 binding (aa29-33) from the φPNJ-6 genome, generating the mutant strain Hoc^Δ29-33-PNJ-6. Unexpectedly, both in vitro and in vivo experiments revealed that the engineered bacteriophage Hoc^Δ29-33-PNJ-6 exhibited significantly enhanced adhesion. Structural analysis of Hoc showed that the deletion altered its conformation and increased the number of L-fucose-binding sites. These changes confer stronger fucose affinity to Hoc^Δ29-33, thereby promoting intestinal colonization by φPNJ-6. Overall, these results indicate that the Hoc-MUC2 interaction does not conform to a simple lock-and-key model and that structural remodeling of Hoc significantly affects the adhesion capacity of φPNJ-6. Our findings provide a strategy for designing long-term mucosa-resident therapeutic bacteriophages and expand the theoretical framework of bacteriophage-host microenvironment interactions.