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.