CRISPR/Cas9-engineered Salmonella phage displaying antimicrobial peptide LL37 for enhanced antibacterial activity.

OBJECTIVES: The increasing prevalence of antibiotic-resistant Salmonella Typhimurium has highlighted the urgent need for alternative therapeutic strategies. This study engineered a lytic S. Typhimurium bacteriophage to present the antimicrobial peptide LL-37 on the virion surface, followed by evaluation of its enhanced antibacterial efficacy. METHODS: A recombinant lytic bacteriophage displaying LL-37 on its capsid was constructed using CRISPR/Cas9-mediated genome editing. The engineered phage was characterized for structural stability, adsorption kinetics, and lytic activity. Antibacterial efficacy was evaluated through bacterial growth inhibition assays, assessment of phage resistance rates, and host-pathogen interaction studies using intestinal epithelial cells. Intracellular bacterial survival was assessed in vitro, and prophylactic efficacy was further examined in a Galleria mellonella infection model. RESULTS: The engineered phage exhibited thermal and pH stability comparable to that of the wild-type phage, while demonstrating enhanced adsorption efficiency. In a cell lysis assay, the engineered phage sustained bacterial suppression for 24-72 h, whereas the wild-type phage permitted bacterial regrowth due to the emergence of phage-resistant mutants. The engineered phage significantly reduced bacterial attachment and intracellular survival in an intestinal epithelial cell model. Furthermore, it improved larval survival in a Galleria mellonella infection model in a dose-dependent manner, without inducing significant cytotoxicity. CONCLUSIONS: LL-37-engineered bacteriophages demonstrated improved antibacterial activity and intracellular infection control against S. Typhimurium. These findings support antimicrobial peptide-armed phages as a promising strategy for enhancing phage therapy and mitigating antibiotic-resistant bacterial infections.