Pepholin, a bacteriophage holin-derived antimicrobial peptide with membrane-disruptive activity and therapeutic efficacy in MDR Pseudomonas aeruginosa burn wounds.

The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa poses a serious threat to burn wound healing, necessitating the development of alternative therapeutics beyond conventional antibiotics. In this study, an artificial intelligence guided design strategy was employed to identify a novel antimicrobial peptide (AMP), Pepholin (12 amino acids, net charge +5) derived from the holin protein of a Pseudomonas bacteriophage, highlighting holins as an underexplored scaffold for AMP discovery. Pepholin demonstrated strong antibacterial and antibiofilm activities against MDR P. aeruginosa, achieving complete bacterial eradication within 90 minutes. Mechanistic investigations confirmed that Pepholin exerts its bactericidal action by permeabilizing bacterial membranes and disrupting membrane integrity in a concentration and time dependent manner. The peptide exhibited high hemocompatibility and minimal cytotoxicity in mammalian cells, resulting in selectivity indices greater than 8. Pepholin retained its antibacterial activity under physiological conditions of salt, serum, and temperature. To explore its therapeutic potential, Pepholin was incorporated into a carbomer-based hydrogel and evaluated in a murine burn wound infection model caused by MDR P. aeruginosa. Topical treatment significantly enhanced bacterial clearance, accelerated wound healing, promoted collagen deposition, and modulated cytokine responses. Collectively, these findings demonstrate that bacteriophage holin derived peptides represent a promising new class of antimicrobial agents and establishes Pepholin as a stable, biocompatible, and mechanistically defined AMP with dual antimicrobial and wound-healing activity, supporting its development as a next-generation therapeutic for infected wound management.