Design and Mechanistic Study of Pf-15: A Linear Antimicrobial Peptide Derived from Gramicidin S Skeleton

Multidrug-resistant bacterial infections threaten public health, and novel antimicrobials are urgently needed. Antimicrobial peptides (AMPs) have emerged as promising candidates for use as natural and safe antimicrobial additives in the food industry. Herein, a series of AMPs derived from Gramicidin S (GS) were rationally designed using strategies such as ring opening, amino acid substitution, peptide truncation, and segment addition to improve their biocompatibility and applicability. Among them, Pf-15, adopting a stable α-helical conformation, exhibited potent, broad-spectrum activity against Gram-positive foodborne pathogens. It demonstrated a low hemolytic activity and high biocompatibility. Mechanistic studies revealed Pf-15 targets lipoteichoic acid in the <i>Staphylococcus aureus</i> cell wall, causing membrane disruption, ATP depletion, and elevated reactive oxygen species. In vivo models of bacteremia and keratitis confirmed that Pf-15 significantly reduces bacterial load without excessive organ toxicity. These findings highlight Pf-15 as a promising, multifunctional antimicrobial additive to improve food safety and extend shelf life.