A flexible antimicrobial peptide achieves pathogen selectivity through lipid-dependent membrane engagement

Antimicrobial peptides (AMPs) are promising candidates for anti-infective development, but their translation is often limited by the difficulty of achieving potent microbial membrane activity without damaging mammalian cells. We report GT-2, a broad-spectrum antimicrobial peptide identified through proteome-informed peptide mining and deep generative sequence optimization, and use it to examine how flexible membrane-active peptides achieve pathogen selectivity. GT-2 inhibited representative fungal, Gram-negative, and Gram-positive pathogens (MIC, 4–32 μg/mL), disrupted microbial membranes, suppressed growth, inhibited biofilm formation, caused less than 5% haemolysis at 500 μg/mL, and reduced Pseudomonas aeruginosa wound burden below the detection limit under the tested topical dosing schedule. Lipid-antagonism, UV-Vis/CD, and all-atom molecular-dynamics analyses showed that GT-2 is largely disordered in buffer but becomes more helical and membrane-engaged in PE- and PI-containing bacterial- and fungal-like membranes than in pure POPC or CHL1/cholesterol-associated contexts. These findings support a lipid-dependent membrane-engagement model in which microbial lipid composition helps convert a flexible peptide into a membrane-active state, whereas the mammalian-like lipid environments analysed here were less permissive for productive insertion.