Lipid-tail modification of the antimicrobial peptide P7 enhances membrane disruption and antibacterial activity against Klebsiella pneumoniae

Antimicrobial peptides (AMPs) are short, host-defense peptides with broad-spectrum antibacterial activity and a low propensity for inducing resistance, positioning them as promising alternatives to conventional antibiotics. The α-helical peptide P7 (KIAKRIWKILRR) previously demonstrated potent antibacterial activity against drug-resistant Salmonella enterica serovar Typhimurium with minimal toxicity. However, its efficacy against ESKAPE pathogens ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter spp.) has not been characterized. In this study, we evaluated P7 and its N-terminal lipidated analogs conjugated with octanoic (C8), pelargonic (C9), and capric (C10) acids against ESKAPE organisms. Lipidation significantly enhanced antibacterial activity, particularly against K. pneumoniae , and promoted α-helical conformations in membrane-mimetic environments. Lipidated peptides also demonstrated improved membrane-permeabilizing and depolarizing capabilities. However, lipidation did not confer enhanced proteolytic stability. C8-P7 exhibited a more favorable activity profile among the analogs. The lipidated variants showed a limited therapeutic index, indicating that further structural optimization is required to improve their selectivity. Flow cytometry and electron microscopy confirmed membrane disruption as the primary mechanism of antibacterial action. Furthermore, additive effects were observed when C8-P7 was combined with gentamicin or ciprofloxacin, as indicated by fractional inhibitory concentration index. These findings highlighted the potential of lipid-tail engineering to enhance antimicrobial potency and membrane-disruptive capacity, offering a foundation for the development of potent lead compounds against multidrug-resistant K. pneumoniae and other ESKAPE pathogens.