Membrane Disruption and Charge Neutralization Drive Synergy Between LL37-Derived Peptides and Polymyxins Against E. coli.

Background/Objectives: Antimicrobial peptides (AMPs) offer promising strategies for combating drug-resistant bacteria such as Escherichia coli (E. coli). This study evaluated the synergistic effects of the human-derived AMP LL37 and its fragments FK16 and FK13, in combination with polymyxin B, colistin, and vancomycin, against three E. coli strains in vitro. Methods: Because the outer membrane of E. coli acts as a barrier to antimicrobial agents, we assessed the membrane-permeabilizing activity of these AMPs. We further examined electrostatic interactions between the cationic AMPs and bacterial surfaces via zeta potential measurements. To simulate physiological conditions, we investigated how Mg2+ and Ca2+ ions, common in blood, stabilize the bacterial outer membrane and influence AMP activity. Additionally, we assessed cytoplasmic membrane permeabilization as a key mechanism of antibacterial action. Finally, to support clinical translation, we evaluated the cytotoxicity of the AMPs on human dermal fibroblasts. Results: Checkerboard assays revealed that the peptide-antibiotic combinations exerted synergistic effects against the tested E. coli strains in both MHB medium and cation-adjusted MHB medium, with FICI values ranging from 0.3125 to 0.5 and <0.375 to 0.5, respectively. All three AMPs (LL37, FK16, and FK13) exhibited strong outer membrane-permeabilizing activity. Zeta potential measurements revealed a correlation between membrane disruption and surface charge neutralization. The presence of Mg2+ and Ca2+ ions was found to stabilize the bacterial outer membrane. Cytoplasmic membrane permeabilization was confirmed as a key antibacterial mechanism. Cytotoxicity assays confirmed the safety profile of these AMPs on human cells. Conclusions: The study demonstrates that LL37 and its fragments enhance the activity of conventional antibiotics against E. coli through membrane disruption and charge neutralization, as evidenced by the FICI values (0.3125-0.5), membrane disruption and charge neutralization. While divalent cations in physiological conditions influence AMP efficacy, these peptides show potential for clinical application against E.coli.