Antimicrobial Peptide Activity in Lipid Bilayers with Smooth-Type Lipopolysaccharides

Antimicrobial resistance has emerged as a global health concern, highlighting the need for alternative therapeutic strategies to conventional antibiotics. Antimicrobial peptides (AMPs) are promising candidates because they kill bacteria mainly by disrupting the membranes and have a lower propensity to induce resistance. Therefore, it is essential to clarify their molecular interactions with bacterial membranes. Although several methods have been proposed to form planar lipopolysaccharide (LPS) bilayers, producing and maintaining stable free-standing LPS-containing membranes remains challenging, particularly for smooth-type LPS bearing the O-antigen. In this study, we establish a microdevice-based droplet contact method that reconstitutes an outer-membrane (OM) model that includes smooth-type LPS. Electrophysiological evaluation of four AMPs (Mag2, Ovi, PG-1, Bac) using this method showed that AMPs interact with the OM mainly through penetration and act on the inner membrane (IM) by inducing both penetration and pore formation. Furthermore, when we scored the electrophysiological parameters for the OM and IM and integrated these scores, the correlation with minimum inhibitory concentration (MIC) reached R = 0.99, whereas the score from either membrane model alone did not reliably predict MIC. These results demonstrate that electrophysiological profiling using integrated IM and smooth-type LPS OM models provides a useful method for quantitative evaluation of membrane-disruptive antimicrobial activity.