Lipopolysaccharide-Phospholipid Separation in the Outer Membrane Vesicle Model Promotes Preferential Binding of Antimicrobial Peptides at Lipid Interfaces.

Multidrug-resistant Gram-negative pathogens pose a major global medical challenge due to the lack of new antibiotics. Outer membrane vesicles (OMVs) in Gram-negative bacteria significantly contribute to their resistance to antimicrobial peptides (AMPs), particularly the lipopeptide polymyxins. However, how the structural organization of the OMV membranes influences AMP binding remains poorly understood. Here, we employed large-scale coarse-grained molecular dynamics simulations and enhanced sampling techniques to explore the structural dynamics of the OMV models and their interactions with polymyxins and other AMPs. Our results demonstrated that the separation of lipopolysaccharides (LPS) and phospholipids (PLs) occurred within the outer leaflet of the OMV models, forming LPS-rich regions, PL-rich regions, and LPS-PL interfaces. Interestingly, small geometric defects appeared at LPS-PL interfaces due to a mismatched orientation between LPS and PL molecules. These defects enhanced polymyxin binding to OMV models with folded conformations, in which their hydrophobic parts inserted into the PL-rich regions while the positively charged residues bound to the exposed phosphate groups of lipid A. Free energy calculations confirmed that polymyxins penetrated OMV models more effectively at LPS-PL interfaces than at LPS-rich regions. Importantly, this biased location at the LPS-PL interfaces was found across six other types of AMPs, including Melittin, LL-37, Magainin 2, Tachyplesin 1, Protegrin 1, and Capitellacin. Our findings suggest that the LPS-PL separation in the OMV models creates distinct microenvironments that favor AMP binding, particularly at LPS-PL interfaces. These mechanistic insights will inspire the design of novel AMPs that can evade the protective effect of the aforementioned OMVs.