Impact of Lipid Composition on LL-37 Interaction with Gram-Negative Bacterial Membrane Models

Gram-negative bacteria have a challenging dual-membrane structure, consisting of an inner membrane (IM) and an outer membrane (OM). The key difference between them lies in their asymmetry and the presence of lipopolysaccharides (LPS) in the OM. This unique architecture gives Gram-negative bacteria distinct physicochemical properties and contributes to their high resistance to external agents such as antibiotics. As antibiotic resistance continues to rise, one promising strategy to overcome it involves the use of antimicrobial peptides (AMPs). AMPs are evolutionarily conserved components of the innate immune system and are less likely to induce resistance compared to conventional antibiotics. Among them, the human AMP LL-37 has drawn attention due to its ability to bind LPS, neutralize its toxicity, and potentially compromise OM integrity. Because AMPs act primarily through direct lipid interactions, several studies have examined them using membrane models such as phospholipid and LPS vesicles. However, these often rely on oversimplified systems, leaving complex membrane interactions poorly understood. To address this gap, the present study investigated how LL-37 binds to and disrupts Gram-negative bacterial membranes using a combination of experimental methods and coarse-grained molecular dynamics (CGMD) simulations. Both a ternary IM model (POPE, POPG, CL) and quaternary OM models (POPE, POPG, CL, LPS) were used to assess the effects of lipid composition and organization on LL-37 interaction. Additionally, an asymmetric OM model (OM-asy) was employed in CGMD simulations to explore the impact of phospholipid distribution across the bilayer. Our findings showed that LL-37 interacts differently with each membrane type: it preferentially targets cardiolipin clusters in the IM and embeds into LPS-rich regions of the OM through a combination of electrostatic and hydrophobic interactions. These interactions lead to structural alterations in the membranes, offering valuable insights into the mechanism of action of LL-37 and underscoring its potential in overcoming Gram-negative bacterial resistance.