Quantified Membrane Permeabilization Indicates the Lipid Selectivity of Membrane-Active Antimicrobials

Most antimicrobial peptides (AMPs) and their synthetic mimics (SMAMPs) are thought to act by permeabilizing cell membranes. For antimicrobial therapy, selectivity for pathogens over mammalian cells is a key requirement. Understanding membrane selectivity is thus essential for designing AMPs and SMAMPs to complement classical antibiotics in the future. This study focuses on membrane permeabilization induced by SMAMPs and their selectivity for membranes with different lipid compositions. We measure release and fluorescence lifetime of a self-quenching dye in lipid vesicles. Apart from the dose-response, we quantify the strength of individual leakage events, and, employing cumulative kinetics, categorize permeabilization behavior. We propose that differing selectivities in a series of SMAMPs arise from a combination of the effect of the antimicrobial agent and the susceptibility of the membrane (with a given lipid composition) for certain types of leakage behavior. The unselective and hemolytic SMAMP is found to act mainly by the asymmetry stress mechanism, mediated by hydrophobic insertion of SMAMPs into lipid layers. The more selective SMAMPs induced leakage events occurring stochastically over several hours. Lipid intrinsic properties might additionally amplify the efficiency of leakage events. Leakage behavior changes with both the design of the SMAMP and the lipid composition of the membrane. Understanding how leakage behavior contributes to the selectivity and activity of antimicrobial agents will aid the design and screening of antimicrobials. An understanding of the underlying processes facilitates the comparison of membrane permeabilization across in vitro and in vivo assays.