Surfactins modulate the lateral organization of fluorescent membrane polar lipids: A new tool to study drug:membrane interaction and assessment of the role of cholesterol and drug acyl chain length

The lipopeptide surfactin exhibits promising antimicrobial activities which are hampered by haemolytic toxicity. Rational design of new surfactin molecules, based on a better understanding of membrane:surfactin interaction, is thus crucial. We here performed bioimaging of lateral membrane lipid heterogeneity in adherent living human red blood cells (RBCs), as a new relevant bioassay, and explored its potential to better understand membrane:surfactin interactions. RBCs show (sub)micrometric membrane domains upon insertion of BODIPY (*) analogs of glucosylceramide (GlcCer*), sphingomyelin (SM*) and phosphatidylcholine (PC*). These domains exhibit increasing sensitivity to cholesterol depletion by methyl-β-cyclodextrin. At concentrations well below critical micellar concentration, natural cyclic surfactin increased the formation of PC* and SM*, but not GlcCer*, domains, suggesting preferential interaction with lipid* assemblies with the highest vulnerability to methyl-β-cyclodextrin. Surfactin not only reversed disappearance of SM* domains upon cholesterol depletion but further increased PC* domain abundance over control RBCs, indicating that surfactin can substitute cholesterol to promote micrometric domains. Surfactin sensitized excimer formation from PC* and SM* domains, suggesting increased lipid* recruitment and/or diffusion within domains. Comparison of surfactin congeners differing by geometry, charge and acyl chain length indicated a strong dependence on acyl chain length. Thus, bioimaging of micrometric lipid* domains is a visual powerful tool, revealing that intrinsic lipid* domain organization, cholesterol abundance and drug acyl chain length are key parameters for membrane:surfactin interaction. Implications for surfactin preferential location in domains or at their boundaries are discussed and may be useful for rational design of better surfactin molecules.