(197h) Mechanistic Studies on Daptomycin-Induced Phase-Transitions on MODEL Lipid Membranes: Effect on Membrane Permeability

Objective: For infections caused by multidrug resistant Gram-positive pathogens the cyclic-lipopeptide antibiotic Daptomycin is considered a potent treatment option. Its bactericidal activity is attributed to a calcium- and PG lipid-dependent disruption in the membrane potential resulting in the release of bacterial contents. Formation of oligomeric-Daptomycin membrane pores has been proposed as an action mechanism. In this study, we propose a different mechanism on model lipid membranes.

Methods: We studied the interactions of Daptomycin with model lipid bilayer vesicles with symmetric and/or asymmetric lipid leaflet compositions. We characterized the release kinetics of fluorescent probes encapsulated in vesicles while systematically varying lipid headgroups and acyl-tails.

Results: Our findings suggest that the increase in membrane permeability by Daptomycin is due to the formation of transient, leaky lipid interfaces that span the bilayer. These interfaces originate right on the boundaries of lateral, phase-separated, well-packed lipid domains induced by Daptomycin and, importantly, are registered across the bilayer. The following two-step process is proposed. In the first step, the binding of Daptomycin on vesicles results in formation of well-packed single-(outer)leaflet lipid-domains via its association with the PG-headgroups only. In this process, a well-packed outer lipid-leaflet domain occurs independent of the extent of unsaturation of the tails of PG-lipids that partition in the Daptomycin-induced domain. In turn, for this initial phase-transition to result in increase of the membrane permeability, the outer lipid leaflet domains need to induce cross-registered phase-transitions of also well-packed lipids in the inner leaflet. This second phase-separated domain occurs only when the tails of the lipids in the inner leaflet are saturated independent of their headgroups. The extent of release via the interfacial domain boundaries depends on the different acyl-tail lengths of lipids across the lateral inner-leaflet-lipid-interfaces.

Conclusions: These results support the hypothesis that Daptomycin increases bilayer membrane permeability via the formation of domains with transiently leaky-interfaces.