Daptomycin Binds but does not Translocate across PC:PG Membranes

Abstract

The lipopeptide daptomycin is a last-resort antimicrobial in multiresistant Staphylococcus aureus infections. Despite its importance, little is understood about the interaction of daptomycin with lipid membranes and the subsequent events leading to antibacterial effects. In this work we asked three fundamental questions: (1) Does daptomycin bind to giant unilamellar vesicles (GUVs) composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG)? (2) Does it cause dye flux across the membrane? (3) Does the lipopeptide itself translocate across the membrane? Fluorescence confocal microscopy was used to visualize the binding of daptomycin to GUVs, dye flux into those vesicles, and peptide translocation across their membranes. First, we found that daptomycin binds to POPC:POPG GUVs in the presence of 0.5, 2.0, and 20 mM Ca2+. Second, we placed the vesicles in a solution containing carboxyfluorescein (CF) to test for influx. However, influx was never observed. Third, GUVs containing inner vesicles were observed to test for the translocation of daptomycin across the outer membrane of the GUVs, and onto the membrane of the inner vesicles. Again, translocation was never observed. However, in solutions containing 2 mM Ca2+ a significant amount (about 50%) of membranes that had bound daptomycin collapsed shortly after binding occurred, resulting in the destruction of the GUVs. Furthermore, at high Ca2+ concentrations (20 mM), the formation of daptomycin-rich clusters on the GUV membrane was observed. At lower concentrations of Ca2+ (2 mM), daptomycin was distributed evenly on the GUV membrane.Supported by NIH Grants GM072507 and AI088567.