Binding of Daptomycin to Lipid Bilayers is not Significantly Altered by the Inclusion of Lysyl-Phosphatidylglycerol

In many bacterial cytoplasmic membranes, including that of Staphylococcus aureus, approximately 20 mol% of the diacylphosphatidylglycerol fraction in the cytoplasmic membrane is esterified to the amino acid lysine. This secondary modification leads to the formation of a net-cationic membrane lipid, which reduces the negative surface charge of the bacterial membrane. Elevated levels of lysyl-phosphatidylglycerol (LPG) are associated with increased bacterial resistance to a number of cationic antibacterial peptides, including the clinically important calcium-dependent lipopeptide antibiotic daptomycin. Inclusion of lysylated phospholipids in mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) has been shown to effectively protect lipid vesicles against leakage of contents induced by the alpha-helical antimicrobial peptides RP-1, mastoparan X, and cecropin A. However, peptide binding to the lipid bilayer was only significantly reduced at LPG fractions exceeding 30 mol%. Here, we investigated whether binding of daptomycin to lipid vesicles containing mixtures of POPG, POPC and LPG is affected in ways similar to that of alpha-helical peptides. We find that daptomycin binds calcium ions in aqueous solution with an apparent dissociation constant on the order of 10 mM. In general, binding of daptomycin to lipid bilayers depends strongly on calcium and the fraction of POPG in the mixture. However, when 20 mol% of POPG are replaced by LPG, daptomycin binding is only minimally affected. We also show that daptomycin does not cross the lipid bilayer into the vesicle lumen in giant lipid vesicles. These data suggest that clinical daptomycin resistance in S. aureus strains is caused by mechanisms beyond an LPG-mediated reduction in daptomycin binding.