Modified Lipid Content Affects Daptomycin-Membrane Interactions

Abstract

Daptomycin is an anionic membrane active antimicrobial lipopeptide used to treat serious infections caused by gram-positive bacteria. It causes target membrane depolarization by forming oligomeric pores that allow leakage of potassium ions, but the complete mechanism of action is unresolved. Antibiotic function is calcium dependant and requires the presence of anionic phospholipids like phosphatidylglycerol (PG) in the target membrane. Because of its efficacy in treating infections resistant to many front-line antibiotics, the recent emergence of strains displaying reduced daptomycin susceptibility is troubling. These quasi-resistant strains have modified membrane lipid content, including an increased presence of cardiolipin (CL) and lysyl-PG, the latter possibly due to the observed up-regulation of mprF, a gene that codes for a protein with lysyl-PG synthase and flippase activity. Using monolayer and bilayer model systems, we sought here to study the effects of the presence of CL on daptomycin binding to PG-containing membranes. Surprisingly, isothermal titration calorimetry (ITC) revealed that daptomycin-membrane affinity increased when small amounts of CL was present and continued to increase until 10 mol%, above which the trend reversed drastically. Results from Langmuir monolayer insertion experiments also show that CL affects the degree of drug insertion into PG-containing lipid films as determined by greater increases in surface pressure after daptomycin was injected into the aqueous subphase when CL was present. Preliminary results from monolayer insertion experiments in which lysyl-PG was included in the films indicate that this lipid also significantly affects drug-monolayer interactions. We hypothesize from these results that alteration of the lipid content of bacterial membranes represents an important component of the potential resistance mechanism and should be considered in the development and formulation of the next generation of membrane targeting antibiotics.