Effects of Cholesterol and Solution pH on Daptomycin-bilayer Interactions

The global challenge of increasing antibiotic resistance development has necessitated the continued development and investigation of novel and existing antimicrobials. Daptomycin is a cyclic lipopeptide isolated from Streptomyces roseosporus, which has been clinically approved to treat a variety of gram-positive infections. Daptomycin is a membrane-active antimicrobial peptide that requires Ca²⁺ ions and phosphatidylglycerol lipids to exert its antimicrobial activity. To better understand the mechanism of action at the biophysical level, daptomycin interactions with bilayers and bacterial membranes were evaluated across different solution pH values and by varying cholesterol content in the model membranes. The results demonstrate that pH 5-8 does not affect daptomycin binding to model membranes or its structural rearrangement upon binding to the bilayer. Similarly, including cholesterol up to 30 mol% in the bilayers did not affect daptomycin binding, with an apparent binding affinity of ~ 1 µM under these conditions. Fluorescence quenching experiments showed that Ca²⁺ binding to daptomycin induced a small but significant shift in the position of Trp in the bilayer, becoming more deeply inserted when calcium was present. Bacterial membrane permeabilization assays confirmed that daptomycin forms stable pores in bacterial membranes large enough to cause leakage of the chromogenic substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). The data demonstrate a remarkable robustness of daptomycin interactions with lipid bilayers.