Membrane permeabilization and peptide-lipid interactions
Membrane permeabilsation was evaluated using propidium iodide assay as previously explained,43 with CTAB as positive control. Peptide–lipid interaction was measured using reconstituted lipid monolayer.44 Total lipid extract from overnight liquid cultures of MRSA USA300 was obtained by Folch extraction, resuspended in chloroform and stored at −20 °C under nitrogen conditions. Pure bacterial and eukaryotic lipids, POPG, cardiolipin, LTA, POPE and POPC (Avanti Polar Lipid USA) were reconstituted in chloroform at 1 mg/ml and stored at −20 °C under nitrogen. Using a 50 µl Hamilton’s syringe, total MRSA lipid extract or pure lipids were spread at the surface of PBS creating a lipid monolayer at the air-water interface until an initial surface pressure of 30 ± 0.5 mN/m was reached. This corresponds to a lipid packing density theoretically equivalent to that of the outer leaflet of the cell membrane.45 After 5–10 min of incubation allowing evaporation of the solvent and stabilization of the initial surface pressure, peptides were injected into the PBS (pH 7.4, volume 800 µl) sub-phase using a 10 µl Hamilton syringe. The variation of the surface pressure caused by peptide injection was then continuously monitored using a fully automated microtensiometer (µTROUGH SX, Kibron Inc., Helsinki, Finland) until reaching equilibrium (maximal surface pressure increase usually obtained after 15–25 min). Critical pressure of insertion of each peptide in the different lipids was also determined by changing the initial pressure of lipid monolayer (from 10 and 30 mN/m) and measuring the variation of pressure caused by the injection of peptide (at 1 µg/ml final concentration). All experiments were carried out in a controlled atmosphere at 20 °C ± 1 °C and data were analyzed using the Filmware 2.5 program (Kibron Inc., Helsinki, Finland). Variation of surface pressure was plotted as a function of initial surface pressure and critical pressure of insertion was calculated as the theoretical value of initial pressure of lipid monolayer not permissive to peptide insertion, i.e., a variation of pressure equal to 0 mN/m. The accuracy of the system under our experimental conditions was determined to be ± 0.25 mNm/1 for surface pressure measurements.