Phosphatidylethanolamine pe

The pellets (5 OD units) of control and AEA (50 μg/mL, 2 h)-treated MDRSA CI-M were resuspended in 100 μL of DDW and sonicated on ice with a probe sonicator (UP200S Ultrasonic homogenizer, Hielscher Ultrasonics, Teltow, Germany) for 1 min with a 30% amplitude and 0.5 s intervals. The lipids from the sonicated samples were extracted according to the Bligh and Dyer method [135 ], and loaded onto a TLC plate (TLC Silica gel 60 F254 20 × 20 cm, Merck, Darmstadt, Germany). As controls, 5 μg of cardiolipin (CL) (Avanti Polar Lipids, Alabaster, AL, USA) and 5 μg of phosphatidylethanolamine (PE) (Sigma, St. Louis, MO, USA) were loaded on the TLC plates. For the first dimension, the plates were run in chloroform-methanol-water solution (65:25:4, v/v/v), and for the second dimension, they were run in chloroform-methanol-acetic acid-water solution (90:15:10:3.5, v/v/v/v). Then, the plates were dried for 5 min and sprayed with Molybdenum Blue reagent (Sigma, St. Louis, MO, USA). Five minutes after spraying, the images of the developed TLC plates were captured using the Fusion Solo S Imager (Vilber, Marne-la-Vallée Cedex 3, France) and the Fusion.CAPT software.

Lipid extraction was conducted as previously described⁵⁷ . Briefly, bacterial cells grown exponentially were washed twice with PBS and resuspended in 1.9 mL of PBS. Bacterial cell suspensions were mixed with 2.4 mL of chloroform and 4.8 mL of methanol to generate a single-phase Bligh and Dyer mixture, incubated at room temperature for 30 min, and subjected to centrifugation at 1500 × g for 20 min. The supernatant was converted into a two-phase solution by adding 2.4 mL of PBS and 2.4 mL of chloroform and centrifuged at 1500 × g for 20 min. The lower phase was removed and dried under a stream of nitrogen gas. For thin-layer chromatography (TLC) to analyze phospholipid, the dried lipid extracts were dissolved in 100 μL of a mixture of chloroform-methanol (2:1). Next, 5 μL of each sample was spotted onto a TLC silica gel 60 plate and the plate was developed in a tank equilibrated with chloroform-methanol-acetic acid (70:20:10 [v/v/v]). After drying the plate, lipids were visualized using iodine vapor. For spot identification, phosphatidylethanolamine (PE) (Sigma, USA), phosphatidylglycerol (PG) (Sigma, USA), and diphosphatidylglycerol (DPG) (Sigma, USA) were spotted onto TLC plate in parallel. After identifying the spots, the spots were scraped and subjected to digestion with perchloric acid. Each PL was quantified by malachite green staining as previously described^{58 (link)}.

Standards of PLs (cardiolipin CL, phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylinositol PI, phosphatidylglycerol PG, phosphatidylserine PS, lysophosphatidylcholine LPC, lysophosphatidylethanolamine LPE, sphingomyelin SPH) were sourced from Sigma-Aldrich (Saint-Quentin Fallavier, France). An authentic ceramide aminoethylphosphonate (CAEP) was kindly donated by Yanic Marty (CNRS, UBO, Brest, France).