Phospholipid Cleavage by LiRecDT1 Toxin

PLD activity was measured using the Amplex Red kit (Thermo Fisher Scientific, Waltham, MA, USA), a sensitive fluorogenic probe for H₂O₂ [22 (link)]. Briefly, LiRecDT1 cleaves SM to yield choline. Choline undergoes oxidation by choline oxidase to generate betaine and H₂O₂. Subsequently, H₂O₂ reacts with the Amplex red reagent in the presence of horseradish peroxidase and yields highly fluorescent resorufin. The assay added LiRecDT1 toxin (1 µg) to the substrate (250 µM/0.1% Triton X-100) and the Amplex red reagent mixture. After incubation (30 min at 37 °C), the resulting fluorescence was measured using a Tecan fluorimeter, Infinite M200, at an excitation wavelength of 540 nm, with emission detected at 570 nm. The same method was used to evaluate the cleavage of all other phospholipid substrates. However, the egg SM contained in the kit was replaced by other synthetic substrates: phospholipids with different numbers of carbon atoms in the fatty acid chains SM 24:0 (d18:1/24:0), SM 18:0 (d18:1/18:0), SM 16:0 (d18:1/16:0), SM 12:0 (d18:1/12:0), SM 06:0 (d18:1/06:0), SM 02:0 (d18:1/02:0), LSM (d18:1/0:0), PC 16:0 (18:0/16:0), LPC 24:0 (24:0/0:0), LPC 18:0 (18:0/0:0), LPC 16:0 (16:0/0:0), LPC 12:0 (12:0/0:0), and LPC 06:0 (06:0/0:0). All phospholipids were acquired from Avanti polar lipids, solubilized in reaction buffer (Tris-HCl 100 mM pH 7.4 containing 10 mM MgCl₂ and 0.01% Triton X-100), at a concentration lower than their critical micellar concentration (CMC). Experiments were performed in triplicates. Alternatively, enzymatic activity determination on different substrates with increasing amounts of Triton X-100. LiRecDT1 wild-type toxin (10 µg) was added to SM, LPC, and PC substrate (250 µM) solubilized in detergent (0.1%, 0.5%, and 1%).