Extended Data Fig. 8: Carboxyfluorescein (CF) assays.
A,B,C) Plectasin does not cause leakage in S. simulans membranes, even at significantly higher concentrations than the MIC (200 nM). Nisin is shown as positive control (MIC towards S. simulans is 75 nM). (A,C) The DiSC2(5) probe becomes fluorescent upon loss of membrane potential22. (B) SYTOX green becomes fluorescent upon binding to dsDNA23 inside bacteria. D-J) All assays were conducted in DOPC vesicles unless specified otherwise. Lipid II concentration is expressed as mol% of total lipid. Experimental details can be found in the methods section. Unless indicated otherwise plectasin was added at 30 seconds and 0.1 v% (final volume) Triton-X100 at 150 seconds. D) Titration of Lipid II concentration. E) same as D) but with Ca2+. F) same as A) but with EDTA. Increasing Lipid II concentration in the membrane promotes permeabilization by plectasin. At high calcium concentrations even membranes with low (and physiologically relevant) Lipid II concentrations are permeabilized. In contrast, in the absence of all calcium (with EDTA) modest leakage could be detected only at the highest Lipid II concentration. G) Vesicles pretreated with EDTA or EGTA were exposed to plectasin. After incubation for 120 seconds CaCl2 or MgCl2 was added. Bivalent cations are essential for plectasin-induced membrane permeabilization. Notably, MgCl2 induced leakage is starkly lower than for CaCl2, irrespective of the use of EGTA or EDTA, hinting at a differential effect of these bivalent cations on the mode of action of plectasin. H) Titration of plectasin to Lipid II vesicles. i, Percentage leakage as a function of plectasin concentration derived from the final leakage % of H). Leakage increases with plectasin concentration until all Lipid II on the outside of the vesicles is occupied (half of the total Lipid II in the sample, as depicted by the dashed line). Leakage thus depends on the formation of a 1:1 Lipid II-plectasin complex. J) The effect of the membrane composition on leakage. While DOPC Lipid II vesicles are efficiently permeabilized by plectasin in the presence of calcium, virtually no leakage is observed when phospholipids native to gram-positive bacteria are included in the membrane. Source data are provided for all graphs A-J).
