Figure 1. hRegIIIα permeabilizes the bacterial membrane.

(a) Listeria monocytogeneswas treated with 25 μM hRegIIIα, pro-hRegIIIα, or BSA or left untreated, and bacterial uptake of SYTOX Green was measured. Results are representative of three independent experiments, and are expressed as a percentage of maximum SYTOX uptake in the presence of 0.2% SDS.

(b) SYTOX Green uptake by L. monocytogenes in the presence of increasing hRegIIIα concentrations. Assays were performed in triplicate. Means±SEM are plotted.

(c) Carboxyfluorescein (CF)-loaded liposomes (10 μM lipid; 85% PC/15% PS) were treated with 1 μM hRegIIIα. 1.0% octylglucoside (OG) was added towards the end to disrupt remaining liposomes. Dye efflux is expressed as percentage of maximal release by detergent. Results are representative of five independent experiments.

(d) 10 μM hRegIIIα was added to CF-loaded liposomes (100 μM lipid;100% PC, 100% PS or 85% PC:15% PS), and dyeefflux was monitored over time. Representative results are shown.

(e) Averaged results from three independent replicates of the experiment shown in (d). ns, not significant; **, p<0.01; ***, p<0.001.

(f) Initial rate of liposome dye efflux (100 μM lipid) as a function of hRegIIIα and pro-hRegIIIα concentration. Results are representative of three independent experiments. *, p<0.05; **, p<0.01.

(g) Intrinsic Trp fluorescence of 1 μM hRegIIIα was measured in the presence of increasing lipid concentrations.

(h) Trp fluorescence of 1 μM hRegIIIα and pro-hRegIIIα as a function of lipid concentration.

(i) Intrinsic Trp fluorescence of 1 μM hRegIIIα was measured in the presence of liposomes (100 μM lipid) of varying lipid composition.

(j) 5.0 μM hRegIIIα or pro-hRegIIIα was added to liposomes (100 μM lipid) incorporating 5% dansyl-PE and dansyl fluorescence was monitored. Assays were performed in triplicate.

(k) FRET efficiency as a function of hRegIIIα and pro-hRegIIIα concentration. Assays were performed in triplicate. Means±SEM are plotted.