Elementary processes of antimicrobial peptide PGLa-induced pore formation in lipid bilayers

Antimicrobial peptide PGLa induces the leakage of intracellular content, leading to its bactericidal activity. However, the elementary process of PGLa-induced leakage remains poorly understood. Here, we examined the interaction of PGLa with lipid bilayers using the single giant unilamellar vesicle (GUV) method. We found that PGLa induced membrane permeation of calcein from GUVs comprised of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) and its rate increased with time to reach a steady value, indicating that PGLa induced pores in the bilayer. The binding of PGLa to the GUV membrane raised its fractional area change, delta. At high PGLa concentrations, the time course of delta showed a two-step increase; delta increased to a value, delta(1), which was constant for an extended period before increasing to another constant value, delta(2), that persisted until aspiration of the GUV. To reveal the distribution of PGLa, we investigated the interaction of a mixture of PGLa and carboxyfluorescein (CF)-labeled PGLa (CF-PGLa) with single GUVs. The change of the fluorescence intensity of the GUV rim, I, over time showed a two-step increase from a steady value, I-1, to another, I-2, concomitant with the entering of CF-PGLa into the lumen of the GUV prior to AF647 leakage. The simultaneous measurement of delta and I indicated that their time courses were virtually the same and the ratios (delta(2)/delta(1) and I-2/I-1) were almost 2. These results indicated that CF-PGLa translocated across the bilayer before membrane permeation. Based on these results, the elementary processes of the PGLa-induced pore formation were discussed.