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Abstract
Gaegurin 4 (GGN4), an antimicrobial peptide isolated from a Korean frog, is five times more potent against Gram-positive than Gram-negative bacteria, but has little hemolytic activity. To understand the mechanism of such cell selectivity, we examined GGN4-induced K+ efflux from target cells, and membrane conductances in planar lipid bilayers. The K+ efflux from Gram-positive M. luteus (2.5 μg/ml) was faster and larger than that from Gram-negative E. coli (75 μg/ml), while that from RBC was negligible even at higher concentration (100 μg/ml). GGN4 induced larger conductances in the planar bilayers which were formed with lipids extracted from Gram-positive B. subtilis than in those from E. coli (p< 0.01), however, the effects of GGN4 were not selective in the bilayers formed with lipids from E. coli and red blood cells. Addition of an acidic phospholipid, phosphatidylserine to planar bilayers increased the GGN4-induced membrane conductance (p<0.05), but addition of phosphatidylcholine or cholesterol reduced it (p < 0.05). Transmission electron microscopy revealed that GGN4 induced pore-like damages in M. luteus and dis-layering damages on the outer wall of E. coli. Taken together, the present results indicate that the selectivity of GGN4 toward Gram-positive over Gram-negative bacteria is due to negative surface charges, and interaction of GGN4 with outer walls. The selectivity toward bacteria over RBC is due to the presence of phosphatidylcholine and cholesterol, and the trans-bilayer lipid asymmetry in RBC. The results suggest that design of selective antimicrobial peptides should be based on the composition and topology of membrane lipids in the target cells.
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Fig. 1.
GGN4-induced membrane currents. (A) Induction of membrane conductance by GGN4 (0.3 μg/ml). Current record shows the changes in membrane conductances induced by GGN4 in a lipid bilayer, composed of PE: PS (1: 1), under 200/0 (cis/trans) mM KCl gradient at 0 mV in the recording solution containing 10 mM HEPES-NMDG (pH 7.2). (B and C) Typical records showing unitary conductances of GGN4 (0.03 μg/ml)-induced pores at symmetrical 100 mM KCl in acidic (PE: PS=3: 7, B) and neutral lipid membranes (PE 100%, C) at 30 mV.
Fig. 2.
Effects of GGN4 on K+ efflux from bacteria and RBC in the solution containing 10 mM HEPES-NaOH (pH 7.0) and 0.15 M NaCl. (A-C) K+ efflux from M. luteus (A), E. coli (B), and RBC (1%, v/v) in the presence of GGN4 (C). (D) Time courses of K+ efflux (Δ) and cell viability (○) after application of GGN4 (12.5 μg/ml) to M. luteus suspension. The extent of K+ efflux was normalized to the total K+ efflux obtained after treatment with 0.5% Triton X-100.
Fig. 3.
Pore-forming activity of GGN 4 in the membranes formed with lipids extracted from G(+), Gram-negative bacteria and RBC in symmetric 25 mM KCl. (A) Current-voltage relation of GGN4-induced channels in the membranes formed with the extracted lipids in response to a ramp pulse (0.03 μg/ml in E. coli lipid membrane and 0.1 μg/ml in B. subtilis lipid membrane). (B) Mean slope conductances of the membranes formed with lipids extracted from B. subtilis, E. coli and RBC. Bars represent standard error of means. n=6.
Fig. 4.
Effects of addition of PC, PS and cholesterol to the lipid bilayers on GGN4-induced conductances. Slope conductances of the membrane at each GGN4 concentration were estimated from the current-voltage relations as shown in Fig. 3. Solid lines are drawn by the linear regression of double logarithmic concentration-conductance relations. Respective slopes of log space (conductance, pS) vs. log. (concentration, μg/ml) are 0.391, 0.546, 0.738 and 0.704 for PE, PE: PC, PE: PC: PS, and PE: PC: PS: CS membranes, respectively. PE, 100% phosphatidylethanolamine; PE: PC, 80% phosphatidylethanolamine and 20% phosphatidylcholine; PE: PC: PS, 80% phosphatidylethanolamine, 10% phosphatidylcholine and 10% phosphatidylserine; PE: PC: PS: CS, 50% phosphatidylethanolamine, 10% phosphatidylcholine, 10% phosphatidylserine, and 30% cholesterol. Symbols and bars represent the means and error bars of slope conductances, respectively, measured from 3~6 bilayers, except the data point at 0.3 μg/ml in PE: PC: PS: CS membrane, where n=2.
Fig. 5.
Transmission electron micrographs of M. luteus and E. coli treated with GGN4 at 37°C for 30 min. M. luteus and E. coli cells (108 cfu/ml) were incubated with 2.5 and 75 μg/ml GGN4, respectively. (A~C) Transmission electron micrographs of M. luteus untreated (A) and treated with GGN4 (B and C). Note the pores (marked by arrows) on the bacterial membranes (B). One of the pores shown in (B) was illustrated at higher magnification to show cytoplasm leaking out through the pore of a diameter of about 55 nm (marked by ∗, C). (D~F) Transmission electron micrographs of E. coli untreated (D) and treated with GGN4 (E and F). Note the layers of outer wall debris were peeled off from the damaged membranes (arrows) and a space developed between outer wall and inner membrane of E. coli at a damaged site (E, arrow). The bars represent 0.5 μm for (A), (B), (D) and (E), and 0.1 μm for (C) and (F).
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