Selective toxicity of antimicrobial peptide S-thanatin on bacteria.

S-thanatin, an analog of thanatin, was synthesized by substituting the 15th amino acid of threonine with serine, which showed a broad antimicrobial activity against bacteria. We reported earlier that membrane phospholipid was found to be the target for S-thanatin with different mechanism from other antimicrobial peptides. In this study, we have performed its structural characterization by circular dichroism (CD) spectroscopy. The CD analysis showed that S-thanatin retained its overall conformation beta-sheet in aqueous buffer, beta-turn in 50% trifluoroethanol (TFE) and beta-hairpin in 0.4 mM POPC-LUVs. In hemolysis assay, S-thanatin exhibited low hemolytic activity and bacteria selectivity. We investigated the effect of the presence of 33 mol percent cholesterol on the interactions of the antimicrobial peptide S-thanatin with phosphatidylcholine (PC) model membrane systems. The results showed that S-thanatin was more potent at disrupting cholesterol-free bacterial than cholesterol-containing eukaryotic membranes. Thus, in all respects, fluorescence dye leakage experiments indicated that cholesterol inhibited the S-thanatin-induced permeabilization of PC vesicles. Finally, flow cytometry was used to monitor changes in bacterial cell membrane potential and cell membrane integrity, with specific fluorescent dyes DiBAC(4)(3) and PI. Adding the respiratory poison CCCP seemed to prevent peptide-induced membrane damage, which suggested that S-thanatin acted at the metabolic level on respiratory chain. These findings might explain why S-thanatin was selective toxicity towards bacteria, but low toxicity towards erythrocytes. It might be due to three factors at least: electrostatic interaction (namely anionic phospholipids); cholesterol; respiratory chain. Copyright 2010 Elsevier Inc. All rights reserved.