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.
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