Indolicidin action on membrane permeability: carrier mechanism versus pore formation

Indolicidin, a 13-residue cationic peptide with extremely high tryptophan content, exhibits broad-spectrum antimicrobial as well as hemolytic activity. To gain insight into the mechanism of indolicidin action on membrane permeability, liposome leakage induced by this peptide was studied by using various probes with vesicles of different lipid compositions. In liposomes containing negatively charged lipids, indolicidin induced rather unselective permeabilization. By contrast, the peptide appeared to be selective in provoking leakage of neutral, egg phosphatidylcholine (PC) liposomes: it effectively induced the release of negatively charged fluorescent dyes, carboxyfluorescein (CF), calcein and sulforhodamine B, but was unable to induce the leakage of a neutral compound, glucose, and that of positively charged doxorubicin. Moreover, organic anions, such as fatty acids, were found to suppress the indolicidin-induced CF leakage of egg PC liposomes. Based on these results, we concluded that indolicidin facilitates the dye release from uncharged lipid vesicles not by formation of membrane pores as it is generally accepted for the majority of antimicrobial peptides but rather via translocation of dye molecules across the membrane in the form of dye-peptide complexes, i.e. indolicidin operates as an organic anion carrier. This conclusion was supported by observing the formation of complexes between indolicidin and pyrenebutyrate in solution. The indolicidin analog having only one arginine was ineffective in pyrenebutyrate binding and CF transport. The mode of action proposed here for indolicidin can be related to that previously postulated for oligoarginine derivatives which are able to carry organic anions across liposomal and bulk phase membranes [Sakai N. & Matile S. J. Am. Chem. Soc. 2003, 125:14348-14356]. The newly identified mechanism of peptide ionophoric activity in uncharged lipid membranes may be involved in hemolytic action of indolicidin via induction of plasma membrane permeability for important anionic metabolites which disturbs regulation of osmotic balance ultimately leading to erythrocyte membrane rupture.