Effects of Pro → Peptoid Residue Substitution on Cell Selectivity and Mechanism of Antibacterial Action of Tritrpticin-Amide Antimicrobial Peptide

Abstract

To investigate the effect of Pro --> peptoid residue substitution on cell selectivity and the mechanism of antibacterial action of Pro-containing beta-turn antimicrobial peptides, we synthesized tritrpticin-amide (TP, VRRFPWWWPFLRR-NH(2)) and its peptoid residue-substituted peptides in which two Pro residues at positions 5 and 9 are replaced with Nleu (Leu peptoid residue), Nphe (Phe peptoid residue), or Nlys (Lys peptoid residue). Peptides with Pro --> Nphe (TPf) or Pro --> Nleu substitution (TPl) retained antibacterial activity but had significantly higher toxicity to mammalian cells. In contrast, Pro --> Nlys substitution (TPk) increased the antibacterial activity but decreased the toxicity to mammalian cells. Tryptophan fluorescence studies indicated that the bacterial cell selectivity of TPk is closely correlated with a preferential interaction with negatively charged phospholipids. Interestingly, TPk was much less effective at depolarizing of the membrane potential of Staphylococus aureus and Escherichia coli spheroplasts and causing the leakage of a fluorescent dye entrapped within negatively charged vesicles. Furthermore, confocal laser-scanning microscopy showed that TPk effectively penetrated the membrane of both E. coli and S. aureus and accumulated in the cytoplasm, whereas TP and TPf did not penetrate the cell membrane but remained outside or on the cell membrane. These results suggest that the bactericidal action of TPk is due to inhibition of the intracellular components after penetration of the bacterial cell membrane. In addition, TPK with Lys substitution effectively depolarized the membrane potential of S. aureus and E. coli spheroplasts. TPK induced rapid and effective dye leakage from bacterial membrane-mimicking liposomes and did not penetrate the bacterial cell membranes. These results suggested that the ability of TPk to penetrate the bacterial cell membranes appears to involve the dual effects that are related to the increase in the positive charge and the peptide's backbone change by peptoid residue substitution. Collectively, our results showed that Pro --> Nlys substitution in Pro-containing beta-turn antimicrobial peptides is a promising strategy for the design of new short bacterial cell-selective antimicrobial peptides with intracellular mechanisms of action.