tachyplesin-peptide--tachypleus-tridentatus and fluorexon

PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolk L-alpha-phosphatidylglycerol/egg yolk L-alpha-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Cell Death; Cell Membrane Permeability; CHO Cells; Circular Dichroism; Cricetinae; Cricetulus; DNA; DNA-Binding Proteins; Escherichia coli; Fluoresceins; Fluorescence; Microbial Sensitivity Tests; Models, Biological; Molecular Sequence Data; Peptides; Peptides, Cyclic; Polyethylene Glycols; Protein Binding; Protein Transport; Staphylococcus epidermidis; Tryptophan; Unilamellar Liposomes

Magainin 2 and tachyplesin I (T-SS) are membrane-permeabilizing antimicrobial peptides discovered from frog skin and horseshoe crab hemolymph, respectively. They are classified into different secondary structural classes, i.e., alpha-helix and cyclic beta-sheet, respectively. We found that F5W-magainin 2 (MG2) and T-SS exhibited marked synergistic effects against Gram-negative and Gram-positive bacteria without enhancing hemolytic activity as a measure of toxicity. Dye release experiments using liposomes suggested that the selective synergism is mainly due to anionic phospholipid-specific synergism in membrane permeabilization. Furthermore, the cyclic structure of T-SS was found to be necessary for synergism because a linear analogue of T-SS did not show good synergism with MG2. These novel observations suggested the possibility of the development of cocktail therapeutic regimens using combinations of antimicrobial peptides.

Topics: Adult; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Binding Sites; Cell Membrane; Circular Dichroism; DNA-Binding Proteins; Drug Synergism; Erythrocytes; Female; Fluoresceins; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Horseshoe Crabs; Humans; Magainins; Peptides, Cyclic; Phospholipids; Protein Conformation; Skin; Xenopus laevis; Xenopus Proteins

Tachyplesin I, isolated from the acid extracts of hemocytes of Tachypleus tridentatus, is a cyclic broad-spectrum antimicrobial peptide forming a rigid, antiparallel beta-sheet because of two intramolecular S-S linkages. The strong binding of the peptide to lipopolysaccharides cannot explain the susceptibilities of gram positive bacteria and fungi to the peptide. We found that tachyplesin I caused a rapid K+ efflux from Escherichia coli cells, concomitant with a reduced cell viability. This result suggests that the peptide-induced permeability enhancement of the bacterial membranes may be a plausible action mechanism. Thus, we studied the interactions of tachyplesin I with various large unilamellar vesicles (LUVs) to reveal the molecular machinery of the antimicrobial activity. Tachyplesin I induced the leakage of calcein, a trapped fluorescent marker, from LUVs of acidic phospholipids, especially phosphatidylglycerol (PG), but not from phosphatidylcholine LUVs. A detailed analysis found that the affinity of the peptide to the PG membranes is very strong and that the binding of one peptide molecule to approx. 200 lipid molecules leads to a significant leakage. The location of tachyplesin I in membranes was estimated by use of the Trp-2 fluorescence of the peptide. The presence of PG LUVs caused a blue shift of the maximum wavelength, an increase in the quantum yield, and a complete protection from fluorescence quenching by an aqueous quencher, acrylamide. Moreover, the degree of fluorescence quenching of the Trp residue by n-doxylstearates was in the order n = 5 greater than 7 greater than 12 approximately equal to 16. These results show that the Trp residue of tachyplesin I seems to locate in a hydrophobic environment near the surface of the PG bilayers.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Biological Transport; Cell Membrane; Cell Membrane Permeability; Cell Survival; DNA-Binding Proteins; Escherichia coli; Fluoresceins; Membrane Lipids; Molecular Sequence Data; Peptides; Peptides, Cyclic; Potassium; Spectrometry, Fluorescence