tritrpticin and fluorexon

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an alpha-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Calorimetry; Cathelicidins; Escherichia coli; Fluoresceins; Kinetics; Molecular Sequence Data; Oligopeptides; Protein Binding; Thermodynamics; Tryptophan; Unilamellar Liposomes

Tritrpticin is a member of the cathelicidin family of antimicrobial peptides. Starting from its native sequence (VRRFPWWWPFLRR), eight synthetic peptide analogs were studied to investigate the roles of specific residues in its biological and structural properties. This included amidation of the C-terminus paired with substitutions of its cationic and Phe residues, as well as the Pro residues that are important for its two-turn micelle-bound structure. These analogs were determined to have a significant antimicrobial potency. In contrast, two other peptide analogs, those with the three Trp residues substituted with either Phe or Tyr residues are not highly membrane perturbing, as determined by leakage and flip-flop assays using fluorescence spectroscopy. Nevertheless the Phe analog has a high activity; this suggests an intracellular mechanism for antimicrobial activity that may be part of the overall mechanism of action of native tritrpticin as a complement to membrane perturbation. NMR experiments of these two Trp-substituted peptides showed the presence of multiple conformers. The structures of the six remaining Trp-containing analogs bound to dodecylphosphocholine micelles showed major, well-defined conformations. These peptides are membrane disruptive and show a wide range in hemolytic activity. Their micelle-bound structures either retain the typical turn-turn structure of native tritrpticin or have an extended alpha-helix. This work demonstrates that closely related antimicrobial peptides can often have remarkably altered properties with complex influences on their biological activities.

Topics: Amino Acid Substitution; Anti-Infective Agents; Escherichia coli; Fluoresceins; Hemolysis; Humans; In Vitro Techniques; Liposomes; Magnetic Resonance Spectroscopy; Micelles; Microbial Sensitivity Tests; Models, Molecular; Oligopeptides; Phosphorylcholine; Staphylococcus aureus; Structure-Activity Relationship

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.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Anti-Bacterial Agents; Bacteria; Cell Membrane; Cells, Cultured; Circular Dichroism; Dose-Response Relationship, Drug; Erythrocytes; Fluoresceins; HeLa Cells; Hemolysis; Humans; Mice; Microbial Sensitivity Tests; Microscopy, Confocal; NIH 3T3 Cells; Oligopeptides; Peptides; Peptoids; Plasmids; Proline; Protein Binding

Tritrpticin, a Trp-rich cationic antimicrobial peptide with a unique amino acid sequence (VRRFPWWWPFLRR), is found in porcine cathelicidin cDNA. Tritrpticin has a broad spectrum of antibacterial and antifungal activities and hemolytic activity comparable to that of indolicidin. To investigate the mechanism of the bacterial killing action of tritrpticin and to identify structural features important for bacterial cell selectivity, we designed several tritrpticin analogs with amino acid substitutions of the Pro and Trp residues. Circular dichroism studies revealed that the substitution of Pro-->Ala (TPA) or Trp-->Phe (TWF) leads to significant conformational changes in SDS micelles, converting the beta-turn to alpha-helix or to poly-L-proline II helix, respectively. Compared to tritrpticin, TPA retained most of its antimicrobial activity, but showed enhanced hemolytic and membrane-disrupting activities. In contrast, TWF showed a 2-4-fold increase in antimicrobial activity against Gram-negative bacteria, but a marked decrease in both hemolytic and membrane-disrupting activities. Taken together, our findings suggest that compared with the beta-turn and alpha-helical structures, the poly-L-proline II helix is crucial for effective bacterial cell selectivity in tritrpticin and its analogs.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteria; Cathelicidins; Circular Dichroism; Dose-Response Relationship, Drug; Fluoresceins; Fluorescent Dyes; Fungi; Hemolysin Proteins; Liposomes; Models, Molecular; Oligopeptides; Protein Conformation; Protein Structure, Secondary