tritrpticin and indolicidin

Drug-resistant microorganisms ('superbugs') present a serious challenge to the success of antimicrobial treatments. Subsequently, there is a crucial need for novel bio-control agents. Many antimicrobial peptides (AMPs) show a broad-spectrum activity against bacteria, fungi or viruses and are strong candidates to complement or substitute current antimicrobial agents. Some AMPs are also effective against protozoa or cancer cells. The tryptophan (Trp)-rich peptides (TRPs) are a subset of AMPs that display potent antimicrobial activity, credited to the unique biochemical properties of tryptophan that allow it to insert into biological membranes. Further, many Trp-rich AMPs cross bacterial membranes without compromising their integrity and act intracellularly, suggesting interactions with nucleic acids and enzymes. In this work, we overview some archetypal TRPs derived from natural sources, i.e., indolicidin, tritrpticin and lactoferricin, summarising their biochemical properties, structures, antimicrobial activities, mechanistic studies and potential applications.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Drug Resistance, Microbial; Humans; Lactoferrin; Models, Molecular; Oligopeptides; Tryptophan

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, a member of the cathelicidin family, is a Trp-rich or Pro/Arg-rich peptide. Since the Trp, Pro and Arg residues are important in membrane disruption and/or cell entry, tritrpticin is a particularly attractive template around which to design novel antimicrobial peptides. Although tritrpticin is effective against a broad spectrum of microorganisms, it also has relatively strong haemolytic activity, which may compromise its therapeutic effects. To identify antimicrobial analogues of tritrpticin that lack cytotoxicity, we have designed and synthesised several molecules based on the amphipathic turn structure of tritrpticin. C-terminal amidation of tritrpticin enhanced its antimicrobial activity, comparable with indolicidin, another Trp-rich peptide. In contrast, the additional insertion of positively-charged amino acids resulted in only small variations in antibiotic activity, suggesting that a total of five positive charges is sufficient for high antimicrobial activity. We found that perfectly symmetric analogues of tritrpticin with C-terminal amidation showed two- to eight-fold improved antimicrobial activity compared with tritrpticin, as well as significantly reduced haemolytic activity. This reduction in cytotoxicity was correlated with decreased permeabilization of the zwitterionic phosphatidylcholine membrane, the major component of the outer leaflet of red blood cells. In addition, we designed a symmetric indolicidin analogue that possessed antimicrobial potency and selectivity. Moreover, we found that these analogues of tritrpticin and indolicidin were effective against several antibiotic-resistant clinical bacterial isolates. Circular dichroism spectroscopy suggested that the structure of these symmetric analogues resembled that of tritrpticin or indolicidin in a membrane mimetic environment. Overall, our findings suggest that these symmetric peptides with an amphipathic turn structure may serve as useful templates for pharmaceutical compounds that may be effective against increasingly antibiotic-resistant microbes.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteria; Bacterial Infections; Cell Membrane Permeability; Circular Dichroism; Drug Design; Drug Resistance, Bacterial; Erythrocytes; Fluorescent Dyes; Hemolysis; Liposomes; Microbial Sensitivity Tests; Oligopeptides

Structural changes for a series of antimicrobial peptides in various solvents were investigated by a combined approach of FTIR and CD spectroscopy. The well-characterized and potent antimicrobial peptides indolicidin and tritrpticin were studied along with several analogs of tritrpticin, including Tritrp1 (amidated analog of tritrpticin), Tritrp2 (analog of Tritrp1 with Arg-->Lys substitutions), Tritrp3 (analog of Tritrp1 with Pro-->Ala substitutions) and Tritrp4 (analog of Tritrp1 with Trp-->Tyr substitutions). All peptides were studied in aqueous buffer, ethanol and in the presence of dodecylphosphocholine (DPC) micelles. It was shown that tritrpticin and its analogs preferentially adopt turn structures in all solvents studied. The turn structures formed by the tritrpticin analogs bound to DPC micelles are more compact and more conformationally restricted compared to indolicidin. While several peptides showed a slight propensity for an alpha-helical conformation in ethanol, this trend was only strong for Tritrp3, which also adopted a largely alpha-helical structure with DPC micelles. Tritrp3 also demonstrated along with Tritrp1 the highest ability to interact with DPC micelles, while Tritrp2 and Tritrp4 showed the weakest interaction.

Topics: Amino Acid Sequence; Amino Acid Substitution; Antimicrobial Cationic Peptides; Buffers; Circular Dichroism; Dimethyl Sulfoxide; Ethanol; Micelles; Oligopeptides; Phosphorylcholine; Solvents; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared; Trifluoroethanol

The interaction of several tryptophan (Trp)-rich cationic antimicrobial peptides with membranes was investigated. These peptides included tritrpticin, indolicidin, lactoferricin B (Lfcin B), and a shorter fragment of lactoferricin (LfcinB4-9). The average environment of the Trp residues of these peptides was assessed from their fluorescence properties, both the wavelength of maximal emission as well as the red edge effect. The insertion of the peptides into vesicles of differing composition was examined using quenching of the Trp fluorescence, with both soluble acrylamide and nitroxide-labelled phospholipids as well as by chemical modification of the Trp residues with N-bromosuccinimide. The results were consistent with the Trp side chains positioned mostly near the membrane-water interface. The extent of burial of the Trp side chains appears to be greater in vesicles containing phospholipids with the anionic phosphatidylglycerol headgroup. Leakage of the aqueous contents of liposomes was also measured using the 8-aminonaphthalene-1,3,6-trisulfonic acid--p-xylene-bis-pyridinium bromide assay. Tritrpticin, which demonstrated the greatest red edge shift, also displayed the largest amount of leakage from liposomes. Taken together, the results illustrate that cationic Trp-rich antimicrobial peptides preferentially disrupt large unilamellar vesicles with a net negative charge following their insertion into the interfacial region of the phospholipid bilayer.

Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Kinetics; Lactoferrin; Lipid Bilayers; Liposomes; Membrane Lipids; Oligopeptides; Pyridinium Compounds; Spectrometry, Fluorescence; Tryptophan