Mechanism of Action and Therapeutic Potential of the ß-Hairpin Antimicrobial Peptide Capitellacin from the Marine Polychaeta Capitella teleta.

Among the most potent and proteolytically resistant antimicrobial peptides (AMPs) of animal origin are molecules forming a ß-hairpin structure stabilized by disulfide bonds. In this study, we investigated the mechanism of action and therapeutic potential of the ß-hairpin AMP from the marine polychaeta Capitella teleta, named capitellacin. The peptide exhibits a low cytotoxicity toward mammalian cells and a pronounced activity against a wide range of bacterial pathogens including multi-resistant bacteria, but the mechanism of its antibacterial action is still obscure. In view of this, we obtained analogs of capitellacin and tachyplesin-inspired chimeric variants to identify amino acid residues important for biological activities. A low hydrophobicity of the ß-turn region in capitellacin determines its modest membranotropic activity and slow membrane permeabilization. Electrochemical measurements in planar lipid bilayers mimicking the E. coli membrane were consistent with the detergent-like mechanism of action rather than with binding to a specific molecular target in the cell. The peptide did not induce bacterial resistance after a 21-day selection experiment, which also pointed at a membranotropic mechanism of action. We also found that capitellacin can both prevent E. coli biofilm formation and destroy preformed mature biofilms. The marked antibacterial and antibiofilm activity of capitellacin along with its moderate adverse effects on mammalian cells make this peptide a promising scaffold for the development of drugs for the treatment of chronic E. coli infections, in particular those caused by the formation of biofilms.

Dodecapeptide Cathelicidins of Cetartiodactyla: Structure, Mechanism of Antimicrobial Action, and Synergistic Interaction With Other Cathelicidins.

In this study, dodecapeptide cathelicidins were shown to be widespread antimicrobial peptides among the Cetruminantia clade. In particular, we investigated the dodecapeptide from the domestic goat Capra hircus, designated as ChDode and its unique ortholog from the sperm whale Physeter catodon (PcDode). ChDode contains two cysteine residues, while PcDode consists of two dodecapeptide building blocks and contains four cysteine residues. The recombinant analogs of the peptides were obtained by heterologous expression in Escherichia coli cells. The structures of the peptides were studied by circular dichroism (CD), FTIR, and NMR spectroscopy. It was demonstrated that PcDode adopts a ß-hairpin structure in water and resembles ß-hairpin antimicrobial peptides, while ChDode forms a ß-structural antiparallel covalent dimer, stabilized by two intermonomer disulfide bonds. Both peptides reveal a significant right-handed twist about 200 degrees per 8 residues. In DPC micelles ChDode forms flat ß-structural tetramers by antiparallel non-covalent association of the dimers. The tetramers incorporate into the micelles in transmembrane orientation. Incorporation into the micelles and dimerization significantly diminished the amplitude of backbone motions of ChDode at the picosecond-nanosecond timescale. When interacting with negatively charged membranes containing phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), the ChDode peptide adopted similar oligomeric structure and was capable to form ion-conducting pores without membrane lysis. Despite modest antibacterial activity of ChDode, a considerable synergistic effect of this peptide in combination with another goat cathelicidin - the α-helical peptide ChMAP-28 was observed. This effect is based on an increase in permeability of bacterial membranes. In turn, this mechanism can lead to an increase in the efficiency of the combined action of the synergistic pair ChMAP-28 with the Pro-rich peptide mini-ChBac7.5Nα targeting the bacterial ribosome.

Marine antimicrobial peptide arenicin adopts a monomeric twisted ß-hairpin structure and forms low conductivity pores in zwitterionic lipid bilayers.

Arenicins are 21-residue ß-hairpin antimicrobial peptides (AMPs) isolated from the marine lugworm Arenicola marina [Ovchinnikova et al., FEBS Lett. 2004;577:209-214]. The peptides have a high positive charge (+6) and display a broad spectrum of antimicrobial activities against bacteria and fungi. Arenicins adopt the monomeric highly twisted ß-hairpin in water or planar ß-structural dimers in anionic liposomes and detergent micelles. Until now, the interaction of cationic ß-structural AMPs with zwitterionic phospholipid bilayers mimicking eukaryotic membranes is not well understood. To study the structural basis of arenicins activity against eukaryotic cells, we investigated arenicin-2 in the solvents of low polarity (ethanol, 4% dioxane) and in zwitterionic soybean PC and PC/PE liposomes by CD and FTIR spectroscopy. It was shown that arenicin-2 adopted the twisted ß-hairpin structure in all the environments studied. Measurements of the Trp fluorescence and H→D exchange in soybean PC liposomes and boundary potential in the planar DPhPC bilayers confirmed the partitioning of the arenicin-2 monomers into interfacial region of the zwitterionic membranes. The low-conductivity (0.12 nS) arenicin-2 pores were detected in the DPhPC bilayers. The lifetime of the open state (up to 260 ms) was significantly longer than lifetime of low-conductivity (0.23 nS) pores previously described in partially anionic membranes (44 ms). The formation of narrow arenicin-2 pores without disruption of the membrane was discussed in the light of the disordered toroidal pore model previously proposed for ß-structural AMPs [Jean - Francois et al. Biophys. J. 2008;95:5748 - 5756]. A novel non-lytic mechanism of the arenicin-2 action was proposed.

Ligand Binding Properties of the Lentil Lipid Transfer Protein: Molecular Insight into the Possible Mechanism of Lipid Uptake.

The lentil lipid transfer protein, designated as Lc-LTP2, was isolated from Lens culinaris seeds. The protein belongs to the LTP1 subfamily and consists of 93 amino acid residues. Its spatial structure includes four α-helices (H1-H4) and a long C-terminal tail. Here, we report the ligand binding properties of Lc-LTP2. The fluorescent 2-p-toluidinonaphthalene-6-sulfonate binding assay revealed that the affinity of Lc-LTP2 for saturated and unsaturated fatty acids was enhanced with a decrease in acyl-chain length. Measurements of boundary potential in planar lipid bilayers and calcein dye leakage in vesicular systems revealed preferential interaction of Lc-LTP2 with the negatively charged membranes. Lc-LTP2 more efficiently transferred anionic dimyristoylphosphatidylglycerol (DMPG) than zwitterionic dimyristoylphosphatidylcholine. Nuclear magnetic resonance experiments confirmed the higher affinity of Lc-LTP2 for anionic lipids and those with smaller volumes of hydrophobic chains. The acyl chains of the bound lysopalmitoylphosphatidylglycerol (LPPG), DMPG, or dihexanoylphosphatidylcholine molecules occupied the internal hydrophobic cavity, while their headgroups protruded into the aqueous environment between helices H1 and H3. The spatial structure and backbone dynamics of the Lc-LTP2-LPPG complex were determined. The internal cavity was expanded from ∼600 to ∼1000 Å3 upon the ligand binding. Another entrance into the internal cavity, restricted by the H2-H3 interhelical loop and C-terminal tail, appeared to be responsible for the attachment of Lc-LTP2 to the membrane or micelle surface and probably played an important role in the lipid uptake determining the ligand specificity. Our results confirmed the previous assumption regarding the membrane-mediated antimicrobial action of Lc-LTP2 and afforded molecular insight into its biological role in the plant.

Effective lipid-detergent system for study of membrane active peptides in fluid liposomes.

The structure of peptide antibiotic gramicidin A (gA) was studied in phosphatidylcholin liposomes modified by nonionic detergent Triton X-100. First, the detergent : lipid ratio at which the saturation of lipid membrane by Triton X-100 occurs (Re (sat)), was determined by light scattering. Measurements of steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene at sublytic concentrations of detergent showed that after saturation of the membrane by Triton X-100 microviscosity of lipid bilayer is reduced by 20%. The equilibrium conformational state of gA in phosphatidylcholine liposomes at Re (sat) was studied by CD spectroscopy. It was found that the conformational state of this channel-forming peptide changed crucially when Triton X-100 induced transition to more fluid membranes. The gA single-channel measurements were made with Triton X-100 containing bilayers. Tentative assignment of the channel type and gA structures was made by correlation of CD data with conductance histograms. Lipid-detergent system with variable viscosity developed in this work can be used to study the structure and folding of other membrane-active peptides.

Molecular mechanism of action of ß-hairpin antimicrobial peptide arenicin: oligomeric structure in dodecylphosphocholine micelles and pore formation in planar lipid bilayers.

The membrane-active, cationic, ß-hairpin peptide, arenicin, isolated from marine polychaeta Arenicola marina exhibits a broad spectrum of antimicrobial activity. The peptide in aqueous solution adopts the significantly twisted ß-hairpin conformation without pronounced amphipathicity. To assess the mechanism of arenicin action, the spatial structure and backbone dynamics of the peptide in membrane-mimicking media and its pore-forming activity in planar lipid bilayers were studied. The spatial structure of the asymmetric arenicin dimer stabilized by parallel association of N-terminal strands of two ß-hairpins was determined using triple-resonance nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles. Interaction of arenicin with micelles and its oligomerization significantly decreased the right-handed twist of the ß-hairpin, increased its amphipathicity, and led to stabilization of the peptide backbone on a picosecond to nanosecond time scale. Relaxation enhancement induced by water-soluble (Mn(2+)) and lipid-soluble (16-doxylstearate) paramagnetic probes pointed to the dimer transmembrane arrangement. Qualitative NMR and circular dichroism study of arenicin-2 in mixed DPC/1,2-dioleoyl-sn-glycero-3-phosphoglycerol bicelles, sodium dodecyl sulfate micelles, and lipid vesicles confirmed that a similar dimeric assembly of the peptide was retained in membrane-mimicking systems containing negatively charged lipids and detergents. Arenicin-induced conductance was dependent on the lipid composition of the membrane. Arenicin low-conductivity pores were detected in the phosphatidylethanolamine-containing lipid mixture, whereas the high-conductivity pores were observed in an exclusively anionic lipid system. The measured conductivity levels agreed with the model in which arenicin antimicrobial activity was mediated by the formation of toroidal pores assembled of two, three, or four ß-structural peptide dimers and lipid molecules. The structural transitions involved in arenicin membrane-disruptive action are discussed.

Physicochemical aspects of the liposome-wool interaction in wool dyeing.

Despite the promising application of liposomes in wool dyeing, little is known about the mechanism of liposome interactions with the wool fiber and dyestuffs. The kinetics of wool dyeing by two dyes, Acid Green 27 (hydrophobic) and Acid Green 25 (hydrophilic), were compared in three experimental protocols: (1) without liposomes, (2) in the presence of phosphatidylcholine (PC) liposomes, and (3) with wool previously treated with PC liposomes. Physicochemical interactions of liposomes with wool fibers were studied under experimental dyeing conditions with particular interest in the liposome affinity to the fiber surface and changes in the lipid composition of the wool fibers. The results obtained indicate that the presence of liposomes favors the retention of these two dyes in the dyeing bath, this effect being more pronounced in case of the hydrophobic dye. Furthermore, the liposome treatment is accompanied by substantial absorption of PC by wool fibers with simultaneous partial solubilization of their polar lipids (more evident at higher temperatures). This may result in structural modification of the cell membrane complex of wool fibers, which could account for a high level of the dye exhaustion observed at the end of the liposome dyeing process.