carbocyanines and 1-palmitoyl-2-oleoylglycero-3-phosphoglycerol

Naturally occurring antimicrobial peptides important for innate immunity are widely studied for their antimicrobial and anticancer activity. The primary target of these AMPs is believed to be the bacterial cytoplasmic membrane. However, the interaction between cytoplasmic membrane and the antimicrobial peptides remains poorly understood. Therefore to focus on the target membrane composition that is required by AMPs to interact with membranes, we have examined the interaction of the antimicrobial and anticancer active 11-residue GA-K4 (FLKWLFKWAKK) peptide with model and intact cell membranes. Effect on the structural conformational properties of GA-K4 peptide was investigated by means of far-UV CD and fluorescence spectroscopic methods. The different conformation of GA-K4 peptide in large unilamellar vesicles (LUV) bilayer and micelle environment suggest that the curvature has an influence on the secondary structure acquired by the peptide. Furthermore, the leakage experiment result confirmed that GA-K4 induced the leakage of cytoplasmic membrane in Staphylococcus аureus bacterial cells. Fluorescence data revealed the interfacial location of GA-K4 peptide in the model membranes. The blue-shift in emission wavelength by tryptophan residues in fluorescence data indicated the penetration of GA-K4 peptide in micelles and phospholipid bilayers. These results showed that the GA-K4 peptide is a membrane-active peptide and its activity depends on membrane curvature and lipid composition. Although further studies are required to confirm the mechanism of action, the data suggest mechanism of toroidal pore formation for the interaction of GA-K4 peptide with membranes. Our studies will be helpful in better understanding of the membrane requirment of peptides to express their therapeutic effects.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Antineoplastic Agents; Benzothiazoles; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Fluorescent Dyes; Kinetics; Lipid Bilayers; Lysophosphatidylcholines; Micelles; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Structure, Secondary; Spectrometry, Fluorescence; Staphylococcus aureus; Unilamellar Liposomes

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an alpha-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The (31)P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC(3)5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Anura; Benzothiazoles; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Dimyristoylphosphatidylcholine; Fluoresceins; Gramicidin; Lipid Bilayers; Membrane Potentials; Phosphatidylcholines; Phosphatidylglycerols; Protein Structure, Secondary; Staphylococcus aureus

The cathelicidin-derived antimicrobial tritrpticin could be classified as either Trp-rich or Pro/Arg-rich peptide. We recently found that the sequence modification of tritrpticin focused on Trp and Pro residues led to considerable change in structure and antimicrobial potency and selectivity, but their mechanisms of microbial killing action were still unclear. Here, to better understand the bactericidal mechanisms of tritrpticin and its two analogs, TPA and TWF, we studied their effect on the viability of Gram-positive S. aureus and Gram-negative E. coli in relation to their membrane depolarization. Although TWF more effectively inhibited growth of S. aureus and E. coli than TPA, only a 30 min exposure to TPA was sufficient to kill both bacteria and TWF required a lag period of about 3-6 h for bactericidal activity. Their different bactericidal kinetics was associated with membrane permeabilization, i.e., TWF showed negligible ability to depolarize the cytoplasmic membrane potential of target cell membrane, whereas we observed significant membrane depolarization for TPA. In addition, while TPA caused rapid and large dye leakage from negatively charged model vesicles, TWF showed very little membrane-disrupting activity. Interestingly, we have looked for a synergism among the three peptides against E. coli, supporting that they are working with different modes of action. Collectively, our results suggest that TPA disrupts the ion gradients across the membrane, causing depolarization and a loss of microbial viability. By contrast, TWF more likely translocates across the cytoplasmic membrane without depolarization and then acts against one or more intracellular targets. Tritrpticin exhibits intermediate properties and appears to act via membrane depolarization coupled to secondary intracellular targeting.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Carbocyanines; Cathelicidins; Cell Membrane; Drug Synergism; Escherichia coli; Membranes, Artificial; Microbial Sensitivity Tests; Oligopeptides; Phosphatidylcholines; Phosphatidylglycerols; Staphylococcus aureus