Koichiro Miyajima

Interactions of an antimicrobial peptide, magainin 2, with outer and inner membranes of Gram-negative bacteria

Magainin peptides, isolated from Xenopus skin, have broad spectra of antimicrobial activity and low toxicities to normal eukaryotic cells, thus being good candidates for therapeutic agents. The mechanism of action is considered to be the permeabilization of bacterial membranes. A number of studies using lipid vesicles have elucidated its molecular detail. However, their interactions with bacteria are not yet well understood. In this paper, we synthesized several magainin analogs with different charges (0 to +6) and hydrophobicities, and systematically studied their interactions with the outer and inner membranes of three species of Gram-negative bacteria (Escherichia coli, Acinetobacter calcoaceticus, Proteus vulgaris). The treatment of the E. coli cells with native magainin 2 (+4) immediately induced the efflux of the intracellular K+ ions and the cell death. A number of blebs were formed on the bacterial surface and the outer membrane became leaky. An increase in the peptides positive charge enhanced the outer membrane permeabilization and the bactericidal activity. The cationic peptides also effectively permeabilized the inner membranes rich in acidic phospholipids, indicating the importance of electrostatic interactions. Substitution of Trp for Phe simultaneously increased the bactericidal activity and the hemolytic activity. A strategy to develop potent antimicrobial peptides was discussed on the basis of these results.

Pore formation and translocation of melittin

Melittin, a bee venom, is a basic amphiphilic peptide, which mainly acts on the lipid matrix of membranes, lysing various cells. To elucidate the molecular mechanism, we investigated its interactions with phospholipid vesicles. The peptide formed a pore with a short lifetime in the membrane, as revealed by the release of an anionic fluorescent dye, calcein, from the liposomes. Our new double-labeling method clarified that the pore size increased with the peptide-to-lipid ratio. Upon the disintegration of the pore, a fraction of the peptides translocated across the bilayer. The pore formation was coupled with the translocation, which was proved by three fluorescence experiments recently developed by our laboratory. A novel model for the melittin pore formation was discussed in comparison with other pore-forming peptides.

Magainin 1-induced leakage of entrapped calcein out of negatively-charged lipid vesicles

Effects of magainin 1, a novel antimicrobial peptide, on the permeability of lipid vesicles were investigated by using calcein as a trapped fluorescent marker. Magainin 1 induces the leakage of calcein specifically out of negatively-charged vesicles. The peptide binds to bovine brain phosphatidylserine sonicated vesicles according to the Langmuir isotherm with a binding constant of 3.8.10(5) M-1 and a binding-site number of 0.10 per lipid molecule. The leakage seems to occur at a critical binding number of approx. 0.03 per lipid molecule. A circular dichroism study revealed that magainin 1 conforms mainly to an unordered structure both in an aqueous solution and in the presence of egg yolk phosphatidylcholine vesicles, whereas to an amphiphilic helix with the phosphatidylserine vesicles. In conclusion, magainin 1 interacts with acidic lipids through electrostatic interactions followed by hydrophobic interactions to form an amphiphilic helix, inducing the leakage.

Interactions of an antimicrobial peptide, magainin 2, with lipopolysaccharide-containing liposomes as a model for outer membranes of Gram-negative bacteria

F12W‐magainin 2 preferentially interacted with lipopolysaccharide‐containing bilayers, permeabilizing the membranes, compared with lipopolysaccharide‐free phosphatidylcholine vesicles. Using this system, we demonstrated for the first time that the magainin peptide forms a helix upon binding to lipopolysaccharide. Incorporation of lipid A into phosphatidylcholine liposomes also enhanced interactions with the peptide. The presence of Mg2+, which nullifies the peptides antibacterial activity against Gram‐negative bacteria, again weakened the interactions between the peptide and lipopolysaccharide‐doped bilayers. This system seems to be useful for investigating the molecular details of peptide‐lipopolysaccharide interactions.

Interactions of an antimicrobial peptide, tachyplesin I, with lipid membranes

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.

Role of saccharides for the freeze-thawing and freeze drying of liposome

Abstract For the preservation of liposomes, freeze-thawing and freeze-drying have been studied by various workers with saccharides (SA) and a freeze-dried liposome preparation is now commercially available. However, the mechanism of stabilizing action of SA in these processes, especially freeze-drying, is not yet fully understood. The interaction of egg yolk phosphatidylcholine (EPC: liquid crystaline state) and DPPC (gel state) liposomes with various glucose oligomers has been studied to elucidate the role of SA in these processes. The stability and property of freeze-dried products were determined by leakage of inner maker, liposomes size and thermal behavior. In the freeze-thawing process of EPC liposome, glucose and maltose are effectively cryoprotective, the large oligomers are not. In the freeze-drying of EPC liposome, glucose is ineffective and maltose is effective, however, oligomers with more than four glucose units are ineffective. As for the DPPC liposome, glucose is less effective and maltose is effective at every SA/PC molar ratio. The large oligomers with more than three glucose units are effective at low SA/PC molar ratio, however, become less effective at high SA/PC molar ratio. As for the DSC thermograms of freeze-dried products of DPPC and DMPC, the phase transition temperatures (Tc) at first scanning seem to be related residual water contents of dried product. At second scanning, Tc values move to certain low temperature almost independent of the kind of SA. The hydrogen bonding must be formed between SA molecule and PC molecule at first scanning. Based on these results, the role of SA will be discussed focusing on the hydrophilic and hydrophobic character of SA.

Correlation between the hydrophobic nature of monosaccharides and cholates, and their hydrophobic indices

Hydrophobic and hydrophilic surface areas, which are the surface areas occupied by CH and CH2 groups and by OH, COO– and ether oxygen groups, respectively, are calculated for monosaccharides and cholates using the computer program developed by Hermann. Hydrophobic indices, defined by the surface-area ratio of the hydrophobic to hydrophilic groups, correlate well with the partition coefficients of the polystyrene–water system for monosaccharides and with values of the critical micelle concentration for cholates. These facts indicate that the concept of hydrophobic index is important in considering hydrophobic interactions of flat molecules in aqueous solutions.

Effects of positive charge density on the liposomal surface on disposition kinetics of liposomes in rats

Abstract The effects of positive charge density on the liposomal surface on the disposition kinetics of liposomes in rats were investigated. The cationic liposomes with zeta potentials of about +15 mV remained in the blood longer than did the neutral liposomes, and the hepatic uptake of these liposomes decreased. The blood clearance of the liposomes with zeta potentials under +10 mV was comparable to that of the neutral liposomes. In contrast, the blood circulation of the liposomes with a higher positive charge density, above +25 mV, was shortened and their hepatic uptake was almost the same as that of the neutral liposomes. The optimum value of positive charge density on the liposomal surface to prolong the residency of liposomes in the blood circulation was thus determined. A liver perfusion experiment showed that the uptake of cationic liposomes with a zeta potential of about +15 mV was effectively suppressed in the presence of erythrocytes, while that of liposomes with a higher zeta potential were little affected. Thus, above +15 mV, the suppressive effect of erythrocytes on the hepatic uptake of cationic liposomes decreased with the increase of the positive charge on the liposomal surface. These results cannot be explained by the binding model, and we therefore propose the ionic atmosphere model: Cationic liposomes surround the erythrocytes with a negative surface charge like the ion atmosphere of the Debye-Huckel theory. The cationic liposomes with a suitable positive charge surround the erythrocytes as an ionic atmosphere and could then escape the reticuloendothelial system (RES). The higher positively charged liposomes were taken up by the liver probably due to the shield of the negative charge of erythrocytes provided by the cationic liposomal atmosphere.

Hypelcin A, an α-aminoisobutyric acid containing antibiotic peptide, induced fusion of egg yolk-l-α-phosphatidylcholine small unilamellar vesicles

Hypelcin A, an α-aminoisobutyric acid-containing antibiotic peptide inducing fusion of egg yolk-l-α-phosphatidylcholine (egg PC) small unilamellar vesicles (SUVs), was investigated by lipid-mixing assay based on resonanceenergy transfer between fluorescent probes, electron microscopy, light scattering, and1H-nuclear magnetic-resonance spectroscopy. At a high peptide-to-lipid ratio of approximately 1:5, the peptide fuses several SUVs of 20–30 nm in diameter into a 40–100 nm vesicle. Under mild conditions where the permeability enhancement (leakage of a trapped fluorescent dye, calcein) of lipid bilayers are observed (peptide to lipid ratios around 1/100), the fusion of the SUVs also occurs, although the fusion requires a somewhat larger amount of the peptide than the leakage does. Furthermore, at higher lipid concentrations, where the aggregation step is sufficiently rapid, the fusion rate is determined by the amount of the membrane bound peptide per lipid molecule, as is the leakage rate. In contrast, for egg PC large unilamellar vesicles (110 nm), hypelcin A induces the leakage, but not the fusion. We conclude that the leakage is not due to the fusion.

Effects of Cholesterol and Cholesteryl Oleate on Lipolysis and Liver Uptake of Triglyceride/Phosphatidylcholine Emulsions in Rats

Emulsions composed of soy bean triglyceride (TG), egg yolk phosphatidylcholine (PC), cholesterol (Chol) or cholesteryl-oleate (CO), labeled with a cholesteryl ether (3H-CHE) and a triglyceride (14C-TO), were injected into rats.14C-TO was removed from plasma faster than 3H-CHE. The 14C-labeled moiety is cleaved by digestion of the TG in the emulsion in plasma and is removed to the endothelial cells (lipolysis). In contrast, the 3H-label remains stably associated and represents circulating emulsion particles. The majority (90%) of the 3H-label disappearing from the plasma accumulated in the liver for all types of emulsions. On the basis of these observations, the lipolysis and the removal of emulsion particles to organs (mainly liver) were determined: 30 mole percent of cholesterol (Chol) at the TG-PC emulsion surface markedly retarded organ uptake, but the effect on lipolysis was rather small; 20 mole percent of cholesteryl oleate (CO) in the TG-PC emulsion cores delayed both organ uptake and lipolysis, and induced a rapid increase in organ uptake rate after the initial delay accompanying the gradual progress of lipolysis. Lipolysis led to the enrichment of the cores with CO. Replacement of the core TG by CO, however, induced strong suppression of the liver uptake. These results show that the lipid composition at both surface and core of emulsion particles is a crucial factor in metabolism in the rat.