Régine Maget-Dana

The monolayer technique: a potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes

Erudites of the antiquity already knew the calming effect of oil films on the sea waves. But one had to wait until 1774 to read the first scientific report on oil films from B. Franklin and again 1878 to learn the thermodynamic analysis on adsorption developed by J. Gibbs. Then, in 1891, Agnes Pockels described a technique to manipulate oil films by using barriers. Finally, in 1917, I. Langmuir introduced the experimental and theoretical modern concepts on insoluble monolayers. Since that time, and because it has been found to provide invaluable information at the molecular scale, the monolayer technique has been more and more extensively used, and, during the past decade, an explosive increase in the number of publications has occurred. Over the same period, considerable and ever-increasing interest in the antimicrobial peptides of various plants, bacteria, insects, amphibians and mammals has grown. Because many of these antimicrobial peptides act at the cell membrane level, the monolayer technique is entirely suitable for studying their physicochemical and biological properties. This review describes monolayer experiments performed with some of these antimicrobial peptides, especially gramicidin A, melittin, cardiotoxins and defensin A. After giving a few basic notions of surface chemistry, the surface-active properties of these peptides and their behavior when they are arranged in monomolecular films are reported and discussed in relation to their tridimensional structure and their amphipathic character. The penetration of these antimicrobial peptides into phospholipid monolayer model membranes, as well as their interactions with lipids in mixed films, are also emphasized.

Iturins, a special class of pore-forming lipopeptides: biological and physicochemical properties

Iturins are a family of lipopeptides extracted from the culture media of various strains of Bacillus subtilis. These amphiphilic compounds are characterized by a peptide ring of seven amino acid residues including an invariable D-Tyr2, with the constant chiral sequence LDDLLDL closed by a C14-C17 aliphatic beta-amino acid. They exhibit strong antifungal activities against a wide variety of pathogenic yeasts and fungi but their antibacterial activities are restricted to some bacteria such as Micrococcus luteus. The biological activity of the iturin lipopeptides is modulated by the primary structure of the peptide cycle as illustrated by the methylation of the D-Tyr2 residue which dramatically decreases the activity or by the inversion of the two adjacent Ser6-Asn7 residues which makes mycosubtilin more active than iturin A. The antifungal activity is related to the interaction of the iturin lipopeptides with the cytoplasmic membrane of target cells, the K+ permeability of which is greatly increased. The ability of iturin compounds to increase the membrane cell permeability is due to the formation of ion-conducting pores, the characteristics of which depend both on the lipid composition of the membrane and on the structure of the peptide cycle. From monolayer experiments it has been suggested that these ionic pores are the consequence of the presence of aggregates (lipopeptide aggregates or lipopeptide/phospholipid complex aggregates) in the phospholipid membrane. It has also been shown that, when active, iturins interact strongly with sterols, forming lipopeptide/cholesterol complexes. Therefore, the biologically efficient structure might be a ternary structure: iturin/phospholipid/sterol.

Lichenysin : A more efficient cation chelator than surfactin

The lipopeptide lichenysin (cyclo-[L-Gln1-->D-Leu2-->L-Leu3-->L-Val4--> L-Asp5-->D-Leu6-->L-Ile7-beta-OH fatty acid]) produced by Bacillus licheniformis structurally resembles surfactin from Bacillus subtilis. The main difference is the presence of a glutaminyl residue in position 1 of the peptide sequence in place of glutamic acid in surfactin. This local variation causes significant changes in the properties of the molecule compared to surfactin. Lichenysin has a higher surfactant power, the critical micellar concentration (c.m.c.) being strongly reduced from 220 to 22 microM and a much higher hemolytic activity because 100% hemolysis was observed with only 15 microM instead of 200 microM. Lichenysin is also a better chelating agent because its association constants with Ca2+ and Mg2+ are increased by a factor of 4 and 16, respectively. This effect is assigned to an increase in the accessibility of the carboxyl group to cations owing to a change in the side chain topology induced by the Glu/Gln exchange. Additionally, the propensity of the lipopeptide for extensive hydrophobic interactions, as illustrated by its low c.m.c., contributes to further stabilization of the cation and an increase in the partitioning of lichenysin into the erythrocyte membrane. Our data support the formation of a lichensyin-Ca2+ complex in a molar ratio of 2:1 instead of 1:1 with surfactin, suggesting an intermolecular salt bridge between two lichenysin molecules. Therefore, when Ca2+ ions are present in the solution, micellization occurs via a dimer assembly, with a possible long-range effect on the spatial arrangement of the micelles or other supramolecular structures. Finally, among all the surfactin peptidic variants so far known, lichenysin is the one for which the three tested activities are the most substantially improved.

Interfacial properties of surfactin

Surfactin is a lipopeptide produced by various strains of Bacillus subtilis and is a very powerful surfactant. Here we present the first report on the interfacial behavior of surfactin. The adsorption, Γmax, of surfactin at the interface of diluted solutions (5 × 10−8 to 5 × 10−7M) is around 3 × 1018 molecule m−2, a value indicating that surfactin molecules are in a very packed situation. Surfactin spreads readily at the airwater interface: the equilibrium spreading pressure πe ≈ 30 mN m−1 reaches 45 mN m−1 when electrolytes (KCl or CaCl2) are dissolved in the alkaline subphase. We have plotted the compression isotherm curves and determined surface parameters. These parameters vary only a little with temperature but are very affected by the pH of the subphase. Plotting the transition pressure value πt as a function of pH results in a titration curve from which one can deduce the pK value of surfactin at the interface. This value, pKs ≈ 6 is around 2 pH units higher than the pK of surfactin in solution. The addition of electrolytes (I = 0.15 M) in the alkaline subphase leads to the neutralization of the surfactin monolayer (protonation of the acidic residues Lue5f8Glu1 and Lue5f8Asp5 of the peptide cycle). This neutralization is complete in the case of Ca++ ions but not in the case of monovalent cations (Na+ or K+). When surfactin monolayers are subjected to successive compression—expansion cycles we observe a reproducible hysteresis loop. The surface parameters of surfactin are compared to those of iturins, lipopeptides also extracted from B. subtilis, the structure and properties of which are very similar.

Iturin lipopeptides: interactions of mycosubtilin with lipids in planar membranes and mixed monolayers.

The interactions between the antifungal lipopeptide mycosubtilin and lipids are studied. Mycosubtilin increases the ion permeability of planar lipid membranes by forming ion conducting pores. The lifetime of these pores is greatly increased when the membrane contains cholesterol. In mixed monolayers the interaction between mycosubtilin and DMPC leads to the formation of a mycosubtilin/DMPC 1:2 complex non miscible in the excess DMPC monolayer but miscible in the mycosubtilin monolayer. Mycosubtilin and cholesterol interact strongly in monolayers in all proportions and form a mycosubtilin-cholesterol (1:2) complex. These results are analyzed with reference to the overall view of the activity of iturins and the importance of the lipopeptide conformation is outlined.

The lipopeptide antibiotic A21978C has a specific interaction with DMPC only in the presence of calcium ions

The A21978C group are lipopeptide antibiotics which kill Gram-positive bacteria only in the presence of calcium ions. The calcium requirement of the antibacterial activity of A21978C correlates well with an in vitro calcium-dependent insertion into phospholipid vesicles. In this paper the interaction of A21978C with phosphatidylcholine is investigated in mixed monomolecular films. The spontaneity of the antibiotic-lipid mixing was determined by calculating the free energy change. On a Ca2+ containing subphase there is a specific interaction between the components at all antibiotic-lipid ratios. This is not true on K+ subphases, where specific interactions never occur. On Mg2+ subphases specific interactions occur only in monolayers containing very little lipid. By analysing the fluorescence of the kynurenine residue we have followed the effects of two factors on the penetration of the antibiotic into lipid bilayer vesicles. Firstly, the phospholipid gel to liquid crystalline phase transition which in the absence of calcium leads to an exclusion of the antibiotic from the bilayer. This trend is completely reversed in the presence of Ca2+. Secondly, the role of this lipopeptides lipid tail was clarified by use of a series of versions of increasing fatty acyl chain length. The results indicate that the interaction promoted by calcium is not simply a hydrophobic attraction between fatty acyl chains but is more likely to be a specific interaction between polar headgroups.

Amphiphilic properties of bacterial lipopeptides: self-association and monolayer studies of iturins

Abstract Iturins are a special class of bacterial lipopeptides with antifungal activity. They are amphiphilic compounds with a heptapeptidic cycle linked to a C 10 , C 12 alkyl chain. We have studied the self-association in aqueous solution of iturin A and its O -methyltyrosine derivative that is biologically inactive. We have determined the concentration at which aggregates appear by using diphenylhexatriene as a fluorescent probe or by following the intrinsic fluorescence of the tyrosyl residue. The methylated derivative aggregated at a concentration 10 times lower than iturin A. Iturins formed monolayers at the air—water interface, isotherms of which have been studied. The monolayers of iturin A were not very stable. The isotherm curve presented a transition region that was interpreted as the passage to a tridimensional structure. The monolayers of O -methyltyrosine iturin A were more stable: the equilibrium spreading pressure π c was 27 m N m −1 as opposed to 9 m N m −1 for iturin A. For both compounds, compression—expansion cycles resulted in nonreproducible hysteresis.

Interactions of the lipopeptide antifungal iturin A with lipids in mixed monolayers

The miscibility and the interactions of the antifungal lipopeptide iturin A with lipids, DMPC and cholesterol, are studied in monolayers at the air/water interface and a comparison of the respective behaviour of iturin A and the biologically inactive methylated derivative MeTyr-iturin A is made. Each lipopeptide is miscible with anyone of the lipids. This behaviour is revealed by the dependence of the transition pressure upon composition and by deviations from the additivity rule of the mean molecular area. The thermodynamic properties of the mixed systems are studied by the method of Goodrich. The mixed monolayers are always more stable than the two separate components, subsequently there are interactions between the components. However, the excess free energy of mixing delta Gexm is positive for the iturin A/DMPC system which is an indication that the interactions between lipopeptide and lipid molecules are weaker than the interactions between the pure components themselves. This is compatible with the presence of self-associated lipopeptide molecules. However, delta Gexm is highly negative for the iturin A/cholesterol system giving evidence of the formation of a specific complex between iturin A and cholesterol which is not the case with the methylated derivative. These data are analysed in connection with previous results concerning the pore-forming properties of these lipopeptides and the lack of biological activity of MeTyr-iturin A.

Methylation of the antifungal lipopeptide iturin A modifies its interaction with lipids

Iturin A, extracted from the culture media of Bacillus subtilis, is an antifungal lipopeptide, the peptide cycle of which includes a D-Tyr residue in position 2. The antibiotic strength of iturin A is related to a change in the permeability of the membrane cells which leads to a leakage of K+ from the intracellular medium. Methylation of the D-Tyr residue dramatically decreases the biological activity of iturin A. Using the intrinsic fluorescence of D-Tyr we have shown that both iturin A and O-methyl-tyrosine iturin A enter the lipid membranes. When dimyristoylphosphatidylcholine vesicles contain iturin A we observe a change in the order degree of the lipid phase and an increase in the transition temperature. The methylated derivative has no effect. Two model membranes have been used to study the permeability changes induced by iturin A and O-methyltyrosine iturin A. Studying ionic permeability we have found that the conductance of a planar lipid membrane increases very much less when the lipopeptide is methylated. On the other hand, the release of carboxyfluorescein trapped in lipid vesicles is less upon addition of O-methyltyrosine-iturin A. We conclude that the Tyr residue of the peptide cycle plays a role in determining the interactions of iturin A with lipid membrane.

Interfacial properties of the antifungal iturins on various electrolyte solutions

Abstract We have studied the interfacial behavior of antifungal iturins (principally bacillomycin F, mycosubtilin, and iturin A) in solution and spread as monomolecular films on aqueous phases containing various chloride salts. The presence of electrolytes lowers the interfacial concentration (surface excess) of iturin solutions. The compression isotherm curves are not superimposable, and for all the compounds the stability of the monolayer is higher on KCl as attested by the transition pressure (for the liquid-expanded part) and by the collapse pressure values. The reduction of the molecular free energy of spreading ΔGs, that measures the affinity of the polar cycle of the lipopeptide for the aqueous phase components, is maximum on KCl in the case of mycosubtilin. The energy of compression ΔGc, that measures the intermolecular forces between the film forming molecules, is generally higher on KCl. The compression leads to more ordered structures when mycosubtilin and iturin A are spread on KCl. When spread on CaCl2, iturin A and bacillomycin F molecules can adopt only two kinds of arrangements, as shown by the convergence of the LE parts of their isotherms. These data are interpreted as interactions between iturins and ions and, more precisely, as discriminating interactions between iturins and the various cations with a special role for K+.