Françoise Peypoux

A study on the interactions of surfactin with phospholipid vesicles

Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactant and posses several other interesting biological activities. By means of differential scanning calorimetry and X-ray diffraction the effect of surfactin on the phase transition properties of bilayers composed of different phospholipids, including lipids forming hexagonal-HII phases, has been studied. The interactions of surfactin with phosphatidylcholine and phosphatidylglycerol seem to be optimal in the case of myristoyl acyl chains, which have a similar length to the surfactin hydrocarbon tail. Data are shown that support formation of complexes of surfactin with phospholipids. The ionized form of surfactin seems to be more deeply inserted into negatively charged bilayers when Ca2+ is present, also supporting the formation of surfactin-Ca2+ complexes. In mixtures with dielaidoylphosphatidylethanolamine, a hexagonal-HII phase forming lipid, surfactin displays a bilayer stabilizing effect. Our results are compatible with the marked amphiphilic nature of surfactin and may contribute to explain some of its interesting biological actions; for instance the formation of ion-conducting pores in membranes.

Pore-forming properties of iturin A, a lipopeptide antibiotic

The addition of iturin A, a lipopeptide antibiotic extracted from Bacillus subtilis, to a bimolecular lipid membrane (BLM) increases dramatically its electrical conductance. For very low concentration of iturin A, discrete conductance steps are observed which are assigned to the formation of conducting pores. The characteristics of these pores depend on the lipid content of the BLM and they change with time. Cholesterol considerably increases the lifetimes of open states. The pores are slightly anion versus cation selective. These first observations unable us to briefly discuss the pore-forming properties of lipopeptides.

Action of peptidolipidic antibiotics of the iturin group on erythrocytes. Effect of some lipids on hemolysis.

Iturin A, bacillomycin L and bacillomycin L dimethyl ester have a strong lytic activity upon human erythrocytes while iturin C is totally inactive. The hemolytic action of the antibiotics is inhibited by free cholesterol as well as by cholesterol included in mixed liposomes of phosphatidylcholine-cholesterol and to a lesser extent by phosphatidylcholine liposomes. This inhibition is the result of an interaction between the antibiotic and added lipids which diminishes the concentration of free antibiotic available to lyse erythrocytes. The inhibitory effect of liposomes on hemolysis demonstrated the affinity of the antibiotic for artificial membrane, especially those containing cholesterol.

Controlled biosynthesis of Val7- and Leu7-surfactins

SummarySurfactin was found to consist of a mixture of two groups of homologous lipopeptides differing by their peptide sequence; Val7-surfactin was recently characterized as a minor companion of the previously described Leu7-surfactin. The addition of various α-amino acids to the culture media led to variations in the production ratios of the two congeners. The supplementation of l-valine or l-isoleucine to the culture medium resulted in a selective enhancement of the production of the Val7-surfactin whereas this production was very low when l-leucine was the nitrogen source in the culture medium.

Production, isolation and characterization of (Leu4) - and (Ile4)surfactins from Bacillus subtilis

Bacillus subtilis coproduces several surfactin variants that are powerful biosurfactants and have potential applications in biology and industry. A single amino acid substitution in the heptapeptide moiety of surfactins strongly modifies their properties. To better establish structure-activity relationships and to search new variants with enhanced properties, Bacillus subtilis was grown into two modified culture media. Two new variants were isolated by chromatographic methods and studied by NMR spectroscopy. As planned, modifications consisted in the substitution of the l-valine residue at the fourth position by a more hydrophobic residue, i.e., leucine or isoleucine. These [Leu4]- and [Ile4]surfactins have a higher affinity for hydrophobic solvents and a twice improved surfactant power. Structure-property correlations were confirmed by analysis of the hydrophobic residue distribution in the three-dimensional model of the structure of surfactin in solution.

Lipopeptides with Improved Properties: Structure by NMR, Purification by HPLC and Structure–Activity Relationships of New Isoleucyl‐rich Surfactins

The biosynthesis of bacterial isoleucyl‐rich surfactins was controlled by supplementation of L‐isoleucine to the culture medium. Two new variants, the [Ile4,7]‐ and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse‐phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified to β‐hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two‐dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure–activity relationships were discussed in details on the basis of the three‐dimensional model of surfactin in solution. Indeed, both variants have increased surface properties compared with that of surfactin, and this improvement is assigned to an increase of the hydrophobicity of the apolar domain favouring micellization. Furthermore, the additional Leu‐to‐Ile substitution at position 2 in the [Ile2,4,7]surfactin leads to a substantial increase of its affinity for calcium, when compared with that of [Ile4,7]surfactin or surfactin. This effect is assigned, from the model, to an increase in the accessibility of the acidic side chains constituting the calcium binding site. Thus, the propensities of such active lipopeptides for both hydrophobic and electrostatic interactions were improved, further substantiating that they can be rationally designed.

Ionophorous and sequestering properties of surfactin, a biosurfactant from Bacillus subtilis

Abstract Surfactin is a bacterial lipopeptide containing two carboxylic groups. Its sequestering properties (surfactin binds calcium and magnesium ions) were determined in aqueous solution at pH 9.5. The calculated association constants were K = 1.5 × 105 M−1 for Ca2− and 1.9 × 104 M−1 for Mg2+. Its complexing properties were determined by the two-phase distribution system; the aqueous phase contained Ca2+ or Rb+ ions and the lipidic phase contained surfactin. At pH 9, surfactin forms a 1:1 complex with Ca2+ and a 2:1 complex with Rb+ and it shows a higher affinity for divalent cations than for monovalent cations. Surfactin is non-selective in binding monovalent cations but is slightly more effective in binding Ca2+ than Mg2+ or Ba2+. Surfactin is also a mobile carrier to transport monovalent and divalent cations across a solvent barrier and mediates the exchange between H− and inorganic cation.

Mode of action of iturin A, an antibiotic isolated from Bacillus subtilis, on Micrococcus luteus

Abstract Iturin A has an antibacterial activity on M. luteus which is strongly reduced in presence of MgCl 2 . Iturin A lyses M. luteus protoplast, this lysis is enhanced by EDTA and inhibited by MgCl 2 . These results suggest an action of iturin A on cytoplasmic membrane with interactions of both lipophilic and polypeptidic moieties of the antibiotic, respectively with membrane lipids and membrane polar components. Polar interactions involve the participation of mineral ions as magnesium ions have a strong inhibition effect on the activity of iturin A. The effect of iturin A on the incorporation of radio-active thymidine, uracil, isoleucine and alanine seems unspecific and is probably a consequence of the primary action on cytoplasmic membrane.

Bacterial lipopeptides induce ion-conducting pores in planar bilayers

Bacterial lipopeptides, known for their antibiotic activities, have been tested for their ability to interact with lipid membranes. These lipopeptides, Iturin A, Bacillomycin L and D and Peptidolipin NA present analogous structural characteristics: a heptapeptidic cycle is linked to a hydrocarbon chain. We present evidence that these lipopeptides modify the conductance of planar bilayers by forming ion-conducting pores.

Aggregational behavior of aqueous dispersions of the antifungal lipopeptide iturin A

Iturin A, a lipopeptide isolated from Bacillus subtilis, possesses a strong antifungal activity, and has been devoted to a great deal of attention. Since iturin is an amphiphilic compound with a great propensity to self-associate in solution as well as inside the membrane, the question arises to whether its aggregational behavior is dependent on the concentration of the lipopeptide. In order to test this, the ability of iturin suspensions to encapsulate water-soluble molecules has been examined. Iturin was dispersed at different concentrations above its critical micellar concentration, in a buffer containing the water-soluble dye 5,6-carboxyfluorescein. For iturin A micelles, a Stokes radius of 1.3 nm and an aggregational number of 7 was obtained. The results shown in this work clearly demonstrate that iturin dispersions in water, at concentrations of 0.7, 1.4 and 3 mM, i.e. far above the critical micellar concentration (40 microM), are capable of encapsulating carboxyfluorescein, probably by adopting a type of aggregate different from the micelle. Negative-staining electron microscopy shows the presence of vesicles with an average size of 150 nm. By using (14)C-iturin, it is shown that, at 3 mM concentration, 40 % of the iturin molecules adopt this vesicular state. It is proposed that iturin molecules form a fully interdigitated bilayer, where each hydrocarbon tail span the entire hydrocarbon width of the bilayer, resulting in multilamellar vesicles capable of encapsulating an aqueous compartment. The possible implications of these results to the membrane destabilizing effect of iturin A, are discussed according to the dynamic cone-shape of the iturin molecule.