Amphiphilic azobenzenes: Antibacterial activities and biophysical investigation of their interaction with bacterial membrane lipids.

With the emergence of multi-drug resistant bacteria and hospital-acquired infections, there is an urgent need to develop new antibiotics. Here, we report the synthesis, physico-chemical characterizations, and antimicrobial activity assays of four Azo compounds that differ in their alkyl chain length. The molecular mechanism of their antibacterial activity was investigated by complementary in vitro and in silico biophysical studies. The compounds with alkyl chain lengths of four or six carbons showed a low MIC50 against Escherichia coli and Bacillus subtilis. Our investigations into the mechanism of their action revealed that phosphatidylethanolamine in the bacterial plasma membrane plays an important role in their antibacterial activity.

Surfactins modulate the lateral organization of fluorescent membrane polar lipids: a new tool to study drug:membrane interaction and assessment of the role of cholesterol and drug acyl chain length.

The lipopeptide surfactin exhibits promising antimicrobial activities which are hampered by haemolytic toxicity. Rational design of new surfactin molecules, based on a better understanding of membrane:surfactin interaction, is thus crucial. We here performed bioimaging of lateral membrane lipid heterogeneity in adherent living human red blood cells (RBCs), as a new relevant bioassay, and explored its potential to better understand membrane:surfactin interactions. RBCs show (sub)micrometric membrane domains upon insertion of BODIPY analogs of glucosylceramide (GlcCer), sphingomyelin (SM) and phosphatidylcholine (PC). These domains exhibit increasing sensitivity to cholesterol depletion by methyl-ß-cyclodextrin. At concentrations well below critical micellar concentration, natural cyclic surfactin increased the formation of PC and SM, but not GlcCer, domains, suggesting preferential interaction with lipid assemblies with the highest vulnerability to methyl-ß-cyclodextrin. Surfactin not only reversed disappearance of SM domains upon cholesterol depletion but further increased PC domain abundance over control RBCs, indicating that surfactin can substitute cholesterol to promote micrometric domains. Surfactin sensitized excimer formation from PC and SM domains, suggesting increased lipid recruitment and/or diffusion within domains. Comparison of surfactin congeners differing by geometry, charge and acyl chain length indicated a strong dependence on acyl chain length. Thus, bioimaging of micrometric lipid domains is a visual powerful tool, revealing that intrinsic lipid domain organization, cholesterol abundance and drug acyl chain length are key parameters for membrane:surfactin interaction. Implications for surfactin preferential location in domains or at their boundaries are discussed and may be useful for rational design of better surfactin molecules.

Interaction of hexadecylbetainate chloride with biological relevant lipids.

The present work investigates the interaction of hexadecylbetainate chloride (C(16)BC), a glycine betaine-based ester with palmitoyl-oleoyl-phosphatidylcholine (POPC), sphingomyelin (SM), and cholesterol (CHOL), three biological relevant lipids present in the outer leaflet of the mammalian plasma membrane. The binding affinity and the mixing behavior between the lipids and C(16)BC are discussed based on experimental (isothermal titration calorimetry (ITC) and Langmuir film balance) and molecular modeling studies. The results show that the interaction between C(16)BC and each lipid is thermodynamically favorable and does not affect the integrity of the lipid vesicles. The primary adsorption of C(16)BC into the lipid film is mainly governed by a hydrophobic effect. Once C(16)BC is inserted in the lipid film, the polar component of the interaction energy between C(16)BC and the lipid becomes predominant. Presence of CHOL increases the affinity of C(16)BC for membrane. This result can be explained by the optimal matching between C(16)BC and CHOL within the film rather by a change of membrane fluidity due to the presence of CHOL. The interaction between C(16)BC and SM is also favorable and gives rise to highly stable monolayers probably due to hydrogen bonds between their hydrophilic groups. The interaction of C(16)BC with POPC is less favorable but does not destabilize the mixed monolayer from a thermodynamic point of view. Interestingly, for all the monolayers investigated, the exclusion surface pressures are above the presumed lateral pressure of the plasma membranes suggesting that C(16)BC would be able to penetrate into mammalian plasma membranes in vivo. These results may serve as a useful basis in understanding the interaction of C(16)BC with real membranes.

Bacillus sp.: A Remarkable Source of Bioactive Lipopeptides.

Surfactin, one of the best lipopeptide surfactants, was first isolated from Bacillus sp. in 1969. Since then, Bacillus sp. has been a remarkable source of bioactive lipopeptides, with a huge natural biodiversity. Lipopeptides from Bacillus sp. are now divided into three main families: surfactin, fengycin, and iturin. The peptide moiety of these lipopeptides is synthesised by huge multi-enzymatic proteins called nonribosomal peptide synthetases, which are responsible for the peptide biodiversity of these lipopeptides. Moreover, the fatty acid chain also encompasses a high diversity with different ß-hydroxy or ß-amino fatty acid chains of different lengths, isomery, or saturation, which can be incorporated. After describing the mode of synthesis of the different families of lipopeptides produced by Bacillus sp. and their biodiversity, this chapter describes how this lipopeptide biodiversity can be increased using genetic engineering and how the lipopeptides can be overproduced and purified. The high biodiversity of lipopeptides induces a broad range of physicochemical properties, which can be linked to multiple biological activities with many applications in different sectors. The increasing understanding of the mode of biosynthesis of these lipopeptides should lead to the development of novel compounds with increased properties and applications.

Sitosterol and glucosylceramide cooperative transversal and lateral uneven distribution in plant membranes.

The properties of biomembranes depend on the presence, local structure and relative distribution assumed by the thousands of components it is made of. As for animal cells, plant membranes have been demonstrated to be organized in subdomains with different persistence lengths and times. In plant cells, sitosterol has been demonstrated to confer to phospholipid membranes a more ordered structure while among lipids, glycosphingolipids are claimed to form rafts where they tightly pack with sterols. Glucosylceramides are glycosphingolipids involved in plant signalling and are essential for viability of cells and whole plant. The glucosylceramide-sitosterol structural coupling within PLPC membranes is here investigated by Langmuir films, in silico simulations and neutron reflectometry, unveiling that a strong direct interaction between the two molecules exists and governs their lateral and transversal distribution within membrane leaflets. The understanding of the driving forces governing specific molecules clustering and segregation in subdomains, such as glucosylceramide and sitosterol, have an impact on the mechanical properties of biomembranes and could reflect in the other membrane molecules partitioning and activity.

Enzymatically-synthesized xylo-oligosaccharides laurate esters as surfactants of interest.

Lipase-catalyzed synthesis of xylo-oligosaccharides esters from pure xylobiose, xylotriose and xylotetraose in the presence of vinyl laurate was investigated. The influence of different experimental parameters such as the loading of lipase, the reaction duration or the use of a co-solvent was studied and the reaction conditions were optimized with xylobiose. Under the best conditions, a regioselective esterification occurred to yield a monoester with the acyl chain at the OH-4 of the xylose unit at the non-reducing end. Surface-active properties of these pure xylo-oligosaccharides fatty esters have been evaluated. They display interesting surfactant activities that differ according to the degree of polymerization (DP) of the glycone moiety.

Molecular organization of surfactin-phospholipid monolayers: effect of phospholipid chain length and polar head.

Mixed monolayers of the surface-active lipopeptide surfactin-C(15) and various lipids differing by their chain length (DMPC, DPPC, DSPC) and polar headgroup (DPPC, DPPE, DPPS) were investigated by atomic force microscopy (AFM) in combination with molecular modeling (Hypermatrix procedure) and surface pressure-area isotherms. In the presence of surfactin, AFM topographic images showed phase separation for each surfactin-phospholipid system except for surfactin-DMPC, which was in good agreement with compression isotherms. On the basis of domain shape and line tension theory, we conclude that the miscibility between surfactin and phospholipids is higher for shorter chain lengths (DMPC>DPPC>DSPC) and that the polar headgroup of phospholipids influences the miscibility of surfactin in the order DPPC>DPPE>DPPS. Molecular modeling data show that mixing surfactin and DPPC has a destabilizing effect on DPPC monolayer while it has a stabilizing effect towards DPPE and DPPS molecular interactions. Our results provide valuable information on the activity mechanism of surfactin and may be useful for the design of surfactin delivery systems.

Molecular dynamics study of micelles properties according to their size.

Surfactants are molecules able to spontaneously self-assemble to form aggregates with well-defined properties, such as spherical micelles, planar bilayers, cylindrical micelles or vesicles. Micelles have notably several applications in many domains, such as drug delivery or membrane protein solubilization. In this context, the study of micelle formation in relation with the structural and physico-chemical properties of surfactants is of great interest to better control their use in the different application fields. In this work, we use the MD approach developed by Yoshii et al. and extend it to surfactants with different structures. We aim to systematically investigate different micellar properties as a function of the aggregates size by a molecular dynamics approach, to get an insight into the micellar organization and to collect some relevant descriptors about micelle formation. For this, we perform short MD simulations of preformed micelles of various sizes and analyze three parameters for each micelle size, namely the eccentricity of the micelles, the hydrophobic/hydrophilic surface ratio and the hydrophobic tails hydration. If these parameters are known descriptors of micelles, they were not yet studied in this way by MD. We show that eccentricity, used as "validator" parameter, exhibits minimal values when the aggregate size is close to the experimental aggregation number for surfactants that are known to form spherical micelles. This hence indicates that our methodology gives consistent results. The evolution of the two descriptors follows another scheme, with a sharp increase and decrease, respectively, followed by a leveling-off. The aggregate sizes at which this stabilization starts to occur are close to the respective aggregation number of each surfactant. In our approach, we validate the use of these descriptors to follow micelle formation by MD, from "simple" surfactants to more complex structures, like lipopeptides. Our calculations also suggest that some peculiar behavior, like that of TPC, can be highlighted by our approach. In the context of peptidic surfactants, our methodology could further help to improve computer simulations combined to molecular thermodynamic models to predict micellar properties of those more complex amphiphilic molecules.

Effect of the antibiotic azithromycin on thermotropic behavior of DOPC or DPPC bilayers.

Azithromycin is a macrolide antibiotic known to bind to lipids and to affect endocytosis probably by interacting with lipid membranes [Tyteca, D., Schanck, A., Dufrene, Y.F., Deleu, M., Courtoy, P.J., Tulkens, P.M., Mingeot-Leclercq, M.P., 2003. The macrolide antibiotic azithromycin interacts with lipids and affects membrane organization and fluidity: studies on Langmuir-Blodgett monolayers, liposomes and J774 macrophages. J. Membr. Biol. 192, 203-215]. In this work, we investigate the effect of azithromycin on lipid model membranes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Thermal transitions of both lipids in contact with azithromycin are studied by (31)P NMR and DSC on multilamellar vesicles. Concerning the DPPC, azithromycin induces a suppression of the pretransition whereas a phase separation between the DOPC and the antibiotic is observed. For both lipids, the enthalpy associated with the phase transition is strongly decreased with azithromycin. Such effects may be due to an increase of the available space between hydrophobic chains after insertion of azithromycin in lipids. The findings provide a molecular insight of the phase merging of DPPC gel in DOPC fluid matrix induced by azithromycin [Berquand, A., Mingeot-Leclercq, M.P., Dufrene, Y.F., 2004. Real-time imaging of drug-membrane interactions by atomic force microscopy. Biochim. Biophys. Acta 1664, 198-205] and could help to a better understanding of azithromycin-cell interaction.

Imaging mixed lipid monolayers by dynamic atomic force microscopy.

Phase imaging with tapping mode atomic force microscopy (AFM) and force modulation microscopy were used to probe the mechanical properties of phase-separated lipid monolayers made of a mixture (0.25:0.75) of the surface-active lipopeptide surfactin and of dipalmitoylphosphatidylcholine (DPPC). The pi-A isotherms and the result of a molecular modeling study revealed a loose, 2-D liquid-like organization for the surfactin molecules and a closely packed, 2-D solid-like organization for DPPC molecules. This difference in molecular organization was responsible for a significant contrast in height, tapping mode phase and force modulation amplitude images. Phase imaging at light tapping, i.e., with a ratio of the set-point tapping amplitude with respect to the free amplitude A(sp)/A(0) approximately 0.9, showed larger phase shifts on the solid-like DPPC domains attributed to larger Young's modulus. However, contrast inversion was observed for A(sp)/A(0)<0.7, suggesting that at moderate and hard tapping the image contrast was dominated by the probe-sample contact area. Surprisingly, force modulation amplitude images showed larger stiffness for the liquid-like surfactin domains, suggesting that the contrast was dominated by contact area effects rather than by Young's modulus. These data emphasize the complex nature of the contrast mechanisms of dynamic AFM images recorded on mixed lipid monolayers.

Adriamycin, cyclophosphamide and vindesine (ACV) versus mitoxantrone, cyclophosphamide and vindesine (NCV) in advanced breast cancer.

The choice of chemotherapy for advanced breast cancer patients is based on both response rates and anticipated side effects. Two polychemotherapy regimens, ACV and NCV, were compared in an open study with 90 patients of whom 69 were evaluable both for efficacy and toxicity. Both ACV and NCV are active in metastatic breast cancer. ACV leads to more remissions in a relatively short time. NCV treatment results in a larger proportion of stable diseases. ACV causes more important alopecia, nausea and vomiting. NCV gives more anemia. The characteristics of both treatment results give the oncologist the opportunity to choose in particular clinical situations.

The structure of two fengycins from Bacillus subtilis S499.

The structures of the two fengycins, lipopeptides from Bacillus subtilis, were elucidated by spectroscopic methods and chemical degradation. They show a close structural relationship to the plipastatins from Bacillus cereus differing only in the stereochemistry of the Tyr residues.

Penetration behaviour of alkylbetainate chlorides into lipid monolayers.

In this paper, the penetration behaviour of the alkylbetainate chloride surfactants (C(n)BC, n=10-16) into lipid monolayers of dipalmitoylphosphatidylserine (DPPS), dipalmitoylphosphatidic acid (DPPA), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyoleoylphosphatidylcholine (POPC) and cholesterol (CHOL) is investigated using the Langmuir trough technique. The penetration of C(n)BC is followed by measurement of the surface pressure increase (Δπ) at a constant surface area after the injection of C(n)BC into the aqueous phase, underneath the lipid monolayer previously spread at the air-water interface at 25°C and at different initial surface pressures (π(i)). The influence of both the lipid head group and the surfactant hydrocarbon chain length on the effectiveness of C(n)BC penetration into these monolayers is discussed. The results have shown that C(n)BC adsorb at the air-water interface giving evidence of their surface-active properties. The adsorption kinetics of C16BC into different lipid monolayers are lipid head charge and lipid head volume-dependent. The magnitude of the surface pressure increase (Δπ) arises in the following order: DPPA>DPPS≫CHOL≈DPPE>POPC. C(n)BC penetration into negatively-charged (DPPS and DPPA) monolayers does not seem to depend on surfactant alkyl-chain length compared to uncharged (CHOL) and zwitterionic (DPPE and POPC) monolayers for which Δπ increases with a larger alkyl-chain length. Electrostatic interactions are mainly involved in the affinity of C(n)BC with monolayers but the hydrophobic effect plays also a role.

Effect of cholesterol and fatty acids on the molecular interactions of fengycin with Stratum corneum mimicking lipid monolayers.

The combination of atomic force microscopy (AFM) and the Langmuir trough technique was used in this work to investigate the molecular interactions of fengycin with lipid monolayers constituted of the major lipid classes found in human stratum corneum (SC). AFM imaging o f spread SC lipids/fengycin monolayers showed that fengycin preferentially partitions into cholesterol-rich phases surrounding 2D domains mainly constituted of ceramide and fatty acid molecules. Penetration experiments of fengycin from the subphase into SC-mimicking monolayers clearly indicated that the lipopeptide insertion at the lipid interface is enhanced in the presence of cholesterol. AFM analysis of mixed SC lipids/fengycin monolayers obtained after lipopeptide penetration revealed that cholesterol strongly interacts with fengycin and undergoes specific molecular interactions with more disordered, loosely packed ceramide molecules. These results highlight the capacity of fengycin to interact with the lipid constituents of the extracellular matrix of SC and, in particular, with cholesterol.

Penetration of surfactin into phospholipid monolayers: nanoscale interfacial organization.

Atomic force microscopy (AFM) combined with surface pressure-area isotherms were used to probe the interfacial behavior of phospholipid monolayers following penetration of surfactin, a cyclic lipopeptide produced by Bacillus subtilis strains. Prior to penetration experiments, interfacial behavior of different surfactin molecules (cyclic surfactins with three different aliphatic chain lengths--S13, S14, and S15--and a linear surfactin obtained by chemical cleavage of the cycle of the surfactin S15) has been investigated. A more hydrophobic aliphatic chain induces greater surface-active properties of the lipopeptide. The opening of the peptide ring reduces the surface activity. The effect of phospholipid acyl chain length (dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine- (DPPC), and distearoylphosphatidylcholine) and phospholipid polar head (DPPC, dipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylserine) on monolayer penetration properties of the surfactin S15 has been explored. Results showed that while the lipid monolayer thickness and the presence of electrostatic repulsions from the interfacial film do not significantly influence surfactin insertion, these parameters strongly modulate the ability of the surfactin to alter the nanoscale organization of the lipid films. We also probed the effect of surfactin structure (influence of the aliphatic chain length and of the cyclic structure of the peptide ring) on the behavior of DPPC monolayers. AFM images and isotherms showed that surfactin penetration is promoted by longer lipopeptide chain length and a cyclic polar head. This indicates that hydrophobic interactions are of main importance for the penetration power of surfactin molecules.

Nanoscale properties of mixed fengycin/ceramide monolayers explored using atomic force microscopy.

To gain insight into the interactions between fengycin and skin membrane lipids, mixed fengycin/ceramide monolayers were investigated using atomic force microscopy (AFM) (monolayers supported on mica) and surface pressure-area isotherms (monolayers at the air-water interface). AFM topographic images revealed phase separation in mixed monolayers prepared at 20 degrees C/pH 2 and composed of 0.25 and 0.5 fengycin molar ratios, in the form of two-dimensional (2-D) hexagonal crystalline domains of ceramide surrounded by a fengycin-enriched fluid phase. Surface pressure-area isotherms as well as friction and adhesion AFM images confirmed that the two phases had different molecular orientations: while ceramide formed a highly ordered phase with crystalline chain packing, fengycin exhibited a disordered fluid phase with the peptide ring lying horizontally on the substrate. Increasing the temperature and pH to values corresponding to the skin parameters, i.e., 37 degrees C/pH 5, was found to dramatically affect the film organization. At low fengycin molar ratio (0.25), the hexagonal ceramide domains transformed into round domains, while at higher ratio (0.5) these were shown to melt into a continuous fengycin/ceramide fluid phase. These observations were directly supported by the thermodynamic analysis (deviation from the additivity rule, excess of free energy) of the monolayer properties at the air-water interface. Accordingly, this study demonstrates that both the environmental conditions (temperature, pH) and fengycin concentration influence the molecular organization of mixed fengycin/ceramide monolayers. We believe that the ability to modulate the formation of 2-D domains in the skin membrane may be an important biological function of fengycin, which should be increasingly investigated in future pharmacological research.

Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity.

The synthesis of extracellular molecules such as biosurfactants should have major consequences on bacterial adhesion. These molecules may be adsorbed on surfaces and modify their hydrophobicities. Certain strains of Bacillus subtilis synthesize the lipopeptides, which exhibit antibiotic and surface active properties. In this study the high-performance liquid chromatography (HPLC) analysis of the culture supernatants of the seven B. subtilis strains, showed that the lipopeptide profile varied greatly according to the strain. Among the three lipopeptide types, only iturin A was produced by all B. subtilis strains. Bacterial hydrophobicity, evaluated by the water contact angle measurements and the hydrophobic interaction chromatography, varied according to the strain. Two strains (ATCC 15476 and ATCC 15811) showing extreme behaviors in term of hydrophobicity were selected to study surfactin and iturin A effects on bacterial hydrophobicity. The two lipopeptides modified the B. subtilis surface hydrophobicity. Their effects varied according to the bacterial surface hydrophobic character, the lipopeptide type and the concentration. Lipopeptide adsorption increased the hydrophobicity of the hydrophilic strain but decreased that of the hydrophobic. Comparison of lipopeptide effects on B. subtilis surface hydrophobicity showed that surfactin was more effective than iturin A for the two strains tested.

Nanometer scale organization of mixed surfactin/phosphatidylcholine monolayers.

Mixed monolayers of the surface-active lipopeptide surfactin-C(15) and of dipalmitoyl phosphatidylcholine (DPPC) were deposited on mica and their nanometer scale organization was investigated using atomic force microscopy (AFM) and x-ray photoelectron spectroscopy (XPS). AFM topographic images revealed phase separation for mixed monolayers prepared at 0.1, 0.25, and 0.5 surfactin molar ratios. This was in agreement with the monolayer properties at the air-water interface indicating a tendency of the two compounds to form bidimensional domains in the mixed systems. The step height measured between the surfactin and the DPPC domains was 1.2 +/- 0.1 nm, pointing to a difference in molecular orientation: while DPPC had a vertical orientation, the large peptide ring of surfactin was lying on the mica surface. The N/C atom concentration ratios obtained by XPS for pure monolayers were compatible with two distinct geometric models: a random layer for surfactin and for DPPC, a layer of vertically-oriented molecules in which the polar headgroups are in contact with mica. XPS data for mixed systems were accounted for by a combination of the two pure monolayers, considering respective surface coverages that were in excellent agreement with those measured by AFM. These results illustrate the complementarity of AFM and XPS to directly probe the molecular organization of multicomponent monolayers.

Insecticide activity of surfactins and iturins from a biopesticide Bacillus subtilis Cohn (S499 strain).

Surfactin C14, surfactin C15, and iturin C15 are lipopeptides purified from Bacillus subtilis (S499 strain). They were incorporated to artificial diet of the fruit fly Drosophila melanogaster (Meigen) (Diptera, Drosophilidae) to assess their potential insecticide activity. Surfactins with long fatty acid chain (C14 and C15) showed insecticide effect on the fruit fly, D. melanogaster. On the contrary, iturin was not toxic to fruit fly D. melanogaster. At 100 ppm, surfactin C14 and C15 showed respectively 85.4 and 92.6% adults mortality after one-day exposure. F1 progeny fly emergence inhibition by C14 and C15 were respectively 79.8% and 91.3%. To check whether the biocide activity of lipopeptides was due to their surface-active properties, detergent Triton X100, SDS, CTAB and Tween 80 were tested. No adult mortality was recorded with the detergents but Triton X100 and SDS showed F1 progeny emergence inhibition similar to that of surfactins. We showed that there was a dose-response activity with surfactin C15.

The macrolide antibiotic azithromycin interacts with lipids and affects membrane organization and fluidity: studies on Langmuir-Blodgett monolayers, liposomes and J774 macrophages.

The macrolide antibiotic azithromycin was shown to markedly inhibit endocytosis. Here we investigate the interaction of azithromycin with biomembranes and its effects on membrane biophysics in relation to endocytosis. Equilibrium dialysis and 31P NMR revealed that azithromycin binds to lipidic model membranes and decreases the mobility of phospholipid phosphate heads. In contrast, azithromycin had no effect deeper in the bilayer, based on fluorescence polarization of TMA-DPH and DPH, compounds that, respectively, explore the interfacial and hydrophobic domains of bilayers, and it did not induce membrane fusion, a key event of vesicular trafficking. Atomic force microscopy showed that azithromycin perturbed lateral phase separation in Langmuir-Blodgett monolayers, indicating a perturbation of membrane organization in lateral domains. The consequence of azithromycin/ phospholipid interaction on membrane endocytosis was next evaluated in J774 macrophages by using three tracers with different insertion preferences inside the biological membranes and intracellular trafficking: C6-NBD-SM, TMA-DPH and N-Rh-PE. Azithromycin differentially altered their insertion into the plasma membrane, slowed down membrane trafficking towards lysosomes, as evaluated by the rate of N-Rh-PE self-quenching relief, but did not affect bulk membrane internalization of C6-NBD-SM and TMA-DPH. Azithromycin also decreased plasma membrane fluidity, as shown by TMA-DPH fluorescence polarization and confocal microscopy after labeling by fluorescent concanavalin A. We conclude that azithromycin directly interacts with phospholipids, modifies biophysical properties of membrane and affects membrane dynamics in living cells. This antibiotic may therefore help to elucidate the physico-chemical properties underlying endocytosis.