ARTICLE pubs.acs.org/Langmuir
Specific Interactions of Mycosubtilin with Cholesterol-Containing Artificial Membranes Mehmet Nail Nasir†,‡,§,||,^ and Franc-oise Besson*,†,‡,§,||,^ †
Universite de Lyon, Villeurbanne, F-69622, France Universite Lyon 1, Villeurbanne, F-69622, France § INSA de Lyon, Villeurbanne, F-69622, France CPE Lyon, Villeurbanne, F-69616, France ^ ICBMS CNRS UMR 5246, Villeurbanne, F-69622, France
)
‡
ABSTRACT: Mycosubtilin is a natural antimicrobial lipopeptide produced by Bacillus subtilis strains. It is characterized by its hemolytic and strong antifungal activities. Mycosubtilin interacts with the plasma membranes of sensitive cells. However, the molecular mechanisms of its biological activities have not been completely elucidated. Our purpose was therefore to analyze the interactions of mycosubtilin with biological membranes by using biomimetic membranes such as Langmuir monolayers and multilayers. Structural changes of mycosubtilin, involving its peptide backbone and the side chain of its tyrosyl residue, were observed when the lipopeptide was interacting with cholesterolcontaining multilayers. The interactions of mycosubtilin with monolayers constituted by pure lipids and by phosholipid/cholesterol or phospholipid/sphingomyelin/cholesterol were also examined. An original behavior of mycosubtilin toward cholesterolcontaining monolayers was found. However, this original behavior was lost when mycosubtilin was interacting with pure cholesterylacetate monolayers. This suggests the involvement of the alcohol group of cholesterol in mycosubtilin cholesterol interactions within membranes. Moreover, mycosubtilin induced changes in the organization and morphology of cholesterolcontaining monolayers, and large condensed domains with different levels of condensation appeared only in the case of DPPC/ sphingomyelin/cholesterol monolayer.
’ INTRODUCTION Mycosubtilin, produced by Bacillus subtilis, was discovered in 1949 by Walton and Woodruff, on account of its strong antifungal activities.1 Several years later, it was shown that this antifungal compound belongs to the iturin lipopeptide family, and its structure was determined (Figure 1).2,3 This family, which included also bacillomycins L, D, F, and iturin A, presents structural similarities. Indeed, these lipopeptides are characterized by a heptapeptide, with the LDDLLDL R-amino acid configuration, cyclized in a ring with a β-amino fatty acid (βAA). They all contain the sequence βAA-L-Asn-D-Tyr-D-Asn. Furthermore, because the treatments currently used to cure mycoses meet problems such the emergence of pathogenic strain resistance, there is increasing interest for new medical applications of antifungal molecules such as mycosubtilin.4 Nowadays, the production of mycosubtilin is still under optimization.5,6 Despite many works carried out on the optimization of its production and its effect on various fungi or yeast strains,4,7 only a few studies were conducted on the understanding of the molecular mechanisms of its biological activity. It has been demonstrated that mycosubtilin acts on plasma membrane of the target cells,7,8 inducing important modifications of the membrane permeability characterized by the release of biomolecules such as r 2011 American Chemical Society
nucleotides, proteins, and lipids from cells.7 It has been also determined that mycosubtilin has a lytic activity upon erythrocytes.8 This activity was inhibited when phosphatidylcholine/cholesterol multilayers were present in the medium; meanwhile, mycosubtilin remained hemolytic in the presence of pure phosphatidylcholine multilayers. Furthermore, it had been shown that mycosubtilin was able to form a complex with cholesterol in premixed lipopeptide lipid monolayers, while it was not miscible with phosphatidylcholine monolayers in the excess of lipids.9 Therefore, in our previous work, we analyzed the binding and/or the insertion of mycosubtilin into pure phoshatidylcholine monolayers as well as mycosubtilin-induced changes to their morphology.10 Because mycosubtilin seems have an affinity for cholesterol, a detailed investigation of mycosubtilin behavior toward cholesterolcontaining biomimetic membranes is required. Besides, it had been shown that the plasma membrane of erythrocytes contains microdomains, called lipid rafts, which are enriched in cholesterol and in sphingomyelin;11 thus, it seems reasonable to question Received: February 28, 2011 Revised: July 8, 2011 Published: July 18, 2011 10785
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Figure 1. Schematic structure of the lipopeptide mycosubtilin. The β-amino fatty acids (bold letters) are iso C16, n C16, iso C17, or anteiso C17. The alkyl chains are summarized by R.
whether mycosubtilin interacts preferentially with these microdomains. In this work, the interactions of mycosubtilin with multilamellar vesicles containing cholesterol and phospholipids were first examined by FT-IR spectroscopy to evaluate mycosubtilin behavior toward a biomimetic system containing cholesterol. The interactions of mycosubtilin with different pure lipid monolayers then were examined by tensiometry measurements of the lipopeptide adsorption to these monolayers. For each monolayer, the critical pressure value (πc) for mycosubtilin insertion was determined. Afterward, we studied more and more complex monolayers, like binary and ternary systems containing cholesterol. Finally, mycosubtilin-induced changes in the isotherm of cholesterol-containing monolayers (pure cholesterol, DPPC/ cholesterol, and DPPC/sphingomyelin/cholesterol) were analyzed. In parallel, morphological mycosubtilin-induced changes in these monolayers were investigated by Brewster angle microscopy.
’ MATERIALS AND METHODS Chemicals. Bovine brain sphingomyelin (SM), cholesterol (Chol), dimethylsulfoxide (DMSO), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. All organic solvents were analytical grade. Deuterium oxide (99.9%) was purchased from Merck (Darmstadt, Germany). The ultrapure water, purified with a Millipore filtering system (Bedford, MA), had a resistivity of 18.2 MΩ cm. Stock solutions of SM, DPPC, or Chol were obtained by dissolving them at 0.545 mM in hexane ethanol (9/1, v/v). DPPC/Chol and DPPC/ Chol/SM solutions were obtained by mixing the stock solutions at the desired mole ratio. Mycosubtilin was prepared from cultures of Bacillus subtilis strain according to Peypoux et al.2 The purity of the lipopeptide, checked by thin-layer chromatography,12 was about 98%. Mycosubtilin solution was obtained by dissolving it into DMSO at 0.545 mM. Interfacial Film Formations and Surface Pressure Measurements. The film balance was built by R&K (Riegler & Kirstein GmbH, Wiesbaden, Germany) and equipped with a Wilhemy-type surfacepressure-measuring system. The subphase was pure water. All monolayer experiments were performed at 21 C as previously described.13,14 Adsorption Experiments at Constant Surface Area. Adsorption experiments were performed on a small Teflon dish (diameter, 4 cm) with a subphase volume of 10 mL. The subphase was stirred with a magnetic stirrer spinning at 100 rev/min. Lipids (in hexane ethanol at 0.545 mM) were spread at the air water interface to reach the desired final surface pressure. Thirty minutes was required for solvent evaporation and film stabilization.15 Mycosubtilin was then injected at a final concentration of 0.545 μM into the subphase. The lipopeptide adsorption at the air water interface, measured by tensiometry, was followed as an increase in surface pressure. The same volume of pure DMSO was injected under the lipid monolayer as control, and no change in surface pressure was detected.
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Isotherm Measurements. Pure or mixed lipid solutions were spread at the air water interface of the Langmuir trough. Its area was 165 cm2, and its subphase volume was 120 mL. After 30 min of waiting for solvent evaporation, the different lipid films were compressed to obtain π/A isotherms, which will be considered as control isotherms. The compression rate was 5 Å2/molecule/min, corresponding to 6 cm2/min. To examine the influence of mycosubtilin on the different lipid films, the lipopeptide was injected into the subphase after stabilization of the pure or mixed lipid monolayers. The molar ratio between spread lipids and injected mycosubtilin was 1/1. A 40 min delay was then required to allow the lipopeptide to reach the air water interface. The film was then compressed as mentioned above, and the π/A isotherm was recorded. Each isotherm is representative of at least two independent measurements. Because it had been reported that DMSO, the solvent of mycosubtilin, could affect the isotherm of lipid monolayers,16 control assays were performed by injecting the same volume of DMSO without mycosubtilin underneath the lipid monolayer, and no significant effect of DMSO was observed. This could be explained by our injected amount of DMSO, which is about 1000 times less important than that used by Chen et al.16 Brewster Angle Microscopy Measurements. The morphology of lipid and mixed lipid/mycosubtilin monolayers at the air water interface was observed with a Brewster angle microscope (NFT iElli2000, G€ottingen, Germany). This microscope was mounted on an R&K Langmuir trough (Riegler & Kirstein GmbH, Wiesbaden, Germany) with 165 cm2 area filled with 120 mL of subphase. BAM images were acquired at different camera shutter speeds. Indeed, due to the compression-induced increase of the monolayer thickness, it is important to use different shutter speeds for a complete analysis of the relative thickness of the film during its overall compression. The camera shutter timing makes it possible to select the exposure time to adapt to different illumination levels. The thicker monolayers required the shorter exposure times, corresponding to the lower obturation speed (OS), to obtain an image. BAM images, surface pressures, and gray levels (GL) were recorded simultaneously during the compression of the monolayers. The compression rate was 3 Å2/molecule/min, corresponding to 2 cm2/min. The BAM spatial resolution was about 2 μm, and the image size was 430 320 μm. Each image is representative of at least two series of measurements. Because DMSO could affect lipid monolayers, a control assay was performed for each monolayer by injecting the same volume of pure DMSO, as that injected with mycosubtilin, into the subphase. No significant effect of DMSO on the monolayer morphologies was noticed, probably because of the low relative quantity of DMSO used in our work. Preparation of Multilamellar Vesicle Samples. Multilamellar vesicles (MLV) containing DMPC and cholesterol (70/30, molar ratio) were prepared as described in Nasir et al.10 The lipid/mycosubtilin molar ratio was 30/1. In all cases, the different compounds were dissolved in a TFE chloroform solvent mixture. FTIR Spectroscopy. Infrared spectra were recorded by means of a Nicolet 510 M FTIR spectrometer after 128 scans at 4 cm 1 resolution.17 The spectrometer was continuously purged with filtered dry air. All experiments were performed with a demountable temperature-controlled flow-through cell (Harrick) equipped with CaF2 windows. The spectrum of 2H2O alone was subtracted from the sample spectrum taken under the same conditions. In the case of MLV spectra, a 20 μL sample of the different MLV was deposited on the CaF2 window. Each spectrum is representative of at least three independent measurements.
’ RESULTS Mycosubtilin Interactions with Cholesterol-Containing Multilayers. MLV constituted by the DMPC and cholesterol
was prepared in the absence and in the presence of mycosubtilin, and the interactions between the lipopeptide and 10786
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Figure 2. FTIR analyses of the mycosubtilin interaction with phospholipids in MLV. Part (A) shows the CdO ester and amides regions, part (B) shows the CH stretching vibration region, and part (C) shows the phosphate region. MLV contained pure DMPC/Chol (gray lines) or mycosubtilin/DMPC/Chol (black lines). The inset compares the amide I region of the mycosubtilin/DMPC/Chol ( ) with the amide I region of the mycosubtilin/DMPC (- - -).
DMPC/cholesterol MLV were analyzed by FTIR spectroscopy (Figure 2). FTIR spectra of DMPC/cholesterol MLV with or without the lipopeptide show a band centered at 1739 cm 1, corresponding to the vibrations of CdO ester groups (Figure 2A), and another band centered at 1237 cm 1, characteristic of phosphate residues (Figure 2B).18,19 These data suggest that mycosubtilin did not interact strongly with the phospholipid CdO ester and phosphate groups. Besides, mycosubtilin did not induce significant change in the location of the CH stretching vibrations of the DMPC acyl chains (Figure 2C). The 1700 1600 cm 1 regions of the FTIR spectra, corresponding to the amide I and amide II bands,20 22 were analyzed to determine the influence of the lipopeptide insertion into the DMPC/cholesterol multilayers on its peptide ring conformation. Figure 2A indicates that only the spectrum recorded in the presence of mycosubtilin displays an amide I band with two maxima at 1654 and 1643 cm 1 and a shoulder at 1668 cm 1. A residual amide II band was observed at 1554 cm 1, and the band at 1520 cm 1 was due to the absorbance of the tyrosyl residue of mycosubtilin. These bands probe the presence of the lipopeptide in the DMPC/cholesterol MLV. The different IR bands observed with the mycosubtilin DMPC/cholesterol MLV were compared to the previously described ones of mycosubtilin alone or inserted in DMPC MLV10 (inset of Figure 2A and Table 1). No
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significant change in the location of the amide I and tyrosyl residue bands was observed in the FTIR spectrum of mycosubtilin alone or inserted in DMPC MLV. These data suggest that the peptide ring and the tyrosyl residue did not participate to the interactions with DMPC. The amide I band of mycosubtilin inserted in DMPC/cholesterol MLV shows a maximum at 1643 cm 1 (very similar to the 1644 cm 1 band of mycosubtilin alone or inserted in DMPC MLV), and another one at 1654 cm 1 appears only in the case of mycosubtilin/DMPC/ cholesterol MLV, suggesting that there are two populations of peptide backbone CdO groups. The first population of CdO (corresponding to the 1643 cm 1 band) represents the carbonyl groups, which are involved in intramolecular hydrogen bonds in the pure mycosubtilin. The second population of CdO (corresponding to the band located at 1654 cm 1), corresponding to carbonyl groups less involved in hydrogen bonds, are generated during the interaction of the mycosubtilin with the lipid molecules.22 The second population, appearing only in the presence of cholesterol, could suggest that the less hydrogenbounded CdO arise from interactions between mycosubtilin and cholesterol. Furthermore, the tyrosyl residue band (due to the phenol ring)17 shifted from 1517 cm 1, for mycosubtilin alone or inserted in DMPC MLV, to 1520 cm 1 for mycosubtilin/DMPC/cholesterol MLV. This shift could be explained by the involvement of the tyrosyl residue in the interaction with cholesterol or DMPC/cholesterol complex. Mycosubtilin Adsorption to Interfacial Monolayers Constituted by Pure Sphingomyelin, Phosphatidylcholine, or Cholesterol. The involvement of the interfacial properties of mycosubtilin in its biological activities upon membranes was studied using the Langmuir balance technology. The lipid specificity of mycosubtilin was analyzed by injecting the lipopeptide under different monomolecular film at a constant area. The resulting interaction was measured as an increase in the surface pressure of monolayers constituted by pure DPPC, cholesterol, or sphingomyelin. The kinetic curves, obtained for an initial surface pressure (πi) of 10 mN/m (Figure 3, inset), show that mycosubtilin induced an increase of about 8 mN/m in the surface pressure of the DPPC or SM monolayer. Faster and greater increases of π were observed when the lipopeptide was injected under a cholesterol monolayer, indicating that the presence of sterol in the monolayer had a positive effect on the mycosubtilin insertion (Figure 3, inset). To further assess the lipid specificity, the lipopeptide was injected under the different lipid monolayers at various πi values. After reaching the equilibrium, the maxima of the mycosubtilininduced increase in π (Δπ) were plotted versus πi (Figure 3). The values of critical pressure (πc) for mycosubtilin insertion into a SM monolayer (i.e., the theoretical value of πi extrapolated for Δπ = 0 mN/m) were about 25 mN/m. In the case of DPPC, the analysis of the data is more complicated because DPPC exhibits different physical states at the temperature of the measurement. Thus, according to Calvez et al.,23 two curves with different slopes were drawn, and two values of πc, 14 and 28 mN/m, were obtained, corresponding to the LE state and the LC state, respectively. Furthermore, the length of the carbon chain of the phosphatidylcholine did not significantly affect the interaction of the lipopeptide with the glycerophospholipid because DMPC and DPPC had similar πc values (Figure 5). The Δπ versus πi plot obtained with pure cholesterol monolayer reflects an original behavior of mycosubtilin as compared to the plots obtained for pure DPPC and SM. Indeed the Δπ versus 10787
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Table 1. Comparison of the Location (cm 1) of the IR Bands of Pure Mycosubtilin (MS), DMPC, and DMPC/Chol MLV with or without Mycosubtilina pure MS17
a
DMPC MLV10
MS/DMPC MLV10
DMPC/Chol MLV
MS/DMPC/Chol MLV
CH3 asymmetric streching
nd
2955
2955
2955
2955
CH2 asymmetric streching CH2 symmetric streching
nd nd
2917 2849
2917 2849
2919 2850
2919 2850
CdO ester
abs
1735
1736
1739
1739
amide I
1643
abs
1644
abs
1654 and 1643
tyrosyl residue
1517
abs
1517
abs
1520
nd = non determined; abs = absent.
Figure 3. Interaction of mycosubtilin with interfacial monolayers constituted by pure lipids. Influence of the initial surface pressure (πi) on the maximal pressure variation (Δπ) induced by mycosubtilin adsorption onto monolayers. Each point was obtained from an independent experiment. 2 correspond to DPPC, 0 to sphingomyelin, and 4 to cholesterol. Final concentration of mycosubtilin was 0.545 μM. The inset gives the kinetics of mycosubtilin adsorption in the presence of the different pure lipids. The arrow indicates the injection of lipopeptide under the lipid monolayer at about 10 mN/m. Each curve is representative of at least two independent assays.
πi plot, obtained with pure cholesterol, shows two parts. Similar biphasic curves had been obtained with surfactin, another lipopeptide produced by Bacillus subtilis.24 The first one had a positive slope, while the second one had a negative slope. For the lowest πi, the mycosubtilin insertion increases with increasing initial surface pressures of cholesterol monolayer. This can be explained by a specific affinity of mycosubtilin for the lipid. For the highest initial πi, the mycosubtilin insertion decreases with increasing initial surface pressure values as observed in the case of pure DPPC and SM. Nevertheless, there was a critical πi value from which the increase of mycosubtilin insertion into the cholesterol monolayer is stopped, and the estimated value of πc for mycosubtilin insertion into a pure cholesterol monolayer was 50 mN/m. This value of πc, which is higher than the surface pressure supposed to prevail in biological membranes,25 and the original πi versus Δπ plot suggest that cholesterol may have an important role in the mycosubtilin insertion within biological membranes. Mycosubtilin Adsorption to Interfacial Monolayers Constituted by Different Lipids. Up to now, we characterized the mycosubtilin behavior toward pure lipids. We then analyzed more complex biomimetic membranes, such as the binary system constituted by DPPC (or DMPC) and cholesterol in a 7/3 mol ratio and the ternary system constituted by DPPC/SM/Chol in an
Figure 4. Interaction of mycosubtilin with interfacial monolayers constituted by mixed lipids. Influence of the initial surface pressure (πi) on the maximal pressure variation (Δπ) induced by mycosubtilin adsorption onto monolayers. Each point was obtained from an independent experiment. ] correspond to DPPC/cholesterol (7/3, mole ratio), and [ correspond to DPPC/cholesterol/sphingomyelin (1/1/1, mole ratio). Final concentration of mycosubtilin was 0.545 μM. The inset gives the kinetics of mycosubtilin adsorption in the presence of the different mixture of lipids. The arrow indicates the injection of lipopeptide under the lipid monolayer at about 10 mN/m.
Table 2. Comparison of the Initial Velocity of the Mycosubtilin Adsorption to Different Lipid Monolayers initial velocitya (mN/m/min) DPPC
0.39
SM
0.81
Chol DPPC/Chol
1.90 0.52
DPPC/SM/Chol
1.40
a
Initial velocities were determined by linear regression of the linear part of the adsorption curves of Figures 3 and 4.
equimolar ratio. The kinetic curves (Figure 4, inset) show the increase of surface pressure after mycosubtilin adsorption onto DPPC/cholesterol monolayers at an initial surface pressure of 10 mN/m. Comparatively to the adsorption kinetics obtained with pure lipid monolayers, the adsorption of mycosubtilin to the DPPC/cholesterol monolayer was faster than those obtained with the pure DPPC or SM monolayers, while it was slower than those obtained with pure cholesterol monolayer (Table 2). Besides, the mycosubtilin adsorption to the DPPC/SM/cholesterol monolayer was similar to those obtained with the pure cholesterol monolayers and was faster than those obtained with the 10788
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Figure 5. Influence of the lipid composition of the interfacial monolayers on the exclusion surface pressures. The estimated surface pressure of biological membranes between 25 and 30 mN/m was indicated by dashed lines. The exclusion pressures (πc) were obtained by linear regression (y = 0.539x + 16.692 with R2 = 0.9771 for DMPC; y = 0.4441x + 12.487 and R2 = 0.9175 for DPPC; y = 0.5571x + 14.208 and R2 = 0.6891 for SM; y = 1.0233x + 51.296 and R2 = 0.9848 for Chol; y = 0.5506x + 19.578 and R2 = 0.9844 for DMPC/Chol; y = 0.5924x + 25.479 and R2 = 0.9928 for DPPC Chol; y = 0.3568x + 18.347 and R2 = 0.9945 for DPPC/Chol/SM). The errors on πc were calculated using IGOR Pro software.
Figure 6. Influence of the acetylation of the secondary alcohol residue of cholesterol on the mycosubtilin adsorption to interfacial monolayers. [ correspond to the adsorption of mycosubtilin to pure cholesteryl acetate monolayer, and the data obtained with pure cholesterol (4), shown in Figure 4, were given for helping the comparison. The inset compares the exclusion pressure of mycosubtilin from the same phospholipids. The exclusion pressure (πc) of mycosubtilin from pure cholesteryl acetate monolayer was obtained by linear regression (y = 0.7568x + 16.832 and R2 = 0.9313). The error on πc was calculated using IGOR Pro software.
monolayers constituted by DPPC/cholesterol or by pure DPPC or SM (Table 2). Figure 4 shows the estimation of the πc for mycosubtilin insertion into mixed lipid monolayers. Both curves were biphasic, as in the case of pure cholesterol monolayer, and πc values are estimated to 43 mN/m for DPPC/Chol monolayer and to 52 mN/m for DPPC/SM/Chol monolayer. These πc values are higher than those obtained with pure phospholipid (DPPC and SM) monolayers, and the πc value obtained with DPPC/SM/ Chol monolayer was very close to that obtained with pure cholesterol monolayer. Figure 5 compares the πc values of mycosubtilin for the different monolayers. The πc values obtained with pure cholesterol and mixed lipid monolayers are higher than the estimated surface pressure values of biological membranes.25
Figure 7. Π/A isotherms of Chol (A), DPPC/Chol (B), or DPPC/ SM/Chol (C) monolayer alone or in the presence of mycosubtilin. “- - -” and “ ” correspond to the isotherm of the lipid monolayer measured before and 40 min after mycosubtilin injection, respectively.
Thus, mycosubtilin inserts more easily into cholesterol-containing monolayers, but this behavior seems to be dependent on the phospholipid composition of the cholesterol-containing monolayers. Indeed, even though the DMPC/Chol, DPPC/Chol, and DPPC/SM/Chol monolayers contain similar mole ratios of cholesterol (about 30%), mycosubtilin was better inserted into the DPPC/SM/Chol monolayer. This led us to consider a synergic effect when cholesterol was in the presence of phosphatidylcholine and sphingomyelin. Mycosubtilin Adsorption to Interfacial Monolayers Constituted by Cholesteryl Acetate. Previous in vivo studies suggested 10789
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Figure 8. Influence of mycosubtilin on the organization of cholesterol monolayer. BAM images before (left column) or 40 min after mycosubtilin injection (right column). Images were taken at different obturation speeds (OS).
a possible involvement of the secondary alcohol residue of cholesterol.26 Thus, the mycosubtilin adsorption toward cholesteryl acetate monolayers was then analyzed at different initial surface pressures (data not shown). The experimental Δπ values obtained with pure cholesteryl acetate monolayers were plotted versus πi (Figure 6). The resulting πc value was significantly lower than the πc value previously determined for pure cholesterol monolayer. Furthermore, the cholesteryl acetate curve was monophasic, showing that the original behavior of mycosubtilin toward cholesterol-containing monolayer needs the presence of the secondary alcohol residue in the cholesterol molecule (Figure 6). Influence of Mycosubtilin on the Lipid Monolayer Isotherm and Morphology. The interactions between mycosubtilin and the different monolayers were examined by comparing the isotherms of pure cholesterol, DPPC/Chol, or DPPC/SM/ Chol monolayers in the presence and in the absence of the lipopeptide. These lipid monolayers were chosen because of the original behavior of mycosubtilin toward them during the adsorption experiments. In parallel, the morphology of the different monolayers was investigated by Brewster angle microscopy. The π/A isotherms of cholesterol and cholesterol mycosubtilin monolayers are shown in Figure 7A. The isotherm of pure cholesterol monolayer depicts one condensed phase. The extrapolation of the isotherm curve to π = 0 gave a molecular area (A0) of ∼40 Å2/molecule in agreement with previous results.27 In the presence of mycosubtilin, the isotherm curve was parallel to those of pure cholesterol and shifted toward high molecular area (A0 = 49 Å2/molecule) due to the insertion of mycosubtilin into the cholesterol monolayer. The shift was still observed up to 30 mN/m (the supposed surface pressure within biological membranes), revealing the stability of cholesterol mycosubtilin monolayer. Figure 8 shows the BAM images at different surfaces pressures of the cholesterol monolayer with or without mycosubtilin. At 0 1 mN/m, pure cholesterol monolayer showed white circle-shaped domains with different sizes on a dark background. In the presence of mycosubtilin, for lowest surface pressures, similar circle-shaped domains were observed. These data are consistent with the coexistence of gaseous and solid phases of cholesterol monolayer and agree with previous data.28 When the surface pressure reached about 5 mN/m, the film of pure
Figure 9. Influence of mycosubtilin on the organization of DPPC/Chol monolayer. BAM images before (left column) or 40 min after mycosubtilin injection (right column). Images were taken at different obturation speeds (OS).
cholesterol became homogeneous, pointing out only a solid phase. In the presence of mycosubtilin and at similar surface pressures, the film was not homogeneous, and small brighter dots appear. Furthermore, the gray level values, correlated to the thickness of the film, increased in the presence of the lipopeptide. This confirms again the insertion of the lipopeptide into the monolayer. The π/A isotherms of DPPC/Chol monolayer recorded in the absence and in the presence of mycosubtilin are shown in Figure 7B. No transition phase was observed with the DPPC/ Chol monolayer, and an A0 of about 85 Å2/molecule can be deduced from the isotherm. In the presence of mycosubtilin, the isotherm curve shifted toward higher molecular areas (A0 = 100 Å2/molecule), and the shift was still observed at 30 mN/m. Figure 9 shows the mycosubtilin-induced morphological changes in monolayers constituted by DPPC/cholesterol (7/3, mol ratio). At 1 mN/m, the DPPC/cholesterol monolayer was inhomogeneous and contained small circular spots on a dark background. These images are consistent with the coexistence of liquid expanded liquid condensed transition phases.28 When the surface pressure reached about 5 mN/m, the film became more and more homogeneous, indicating the existence of a condensed phase. When the compression increased, the thickness of the film increased. In the presence of mycosubtilin, the 10790
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mycosubtilin into these lipids. The shift at the lowest compressions was greater for DPPC/SM/Chol monolayer, as compared to the others, agreeing with a greater interaction of the lipopeptide with this monolayer at the lowest surface pressures. The progressive decrease of the mycosubtilin-induced shift that was observed with DPPC/Chol and DPPC/SM/Chol monolayers could be explained by the expulsion of the lipopeptide from the monolayer or by the condensation of lipids or mycosubtilin lipid molecules during the compression. In this case, mycosubtilin would remain in the monolayer along the compression, while the molecular area decreases because of the condensation of the molecules. The morphology of the DPPC/SM/Chol monolayer containing the three lipids at equimolar ratio was examined in the absence and in the presence of mycosubtilin (Figure 10). Below 5 mN/m, DPPC/SM/cholesterol monolayer was heterogeneous, and bright strips separated by a dark background and the small bright circle-shaped domains were observed. For pressures higher than 7 mN/m, the monolayer forms a homogeneous film, and the thickness of the film increased during its compression. When mycosubtilin was interacting with a DPPC/SM/cholesterol monolayer, the BAM images were different from those of the monolayer containing only lipids. The mycosubtilin/DPPC/ SM/cholesterol film remained heterogeneous all along the compression, and the strips at different brightness were still observed. This might be due to the coexistence of several domains formed by different lipids, by mycosubtilin and some lipids, or by mycosubtilin molecules themselves. Furthermore, at each surface pressure, the gray level decreased in the presence of mycosubtilin, pointing out that mycosubtilin could fluidize the monolayer.
Figure 10. Influence of mycosubtilin on the organization of DPPC/ SM/Chol monolayer. BAM images before (left column) or 40 min after mycosubtilin injection (right column). Images were taken at different obturation speeds (OS).
’ DISCUSSION Mycosubtilin belongs to the iturin lipopeptide family, as iturins and bacillomycins. Among them, which all exhibit strong antifungal activities, mycosubtilin is the most active.26 Previous works showed that these antifungal activities could be inhibited by adding free sterol in the culture medium of the sensitive cells and that the phenolic OH of their tyrosine residues was involved in their antifungal activities.26 Furthermore, the biological activities of mycosubtilin, as those of the other iturinic lipopeptides, had been correlated to its interactions with the plasma membrane of the sensitive cells, which all contain a sterol. To better understand the behavior of mycosubtilin toward biological membranes, these interactions were modeled using biomimetic membrane systems.
heterogeneity of the mycosubtilin/lipid mixed film disappeared later during the compression, as compared to the DPPC/cholesterol monolayer, and the relative thickness of the film decreased. This might be due to a possible fluidizing effect of mycosubtilin on the lipid monolayer. The isotherms of DPPC/SM/Chol monolayer with and without mycosubtilin are shown in Figure 7C. The compression of lipid monolayer alone resulted in an isotherm with no phase transition, and A0 can be estimated to about 59 Å2/molecule. In the presence of mycosubtilin, the isotherm curve shifted toward higher molecular area (A0 = 81 Å2/molecule), and meanwhile both curves converged all along the compression, yet the shift was still observed at 30 mN/m. The isotherms of pure cholesterol, DPPC/Chol, and DPPC/SM/Chol monolayers, showing a shift toward higher molecular area, suggest an insertion of
containing pure lipid allowed us to determine that mycosubtilin exhibits preferential affinity for cholesterol because its exclusion pressure from pure cholesterol monolayer was superior to the postulated pressure prevailing within the biological membranes. Furthermore, the secondary alcohol residue of cholesterol seems to be involved in its interaction with mycosubtilin because its exclusion pressure from pure cholesteryl acetate monolayer was significantly lower than the exclusion surface pressure from cholesterol monolayer. In the presence of cholesterol-containing bilayers, a shift of the IR absorption band, due to the tyrosine residue of the lipopeptide, was observed, suggesting that the phenol ring of the tyrosine residue of mycosubtilin interacted with the sterol. This hypothesis could be supported by a previous study, showing that the derivation (methylation or acetylation) of the phenolic OH of the tyrosine residue of mycosubtilin
Activity Structure Relationship during Mycosubtilin Membrane Interactions. Using different interfacial monolayers
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Langmuir abolished its biological activities.8,26 Thus, the changes in the amide I region of the mycosubtilin IR spectrum measured in the presence of cholesterol-containing bilayers could be explained by cholesterol-induced conformational modifications. Furthermore, previous NMR data analysis of mycosubtilin29 showed that the peptide ring is rather rigid in the region of L-proline, while the neighborhood of D-tyrosine is more flexible. This flexibility could favor the interactions between the phenol ring of the tyrosine residue of mycosubtilin and cholesterol. Are the Lipid Rafts the Cell Membrane Target of Mycosubtilin? Thus, in a first approach, the action of mycosubtilin upon biological membranes could be explained by a specific affinity of mycosubtilin toward cholesterol, but cholesterol of the plasma membranes is not alone and is surrounded by other lipids. Thus, an investigation strategy based on increasing step-by-step the complexity of system allowed us to determine the influence of the lipid composition of the monolayers on the lipopeptide interaction. Mycosubtilin inserted similarly into DPPC/sphingomyelin/cholesterol and pure cholesterol monolayers (similar exclusion surface pressure values), while it inserted more easily into DPPC/sphingomyelin/cholesterol monolayer than into DPPC/ cholesterol monolayer, even though these two monolayers contain the same amount of sterol. This suggests an improvement of mycosubtilin interactions with sterol when the latter is in the presence of DPPC and sphingomyelin in a more ordered state. In fact, the lipid composition of the DPPC/sphingomyelin/cholesterol used in this work could be considered to mimic the lipid composition of some plasma membrane domains, which are clusters of lipids in a more ordered state now referred to as lipid rafts.30 These lipid rafts are enriched in sterol, glycosphingolipids, and phospholipids with saturated acyl chains. Thus, the role of sterol seems to be essential in mycosubtilin membrane interaction, but the lipopeptide may have a preference for interacting with sterol in lipid rafts. It is then reasonable to question whether mycosubtilin acts on the lipid rafts of the sensitive cells. Indeed the iturinic lipopeptides are hemolytic compounds, and it has been shown that iturin A, another iturinic lipopeptide differing from mycosubtilin only by the inversion of two amino acids,22 induces the hemolysis of erythrocytes with concomitant release of vesicles. These vesicles are enriched in cholesterol, sphingomyelin, and phosphatidylcholine.31 Such data might suggest that the membrane target of other mycosubtilin-sensitive cells might be the lipid rafts. This hypothesis could be confirmed by in vivo experiments by looking for mycosubtilin-induced damages in the lipid rafts of sensitive cells.
ARTICLE
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’ AUTHOR INFORMATION Corresponding Author
*Tel.: +33 04 26 23 44 13. Fax: +33 04 72 43 15 43. E-mail:
[email protected].
’ ACKNOWLEDGMENT We thank Dr. Gladys Matar for fruitful discussions. M.N.N. is the recipient of a Ph.D. fellowship from the French Ministere de l’Enseignement Superieur et de la Recherche. This work was supported by the Centre National de la Recherche Scientifique. ’ REFERENCES (1) Walton, R.; Woodruff, H. J. Clin. Invest. 1949, 28, 924. 10792
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