Article pubs.acs.org/Langmuir
Interaction of the Lipopeptide Biosurfactant Lichenysin with Phosphatidylcholine Model Membranes Jonathan R. Coronel,† Ana Marqués,‡ Á ngeles Manresa,‡ Francisco J. Aranda,§ José A. Teruel,§ and Antonio Ortiz*,§ †
Escuela Superior Politécnica del Litoral, ESPOL, Facultad de Ingeniería Mecánica y Ciencias de la Producción, Campus Gustavo Galindo, P.O. Box 09-01-5863, Guayaquil, Ecuador ‡ Laboratorio de Microbiología, Facultad de Farmacia, Universidad de Barcelona, Joan XXIII s/n, E-08028 Barcelona, Spain § Departamento de Bioquímica y Biología Molecular-A, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, E-30100 Murcia, Spain ABSTRACT: Lichenysins produced by Bacillus licheniformis are anionic lipopeptide biosurfactants with cytotoxic, antimicrobial, and hemolytic activities that possess enormous potential for chemical and biological applications. Through the use of physical techniques such as differential scanning calorimetry, small- and wide-angle X-ray diffraction, and Fourier-transform infrared spectroscopy as well as molecular dynamics simulations, we report on the interaction of Lichenysin with synthetic phosphatidylcholines differing in hydrocarbon chain length. Lichenysin alters the thermotropic phase behavior of phosphatidylcholines, displaying fluid-phase immiscibility and showing a preferential partitioning into fluid domains. The interlamellar repeat distance of dipalmitoylphosphatidylcholine (DPPC) is modified, affecting both the phospholipid palisade and the lipid/water interface, which also experiences a strong dehydration. Molecular dynamics confirms that Lichenysin is capable of interacting both with the hydrophobic portion of DPPC and with the polar headgroup region, which is of particular relevance to explain much of its properties. The results presented here help to establish a molecular basis for the Lichenysin-induced perturbation of model and biological membranes previously described in the literature.
1. INTRODUCTION Biological surfactants, or biosurfactants, are commonly described as chemical compounds of microbial origin that exhibit surface activity. The interest in these compounds has been growing lately, on the one hand, because of the diversity of their physiological roles, increasing accessibility to substrates, participating in defensive actions, or contributing to biofilm formation1−4 and, on the other hand, due to the rich diversity of chemical structures that can be found, which results in an important set of remarkable biological properties and potential applications.5,6 Because of these interesting biological actions the number of biosurfactants described in recent years has increased, mainly within the group of glycolipids and lipopeptides.5,6 Accordingly, research on biosurfactants is experiencing a growing interest to the point that some of them, like Pseudomonas aeruginosa rhamnose glycolipids, are being commercialized as antifungal compounds for agriculture (Zonix, Jeneil Biosurfactant Company, USA). Biosurfactants with lipopeptide structure are among the most remarkable biosurfactants found so far.7 The surfactin family, composed of heptapeptides interlinked with a β-hydroxy fatty acid to form a cyclic lactone ring structure,8 is characterized by powerful surfactants with exceptional properties.9 Following © XXXX American Chemical Society
surfactins, iturins, bacillomycins, mycosubtilins, or fengycins also display a strong in vitro antifungal action against a wide variety of yeast and fungi but only limited antibacterial and no antiviral activities described so far.10−13 The fungal toxicity of iturins has been attributed to its interaction with target membranes, and the underlying mechanism has been established on the basis of an osmotic perturbation through formation of ion-conducting pores,14 differing from the disruption or solubilization of the target membrane caused by surfactin.15 A third group of very interesting lipopeptide biosurfactants is formed by lichenysins. Lichenysins A, B, C, D, and G produced by Bacillus licheniformis are anionic surfactants due to the presence of Asp or Glu residues.16−20 Although lichenysins structurally could be placed within the family of surfactin, the presence of glutamine in position 1 replacing the glutamic acid of surfactin gives rise to distinct physicochemical and biological properties that deserve experimental investigation. Received: June 1, 2017 Revised: August 12, 2017
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DOI: 10.1021/acs.langmuir.7b01827 Langmuir XXXX, XXX, XXX−XXX
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detector (PSD; M. Braun, Garching, Germany), monitoring the s range (s = 2 sin θ/λ, 2θ = scattering angle, λ = 1.54 Å) between 0.0075 and 0.07 Å−1. Nickel-filtered Cu Kα X-rays were generated by a Philips PW3830 X-ray generator (Eindhoven, The Netherlands) at 50 kV and 30 mA. The calibration of the detector position was performed by using silver stearate (d-spacing at 48.8 Å) as reference material. Background-corrected SAXD data were analyzed using the program GAP (Global Analysis Program) written by Georg Pabst and obtained from the author.27,28 This program allowed us to retrieve the membrane thickness, dB = 2(zH + 2σH) from a full q-range analysis of the SAXD patterns.29 The parameters zH and σH are the position and width, respectively, of the Gaussian used to describe the electron dense headgroup regions within the electron density model. 2.4. Fourier Transform Infrared Spectroscopy. For the FTIR, multilamellar vesicles were prepared, essentially as described above, in 50 μL of a D2O buffer containing 150 mM NaCl, 0.1 mM EDTA, 5 mM Hepes pD 7.4. CaF2 windows (25 × 2 mm) separated by 25 μm Teflon spacers were mounted in a thermostated Symta cell holder. Infrared spectra were collected in a Nicolet 6700 FTIR spectrometer (Madison, WI). The equipment was continuously purged with dry air to minimize the contribution arising from the peaks of atmospheric water vapor. The sample holder was thermostated using a Peltier device (Proteus system from Nicolet). Each spectrum was obtained by acquiring 128 interferograms at a resolution of 2 cm−1. Spectra were collected at 2 °C intervals, allowing 5 min of equilibration between temperatures. The D2O buffer spectra taken at the same temperatures were subtracted interactively using either Omnic or Grams (Galactic Industries, Salem, NH) software. 2.5. Molecular Dynamics. DPPC topology file was obtained from Chiu and coworkers.30 The Lichenysin molecule (Figure 1) and the
Given the characteristics of lichenysins, a better understanding of the molecular events underneath their biological actions is essential to explain their properties and to develop new applications. However, as compared with the vast amount of information available on the physicochemical and biological properties of surfactins,9,21 few works have addressed the study of lichenysins. So far, Lichenysin G has been shown to have a higher surface activity than surfactin given its much lower critical micellar concentration,17 which perhaps might be related to the reported cytotoxic,22 antimicrobial,23 or hemolytic activities18,22,24 of this lipopeptide. Recently, we have shown that lipid composition is essential for Lichenysin action on membranes and that the presence of Lichenysin in the membrane increases its permeability to hydrophilic molecules, which can then easily cross the lipid palisade.24 With the aim of gaining deeper insight into the physicochemical basis of these findings, here we present a detailed biophysical study on the interaction of Lichenysin with various phosphatidylcholine model membranes, showing molecular details of the phospholipid/lipopeptide interactions and the perturbations exerted by Lichenysin on the phospholipid bilayer.
2. EXPERIMENTAL SECTION 2.1. Materials. Dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC) were from Avanti Polar Lipids (Birmingham, AL). All inorganic salts and buffers were of analytical grade. The bacterial lipopeptide Lichenysin was produced, purified, and characterized as described before.25 Purified water was deionized in a Milli-Q equipment (Millipore, Bedford, MA) (resistivity 18 MΩ). Stock solutions of the various phospholipids were prepared in chloroform/ methanol (2:1) and stored at −80 °C. Phospholipid phosphorus was determined according to the method of Böttcher.26 Water and all the buffer solutions used were filtered through 0.2 μm filters prior to use. The osmolarity of all the buffers and solutions was routinely checked using an Osmomat 030 osmometer (Gonotec, Berlin, Germany). 2.2. Differential Scanning Calorimetry. The lipid mixtures for differential scanning calorimetry (DSC) measurements were prepared by mixing organic solvent solutions containing the phospholipids DMPC, DPPC, or DSPC, or their mixtures as indicated, and Lichenysin at the appropriate proportions. Dry N2 was used to remove the organic solvent, and the last traces were eliminated by a further 3 h of evaporation in a vacuum chamber. To the dry samples, 2 mL of a buffer containing 150 mM NaCl, 0.1 mM EDTA, 5 mM Hepes pH 7.4 was added, and vesicles were formed by vortexing the mixture, keeping the sample temperature above the gel to liquidcrystalline phase-transition temperature of the phospholipid or phospholipid mixture. Experiments were performed using a MicroCal MC2 calorimeter (MicroCal, Northampton, USA) at a final phospholipid concentration of 1 mg mL−1 and a heating scan rate of 60 °C h−1. Partial phase diagrams were elaborated using data from heating thermograms for a given mixture of phospholipids and lipopeptide at various proportions. The onset and completion temperatures for each transition peak were plotted as a function of the molar fraction of Lichenysin, which allowed us to define the boundary lines of the partial temperature−composition phase diagram. 2.3. X-ray Diffraction. Samples for X-ray diffraction analysis were prepared essentially as described above for DSC, centrifuged in a bench microfuge and placed in the sample holder with the aid of a spatula. A steel holder with cellophane windows was used, which provided an excellent thermal contact to the Peltier heating unit. An exposure time of 5 min was used, with a 10 min equilibration period prior to each measurement. Small-angle (SAXD) and wide-angle (WAXD) X-ray diffraction measurements were carried out simultaneously using a Kratky compact camera (M. Braun-Graz Optical Systems, Graz, Austria), provided with a linear position sensitive
Figure 1. Chemical structure of the Bacillus licheniformis Lichenysin D used in this study, containing the sequence QLIVDIL linked to a βhydroxymyristoyl group. molecular dynamics simulations were carried out as we have described recently.24 The pure DPPC lipid bilayer was composed of two monolayers containing 36 DPPC molecules per monolayer hydrated with a total of 3349 molecules of water. The DPPC/Lichenysin bilayer contained 66 DPPC molecules, 2 Lichenysin molecules, and 2 Na+ ions, yielding a 33:1 DPPC/Lichenysin ratio (3.0 mol %). This configuration allowed us to get insight into DPPC−Lichenysin interactions for which an equilibration time in the nanosecond to microsecond scale was appropriate.
3. RESULTS AND DISCUSSION 3.1. Lichenysin Alters the Thermotropic Phase Behavior of Phosphatidylcholines. Figure 2 shows the DSC profiles of mixtures of Lichenysin with DMPC (panel A), DPPC (panel B), and DSPC (panel C), which have the same polar headgroup and differ in the hydrocarbon chain length. As a control a pure Lichenysin suspension prepared at the same concentration of the phospholipids was checked, and no B
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Figure 2. Differential scanning calorimetry profiles of mixtures of Lichenysin with DMPC (A), DPPC (B), and DSPC (C). The thermograms correspond to heating scans carried out at 60 °C h−1. Total phospholipid concentration was 1.3 mM. The concentration of Lichenysin (mol %) is given on the profiles.
thermotropic transitions were observed within the range of −12 to 70 °C (not shown), indicating that all of the transitions described below must be attributed to the phospholipids. In Figure 2A, it can be seen that pure DMPC was characterized by a low-cooperativity low-enthalpy endothermic transition with a Tc at ∼12.5 °C (known as the pretransition), which corresponded to the change from the gel lamellar phase (Lβ′) to the ripple gel phase (Pβ′), and a high-cooperativity highenthalpy main transition with a Tc at ca. 23.5 °C and a Tm of 24.3 °C, from the gel phase (Pβ′) to the liquid-crystalline phase (Lα).31,32 The pretransition was completely abolished upon the incorporation of Lichenysin at a concentration as low as 1 mol %. The main gel to liquid-crystalline phase transition was progressively shifted to lower temperatures and broadened as the concentration of the lipopeptide was increased, indicating an interaction of Lichenysin with the hydrophobic portion of the phospholipids, which decreased the van der Waals interactions between the acyl chains and thus reduced the cooperativity and shifted transition temperatures to lower values. At 5 mol % of Lichenysin a second peak above the main transition could be observed. This peak became more prominent as the concentration of the lipopeptide was increased, so that at 7 mol % two well-differentiated transitions were clearly observed, which were better resolved at 10 and 15 mol %. These new peaks must be due to mixed phospholipid/ lipopeptide phases because, as commented on above, pure Lichenysin did not show any thermotropic transition within this range of temperature. The maximum concentration of Lichenysin shown was 20 mol %, at which a much broadened transition with very low enthalpy was still detected, the transition being abolished at higher concentrations. The pattern obtained for DPPC and DSPC bilayers (Figure 2B,C) was qualitatively similar: The pretransition was abolished at low concentrations, and new peaks were present in the thermograms upon increasing Lichenysin concentration. The complex thermotropic behavior observed for the three species of phosphatidylcholine was indicative of a lateral segregation of phases of different phospholipid/Lichenysin stoichiometry within the plane of the bilayer.
The calorimetric data obtained from the endothermic scans shown in Figure 2 allowed the construction of partial phase diagrams for the phospholipid component (Figure 3). For the sake of simplicity the pretransitions were omitted from the phase diagrams. The partial phase diagrams for the three phospholipids (Figure 3A−C) showed a near-ideal behavior for the solidus line, which progressively was shifted to lower temperatures as the concentration of Lichenysin was increased, indicating good miscibility between the phospholipids and the lipopeptide. In the diagram for DMPC (Figure 3A) the f luidus line showed an upward trend with Lichenysin concentration, indicating that fluid phase immiscibility was taking place for all Lichenysin concentrations examined. Thus in the region of the phase diagram denoted as G + F there are at least two fluid phases (F + F′) coexisting with the gel phase (G) because at least two peaks were observed in the thermograms at Lichenysin concentrations above 5 mol % (Figure 2A). Fluid phase immiscibility was also observed for the mixtures with DPPC, indicated by a horizontal f luidus line along the whole range of concentration studied (Figure 3B). However, the phase diagram for the system Lichenysin/DSPC (Figure 3C) was slightly different, with the region of fluid immiscibility being shorter, only up to a concentration of 7 mol %. At concentrations above this value both the solidusand f luidus lines also showed a downward trend with increasing Lichenysin concentrations, indicating good miscibility in the liquidcrystalline state as well and a near-ideal behavior for the system formed by these two lipids. These results clearly showed that Lichenysin/phosphatidylcholine interactions led to lateral phase segregation of complexes of defined stoichiometry, that is, domain formation. This type of domain formation has also been described for other biosurfactants like the lipopeptide surfactin33 or the glycolipid dirhamnolipid,34 which also exhibited a fluid phase immiscibility in mixtures with DMPC, which was suggested to be behind the membrane permeabilization activities of these compounds.35 The driving force leading to phase separation in membranes has been calculated from theoretical studies36 and accordingly in our case could be attributed to the hydrophobic mismatch between Lichenysin and phosphatidylcholine, favoring self-assemblies over assemC
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Figure 4. DSC thermograms for aqueous dispersions of Lichenysin with an equimolar mixture of DMPC and DSPC. Mol% of Lichenysin with respect to the total phospholipid is given on the curves.
component was very little affected up to this same concentration. The Tc of the lower melting component was shifted from 25.4 °C, in the absence of Lichenysin, to 20.3 °C for the sample with 20 mol % Lichenysin. This smaller effect as compared with that found for mixtures with pure DMPC (Figure 3) was due to the fact that although Lichenysin preferentially partitioned into the DMPC-rich component, some of the lipopeptide was also associated with the higher melting one (DSPC), as indicated by the progressive shift of the Tm of this component to lower temperatures (42 °C at 20 mol % Lichenysin). The complex phase behavior observed for the three pure individual phospholipids was also observed here, with the appearance of a shoulder at the low-temperature side of the low-melting component at concentrations of 3 mol % and above and a shoulder at the high-temperature side of the high-melting component at 20 mol %. These results indicated that Lichenysin, or phosphatidylcholine/Lichenysin complexes of a given stoichiometry, preferentially localized into fluid domains within a membrane displaying monotectic behavior. Free oleic and linoleic acids,39 two unsaturated fatty acids with cis-double bonds, and other biomolecules as relevant as cholesterol38 or α-tocopherol40 have also been found to partition preferentially into fluid domains. The main similarity between these lipid molecules and Lichenysin, with its amino acid ring, is probably their bulky shapes, making it difficult to fit in the hexagonal packing of the hydrocarbon chains of the phosphatidylcholines in the gel phase and thus better accommodating into fluid domains. This preferential partitioning into fluid domains is of biological relevance because Lichenysin has been shown to induce permeabilization of model and biological membranes in the fluid state.24 A similar effect has been recently described for the interaction of ultrashort lipopeptides with negatively charged phospholipid membranes,41 indicating that this behavior could be more general as far as lipopeptides are concerned. 3.2. Lichenysin Modifies the Structural Parameters of DPPC Bilayers. X-ray diffraction measurements were used to provide information on the structural characteristics of DPPC/ Lichenysin bilayers. Multilamellar phospholipid structures show X-ray reflections with relative positions of 1:1/2:1/3.42 Figure 5A shows the SAXD pattern profiles corresponding to pure DPPC and DPPC containing Lichenysin at temperatures below
Figure 3. Partial phase diagram for the mixtures of Lichenysin with DMPC (A), DPPC (B), and DSPC (C). The data for the solidus line (closed circles) and the f luidus line (open circles) were obtained from the onset and completion temperatures, respectively, of the thermograms shown in Figure 2. F and F′ denote fluid (liquid-crystalline, Lα) phases, and G denotes the gel (solid, Lβ) phase.
blies between the lipopeptide and the phospholipids, therefore leading to domain formation. To establish the preference of Lichenysin for solid or fluid domains, a system composed of DMPC and DSPC, which showed phase separation, was used. Figure 4 presents the effect of Lichenysin on the thermotropic behavior DMPC/DSPC 1:1, which is known to display a monotectic behavior.37,38 In the absence of Lichenysin, two well-defined transitions with low cooperativity were observed. The first transition, with a Tm of 31.2 °C (higher than the 24.3 °C observed for pure DMPC), corresponded to the gel to liquid-crystalline phase transition of a component rich in DMPC, whereas the second transition, with a Tm of 48.8 °C (i.e., lower than the one of pure DSPC: 56.5 °C), corresponded to a component rich in DSPC. It can be seen that the presence of Lichenysin mainly affected the first transition, that is, the lower melting component, which almost totally had disappeared at 15 mol %, whereas the higher melting D
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somehow larger separation.47 In the presence of Lichenysin the system showed the characteristic pattern of the ripple phase (4.15 Å), indicating that the disappearance of the pretransition observed by DSC was due to a lowering of the pretransition temperature and broadening of the peak. At 50 °C, the pattern for the Lα phase consisted of a single broad diffuse reflection, and the presence of Lichenysin did not affect the arrangement of the DMPC acyl chains in this liquid-crystalline phase. Background-subtracted SAXD patterns for DPPC and DPPC/Lichenysin at 5 mol % and 50 °C were subsequently analyzed using the global analysis program (GAP) to obtain the component parameters of the d value, that is, membrane thickness (dB) and the thickness of the water layer (dW): d = dB + dW. Figure 6 shows the corresponding 1D electron density
Figure 5. X-ray diffraction of DPPC/Lichenysin systems. Panel A: SAXD profiles of DPPC in the absence (upper) and presence of 5 mol % Lichenysin (lower); panel B: WAXD profiles of DPPC in the absence (upper) and presence of 5 mol % Lichenysin (lower), at 25 °C (in the gel state) and 50 °C (in the liquid-crystalline state). Figure 6. One-dimensional electron density profiles calculated from SAXD profiles of pure DPPC (solid line) and DPPC containing Lichenysin at 5 mol % (dashed line) at 50 °C using the GAP program.
and above the main gel to liquid-crystalline phase transition. The reflections observed for pure DPPC, with relative distances of 1:1/2:1/3, were consistent with a multilamellar organization. From the diffractograms it was possible to obtain the interlamellar repeat distance in the lamellar phase, which is given by the largest first-order reflection (d value) and which is a summation of the bilayer thickness and the thickness of the water layer between bilayers. DPPC gave rise to a first-order reflection with a d value of 62.5 Å in the Lβ′ gel state (25 °C) and 64.1 Å in the Lα liquid-crystalline state (50 °C), in agreement with previous data.43−45 The sample containing Lichenysin showed two reflections that related as 1:1/2 at both temperatures, confirming that the presence of the lipopeptide did not alter the lamellar structural organization of DPPC; however, the interlamellar repeat distance was increased: At 25 °C, the system containing Lichenysin showed a d value of 79.4 Å, close to that found for the pure phospholipid in the rippled phase,45 whereas at 50 °C, the d value was 70 Å, higher than that obtained for the pure phospholipid. The formation of a rippled phase, induced by the structurally related lipopeptide surfactin, in DPPC bilayers at temperatures below the pretransition temperature has been visualized by AFM.46 Because this effect could be due to a modification of bilayer thickness or the thickness of the water layer between bilayers, further investigation was carried out (see below). Experiments in the wide angle region (WAXD) were also carried out to obtain information on the chain lattice and thus about the packing of the DPPC acyl chains in the presence of Lichenysin. Figure 5B shows the WAXD pattern corresponding to DPPC in the absence and presence of 5 mol % Lichenysin at temperatures below and above the main transition. Pure DPPC at 25 °C showed a sharp reflection centered at 4.2 Å and a broad one at 4.1 Å. This was the typical pattern of an Lβ′ phase and corresponded to a quasihexagonal lattice, with the acyl chains tilted with respect to the bilayer normal forming one group of four closely spaced chains and two chains at a
profile along the bilayer normal calculated from the SAXD diffractograms. The profile for pure DPPC contained a central region of relative low electron density corresponding to the hydrocarbon chains of the phospholipid, a region of relatively high electron density corresponding to the headgroups, which symmetrically border the hydrocarbon region, and an interstitial solvent-rich layer with electron density values intermediate between those of the first two regions. The bilayer was centered at the origin so that the low electron density depression at 0 Å corresponded to the terminal methyl groups at the center of the bilayer. The addition of Lichenysin resulted in a higher rho value of the central region, between −15 and +15 Å, indicating that the lipopeptide was localizing here. It was also obtained that the increase in d value shown above was mainly due to an increase in the thickness of the water layer between bilayers, dW, from 17.5 to 23.6 Å, because dB only showed a small decrease for the sample with Lichenysin (45.4 Å) as compared with the pure phospholipid (45.8 Å). Other compounds such as curcumin, a bioactive pigment bearing two hydroxyl groups, have also been shown to increase DPPC bilayer thickness.43 On the contrary, because Lichenysin is negatively charged at pH 7.4 due to the Asp residue (Figure 1; pKa ca. 4.0), it gives a negative charge to the bilayer surface, increasing repulsion between adjacent bilayers and inducing the observed swelling. A similar result has been shown for the antihypertensive drug valsartan,48 which also contains an ionized carboxyl group. Nevertheless, it has to be considered that other factors, like a distinct penetration of Lichenysin into the bilayer, can also modify electrostatic repulsions and thus bilayer thickness. 3.3. Effect of Lichenysin on the Fluidity and Hydration of the Bilayer. FTIR was used to investigate the molecular E
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Langmuir interactions between Lichenysin and phospholipids, either at the polar region of the bilayer through analysis of the ester carbonyl stretching band (νCO) or at the acyl chains palisade through the methylene stretching bands (νCH2), to obtain information about its effect on the fluidity and hydration of the bilayer. Figure 7 shows the maximum of the νCH2 symmetric
stretching band of pure DPPC and mixtures with Lichenysin at various proportions as a function of temperature because through the study of shifts in the frequency of this band it is possible to obtain information about the phase behavior of phospholipid membranes.49 Incorporation of Lichenysin into DPPC shifted the maxima of the νCH2 band to higher values in the whole temperature range, that is, both below and above the main gel to liquid-crystalline phase transition, indicating that the lipopeptide alters the ratio of all-trans/gaucheconformers, resulting in an increase in acyl chain mobility, or fluidity, in agreement with the DSC results shown above. This interpretation in terms of conformational order is compatible with the increase in d value shown above.50 It is interesting to mention that a similar increase in membrane fluidity has been reported for short synthetic lipopeptides incorporated into negatively charged PE/PG membranes.41 To study the effect at the level of the polar lipid/water region, the νCO stretching band of the phospholipids is most convenient. Diacylphospholipids in lipid vesicles contain hydrated and dehydrated CO groups, and their ratio depends on the physical state of the bilayer.51,52 Thus the νCO band of a pure phospholipid is the result of two component bands centered at about 1742 and 1727 cm−1 and assigned to dehydrated and hydrated CO groups, respectively.49 Figure 8 shows the spectra of pure DPPC and DPPC containing 5 mol % Lichenysin at 30 and 50 °C (solid lines). These spectra were subjected to band fitting using a Gaussian−Lorentzian function, and the component bands were obtained (dashed lines), allowing us to determine the proportions of the dehydrated and
Figure 7. Temperature dependence of the maximum frequency of the νCH2 symmetric stretching band of DPPC. Plots correspond to pure DPPC (closed circles) and DPPC in the presence of Lichenysin at 0.5 mol % (open circles), 1 mol % (closed squares) and 5 mol % (open squares).
Figure 8. CO stretching band of DPPC in the absence and presence of Lichenysin. The bands are shown at 30 °C (phospholipid in the gel phase) and 50 °C (phospholipid in the liquid-crystalline phase). The dashed lines correspond to the component bands obtained by band fitting, as explained in the text. F
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Figure 9. Molecular dynamics simulation on the interaction between Lichenysin and DPPC bilayers. (A) Mass density profiles of a DPPC bilayer in the absence (a) and presence (b) of Lichenysin and the DPPC CO groups (c) and the Lichenysin molecule (d) in the DPPC/Lichenysin bilayer. (B) Snapshot of a DPPC bilayer containing Lichenysin. Blue lines correspond to DPPC molecules and green lines to Lichenysin molecules. Water molecules are shown in red and white.
between the DPPC ester CO groups and water was calculated from the MD simulations, showing that the presence of 3 mol % Lichenysin reduced hydrogen bonding by 9%, in accordance with the FTIR data commented on above. The high degree of correlation between the MD simulations and the experimental data provided a solid basis to obtain a model on how Lichenysin is distributed within the membrane in a Lichenysin/DPPC system (Figure 9 shows a snapshot). It was observed that Lichenysin was capable of interacting with the hydrophobic portion of DPPC as well as with the polar headgroup region, confirming the perturbations on the bilayer described by the experimental techniques. This localization and the concomitant modification of the bilayer properties is of particular relevance to explain Lichenysin-induced permeabilization of fluid phospholipid bilayers that has been previously described.24
hydrated components. It was observed that incorporation of just 5 mol % of Lichenysin increased the proportion of the dehydrated component at temperatures below (from 19.6 to 32%) and above the gel to liquid-crystalline phase transition (from 15.3 to 23.6%). These results indicated that Lichenysin exerted a rather intense perturbation of the polar region of the bilayer, decreasing the hydrogen bonding of the phospholipid CO groups with the water molecules of the hydration layer. The stronger effect observed when the phospholipids were in the gel phase must be due to the fact that under these conditions the lipopeptide was near-ideally mixed with DPPC, as shown above by DSC, whereas the lateral segregation taking place in the fluid phase due to the immiscibility shown above would result in a considerable reduction of its interactions with the phospholipid molecules. 3.4. Molecular Dynamics Simulations of Lichenysin/ DPPC Bilayers. Molecular dynamics simulations were carried out to study Lichenysin/DPPC interactions and obtain information on the location of the lipopeptide in the bilayer. The density profiles of the complete bilayer and some of the components along the z-axis, which is normal to the DPPC bilayer, were analyzed to obtain their precise location (Figure 9A). The mass density profile of pure DPPC showed the typical shape for this phospholipid bilayer.33 In the presence of Lichenysin the behavior showed a good coincidence with that described for SAXD (Figure 6), observing a slight and somehow non significant decrease in bilayer thickness as well as a significant increase in mass density between −15 and +15 Å. Accordingly, the mass density profile corresponding to the Lichenysin molecule (Figure 9A, curve d) showed that the lipopeptide was localized in that region, corresponding mostly to the DPPC hydrocarbon chains. From these MD simulations the number of DPPC acyl chain gauche conformers was calculated,with the presence of Lichenysin producing a slight increase from 26.5 to 27.1%, indicating an increase in fluidity, in agreement with the shift to higher frequencies of the FTIR νCH2 stretching band shown above (Figure 7). It can be seen as well that there was an overlap between the profile of Lichenysin and that of the DPPC carbonyl groups (Figure 9A, curve c), indicating that the lipopeptide could also interact with the polar region of DPPC, which is important to explain the dehydrating effect of the CO groups described by FTIR (Figure 8). To sustain this issue the hydrogen bonding
4. CONCLUSIONS The cytotoxic,22 antimicrobial,23 and hemolytic18,22,24 activities of the lipopeptide biosurfactant Lichenysin, together with the finding that lipid composition is essential for the Lichenysininduced membrane permeabilization action in model membranes,24 strongly suggest that biological membranes could well constitute a primary site of its action. In the case of the antimicrobial activities of Lichenysin,23 it has to be taken into consideration that phosphatidylcholine is not the major phospholipid component of bacterial membranes; therefore, extrapolation of the results presented here to in vivo situations with living bacteria cannot be straightforward. With this idea in mind, and through the use of various complementary biophysical techniques, we have shown here that the interaction between the biosurfactant Lichenysin and various saturated phosphatidylcholines of different acyl chain length affects the structure of the lamellar phase formed by the pure phospholipids, which may occur by changing the van der Waals interactions between the phospholipid hydrocarbon chains and also by modifying the headgroup region. The lipopeptide increases phospholipid acyl chain motion and bilayer thickness and dehydrates the phospholipid/water interface. Molecular dynamics calculations show that Lichenysin prefers to locate in the interior of DPPC bilayers, and in this way it perturbs the phospholipid palisade and affects the thermotropic phase transition of the phospholipid, altering the thickness of the membrane and decreasing the hydrogenG
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bonding pattern of the interfacial region, in agreement with the experimental data. These simulations have allowed us to obtain a model showing the localization of Lichenysin into a DPPC bilayer, which could explain the biological membranes’ disordering activities commented on above. Thus the permeabilization of phospholipid membranes by Lichenysin shown before24 could be a consequence of the specific interactions and localization described here. To this respect, in that previous work24 it was shown that Lichenysin permeabilized phospholipid membranes without membrane solubilization,24 supporting the “pore” model. This finding is now supported by the DSC results presented here, showing thermotropic phase transitions at lipopeptide concentrations at which membrane permeabilization occurs, which indicates the existence of a functional bilayer and provides additional support for the “pore” hypothesis.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +34 868 884788. Fax: +34 868 884147. E-mail: ortizbq@ um.es. ORCID
Ana Marqués: 0000-0002-4914-0472 Antonio Ortiz: 0000-0001-9645-9614 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partially supported by project CTQ 2014 59632R (Spain). J.R.C. acknowledges the financial support of SENESCYT-ESPOL (Ecuador) for a six month stay at the ́ Departamento de Bioquimica y Biologiá Molecular-A of the University of Murcia to carry out part of this study.
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