Kinetic and Structural Aspects of the Permeabilization of Biological

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Kinetic and structural aspects of the permeabilization of biological and model membranes by lichenysin Jonathan R Coronel, Francisco J. Aranda, Jose A. Teruel, Ana M. Marqués, Angeles Manresa, and Antonio Ortiz Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04294 • Publication Date (Web): 11 Dec 2015 Downloaded from http://pubs.acs.org on December 20, 2015

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Kinetic and structural aspects of the permeabilization of biological and model membranes by lichenysin Jonathan R. Coronel2#, Francisco J. Aranda1, José A. Teruel1, Ana Marqués2, Ángeles Manresa2, and Antonio Ortiz1* 1

Departamento de Bioquímica y Biología Molecular-A, Facultad de Veterinaria,

Universidad de Murcia, E-30100 Murcia, Spain; and 2Laboratorio de Microbiología, Facultad de Farmacia, Universidad de Barcelona, Joan XXIII s/n, E-08028 Barcelona, Spain.

Key words: Lichenysin, Biosurfactant, Erythrocytes, Hemolysis, Phospholipid Vesicles, Molecular Dynamics

*Corresponding author:

Tel.: +34 868 884788 Fax: +34 868 884147 E-mail: [email protected]

#

Present address: Escuela Superior Politécnica del Litoral, ESPOL, Facultad de Ingeniería Mecánica y Ciencias de la Producción (ESPOL-FIMCP), Campus Gustavo Galindo, P.O. Box 09-01-5863, Guayaquil-Ecuador.

ACS Paragon Plus Environment

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ABSTRACT

The various lichenysins produced by Bacillus licheniformis are anionic surfactants with interesting properties. Here it is shown that lichenysin caused hemolysis of human erythrocytes, which varied with lichenysin concentration in a sigmoidal manner. The release of K+ from red blood cells induced by lichenysin preceded the leakage of hemoglobin, and in addition, hemolysis could be impeded by the presence of compounds in the external medium having a size larger than that of PEG 3350, indicating a colloid-osmotic mechanism for hemolysis. Lichenysin also caused permeabilization of model phospholipid membranes, which was a slow process with an initial lag period of 10-20 seconds observed for all lichenysin concentrations. A high cholesterol ratio in the membrane decreased the extent of leakage as compared to that of pure POPC, whereas at lower ratios the effect of cholesterol was the opposite, enhancing the extent of leakage. POPE was found to decrease the extent of leakage at all the concentrations assayed; and inclusion of DPPC resulted in a considerable increase in CF leakage extent. From this scenario it was concluded that lipid membrane composition plays a role in the target membrane selectivity of lichenysin. Molecular dynamics simulations indicated that lichenysin is well distributed along the bilayer, and Na+ ions can penetrate inside the bilayer through the lichenysin molecules. The presence of lichenysin in the membrane increases the permeability of the membrane to hydrophilic molecules facilitating its flux across the lipid palisade. The results presented in this work contribute to understand the molecular mechanisms which explain the biological actions of lichenysin related to biomembranes.


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1. INTRODUCTION

The word biosurfactant is used to refer to surface active molecules produced by bacteria and fungi, with a rich diversity in chemical structures and an increasing list of interesting physicochemical and biological activities. The physiological roles of biosurfactants of microbial origin are directed towards increasing access to substrates, defensive actions, and biofilm formation.1-4 Due to the outstanding biological actions of these compounds,5,6 a number of biosurfactants have gained interest and some of them, for instance rhamnolipids, are currently commercialized as antifungal for agriculture (ZonixTM, Jeneil Biosurfactant Company, USA), emphasizing the current relevance of this field of research. Lipopeptides are among the most interesting biosurfactants known so far.7 Within this group surfactins, formed by an heptapeptide ring linked to a β-hydroxy fatty acid, forming a lactone ring,8 are potent surfactants with very interesting properties.9,10 Iturins, bacillomycins and mycosubtilin are also heptapeptides with a 14-17 carbons β-amino fatty acid. Their biological activity is different to that of surfactins: they behave as strong in vitro antifungals towards various yeast and fungi, but their antibacterial activity is limited, and have not shown antiviral activities.11-13 The fungal toxicity of iturins is very likely to rely on their interaction with membranes, and it has been shown that the mechanism of action is based on osmotic perturbation, including ion-pore formation, but not the disruption of the membrane or its solubilization, as has been shown for surfactins.14,15 Another group of lipopeptide biosurfactants is formed by lichenysins. Lichenysins A, B, C, D, and G produced by B. licheniformis, are anionic surfactants due to the presence of Asp and/or Glu residues.16-19 Lichenysins belong structurally to the B. subtilis surfactin family, but the presence of glutamine in position 1 instead of the glutamic acid of surfactin, is the origin of quite different properties. However, whereas there is a vast amount of information on the physicochemical and biological properties of surfactin,9,20 just a few works have addressed the properties of lichenysin. The cmc of lichenysin G has been shown to be at least one order of magnitude lower than that of the related compound surfactin, indicating a higher surface activity17. In addition lichenysin has been shown to display potent antimicrobial,21 cytotoxic22 and hemolytic activities.22,23 On the other hand, lichenysins and related compounds have been also shown to be of great potential in environmental remediation and petroleum recovery.24,25 On the light of all this information it is clear that a better understanding of


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the molecular basis of the mode of action of lichenysin will be valuable to explain some of this activities and for developing new applications. Thus, this work focuses on the molecular mechanism underlying permeabilization of model and biological membranes by the lipopeptide biosurfactant lichenysin, in an effort to explain the basis of the outstanding properties of this lipopeptide. Our results show that lichenysin introduces positive membrane curvature in the target bilayer, which can be exploited for technological applications in nano-systems and liposome-mediated drug delivery.26


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2. EXPERIMENTAL SECTION

2.1. Materials

All lipids, namely 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine (POPE), and cholesterol were from Avanti Polar Lipids Inc. (Birmingham, AL). 5(6)-Carboxyfluorescein (CF) (99% by HPLC), sucrose and the various PEG used were from Sigma-Aldrich (Madrid, Spain). All inorganic salts and buffers were of analytical grade. The bacterial lipopeptide lichenysin was produced, purified and characterized as described before.27 Purified water was deionized in a Milli-Q equipment from Millipore (Millipore, Bedford, MA) and had a resistivity of ca. 18 MΩ. The stock solutions of the phospholipids used prepared in chloroform/methanol (2:1), were stored at -80ºC. For the determination of phospholipid phosphorous was determined following the procedure described by Böttcher et al.28 A buffer containing 150 mM NaCl, 5 mM Hepes pH 7.4 was used unless otherwise is indicated when needed. Water and all other solutions were passed through 0.2 µm filters right before its use. The osmolarity of all the solutions was measured using an Osmomat 030 osmometer (Gonotec, Berlin, Germany).

2.2. Phospholipid vesicles contents release

The CF leakage experiments were carried out as follows. First, the appropriate amounts of the corresponding phospholipids (and cholesterol when indicated) in chloroform solutions were combined. Dry N2 was used to gently evaporate the solvent up to obtain a thin film in a glass tube. The remaining traces of solvent were eliminated by 2-3 h desiccation under vacuum. One ml of buffer (75 mM CF, 5 mM Hepes pH 7.4) was added to the previous samples and multilamellar vesicles were obtained by vortexmixing. Large unilamellar vesicles (LUV) were obtained by 11 timex extrusion of the multilamellar vesicles through polycarbonate filters (0.1 µm pore diameter) (Nuclepore, Pleasanton, CA), which is a well characterized and standardized method.29 The size of the vesicles was routinely checked by dynamic light scattering in a Malvern Autosizer 4800 (Worcester, UK). Occasionally SAXS, carried out using a Kratky compact camera


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(MBraum-Graz-Optical Systems, Graz, Austria) and a linear position sensitive detector (PSD; MBraum, Garching, Germany), was used to confirm that the vesicles were unilamellar in structure. Gel filtration on Sephadex G-75, using 150 mM NaCl, 5 mM Hepes pH 7.4 as elution buffer, was used to separate the vesicles from non-encapsulated material. Lichenysin was added at the appropriate concentrations from a 2 mM stock solution in DMSO. It was thoroughly checked that the volumes of DMSO used did not have any effect on the vesicles.

2.3. Hemolysis

Human erythrocytes were prepared just before the experiments from a red blood cell concentrate obtained from a local blood bank. Cells were washed twice with buffer (150 mM NaCl, 5 mM Hepes, pH 7.4), and suspended in the same volume of buffer before use. All these operations were carried out at 4ºC. For measuring K+ and hemoglobin release, the above erythrocyte concentrate was diluted using 150 mM NaCl, 5 mM Hepes, pH 7.4 buffer to obtain a suspension with A540 = 1. Time-dependent hemoglobin release was determined, upon incubation of red blood cells with lichenysin at 37ºC unless otherwise is indicated, by measuring the absorbance at 540 nm in the clear supernatants after pelleting the membranes by centrifugation for 2 min in a bench microfuge. The total amount of hemoglobin was established by lysing the erythrocytes with distilled water. Lichenysin was added at the appropriate concentrations from a 2 mM stock solution in DMSO. It was thoroughly checked that the volumes of DMSO used did not have any effect on the red blood cells under the same conditions of the experiments. Samples were continuously stirred using a magnetic device, in a jacketed vessel keeping constant temperature using a circulating water bath. For the determination of K+ leakage and aliquot of the above mentioned supernatants diluted 1:5 with distilled water was used. K+ concentration was measured in a sequential atomic emission spectrometer Perkin-Elmer Optima 2000DV. Calibration was carried out using a standard containing 1000 ppm of 23 different elements, with the appropriate dilution to obtain a lineal calibration in the range from 0.5 to 10 ppm of K+ in this case.


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2.4. Osmotic protection experiment

For the osmotic protection experiments, red blood cells were prepared as described above in a buffer solution with 135 mM NaCl, 5 mM Hepes, pH 7.4, supplemented with 30 mM of sucrose, PEG 400, 600, 1000, 3350, 6000 or 10000. Lichenysin was added (from a 2 mM stock solution in DMSO) up to the various final concentrations indicated in each experiment, and hemolysis was determined after incubation at 37ºC for 1 h. The molecular diameters used were: sucrose, 9.8 Å; PEG 400, 11.2 Å; PEG 600, 13.8 Å; PEG 1000, 17.8 Å; PEG 3350, 34 Å, PEG 6000, 54 Å and PEG 10000, 72 Å.30

2.5. Molecular dynamics simulations The lichenysin molecule (Fig. 1) was constructed using PyMOL software,31 with aspartyl residue in the unprotonated form. To build the topology file, it was submitted to the PRODRG server32 and an initial topology file was retrieved. Based on such information, lichenysin structure was described in GROMOS96 43a1 force field parameters.33 Afterwards, the topology was further modified to include some refinements. In order to calculate atomic charges it was submitted to full‑geometry optimization using ab initio quantum‑mechanical computations using Firefly QC package,34 which is partially based on the GAMESS (US)35 source code, with a 6-31G basis set and restricted Hartree‑Fock method. Hessian matrix analysis was used to characterize the optimized structure as true minima on the potential energy surface. This minimal energy conformation was submitted to single-point ab initio procedures to calculate Löwdin atomic charges, and these calculated atomic charges were used to modify the topology file. The topology file was further modified with charge‑groups containing no more than four contiguous atoms, to ensure that charge‑groups spatial extent was small.36 POPC topology file was obtained from Kukol (2009).37 Packmol38 was used to generate the initial configuration of an asymmetric bilayer with a leaflet composition of 30 POPC containing 6 lichenysin randomly distributed, yielding a 5:1 POPC/lichenysin ratio. A total of 2137 water molecules were added to the lipid bilayer containing the lichenysin molecules, resulting in a 35.6 water/lipid ratio. This water/lipid ratio is well above the minimum value necessary to


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create a fully hydrated system.39 Sodium ions were also added as counter‑ions in the water phase, to assure electrical neutrality of the system. Molecular dynamics calculations were performed using GROMACS v4.5 molecular simulation package40 under constant number of particles, pressure and temperature. Water was simulated by the simple point charge (SPC) model. This model was chosen because, for lipid/water interfaces, it provides results closest to experimental observations, and has the added advantage of being computationally expedient.41,42 The lipid bilayer was aligned such that it lay in the XY plane, i.e., the monolayers normal was parallel to the z-axis. Gromacs 43A1-S3 force field, which is an improved force field for lipids based on GROMOS96 43a1, was used.43 All simulations were performed in the NPT ensemble. Pressure control was carried out using the weak‑coupling Berendsen scheme with a time constant for coupling of 0.1 ps and semi‑isotropic pressure coupling (isotropic in the x and y direction, but different in the z direction). Temperature control was carried out with a V‑rescale thermostat with a time constant for coupling of 0.01 ps at 293K.44 Periodic boundary conditions were applied in all directions. All bonds were constraint using the SETTLE algorithm45 for water and the LINCS algorithm for all other bonds.46 A leap frog integration scheme with a time step of 2 fs was used. The Particle Mesh Ewald method was used to correct for long‑range electrostatic interactions,47 and short‑range electrostatic and van der Waals interactions were cut‑off at 1.25 nm.48,49 Molecular dynamic simulations were carried out for POPC and POPC + lichenysin bilayers at a constant pressure of 1 bar and 293 K, i.e. above the main phase transition temperature of POPC. Energy minimization, using the steepest descent algorithm, was done before molecular dynamics simulations,50 to remove any steric clashes or inappropriate geometries. The systems were then simulated for 500 ps in the NPT ensemble using simulated annealing from 0 to 293K with a cut-off distance for the short-range neighbor list of 1.4 nm to assure pre-equilibration of the system at 293K. Then a total of 360 ns molecular dynamic simulations were carried out to allow relaxation and equilibration of the systems. The last 100 ns were collected for all calculations. Viewers VMD 1.8.251 and PyMOL 1.5.0.131 were used to carry out a rough inspection of the arrangement of the lichenysin molecules in the bilayer, and to capture images throughout the corresponding trajectories.


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The permeability of the bilayer to a polar penetrant molecule, characterized by the excess chemical potential of a water molecule across the bilayer, was calculated using Widom’s particle insertion technique. The penetrant particle was inserted in the bilayer, to calculate the potential profile, as proposed by Notman et al.52 A slightly modified version of the test particle insertion algorithm implemented in GROMACS was used. The modification consisted in dividing the system into 20 partitions along the z-axis, performing the insertions in separate runs. Totally, 103 insertions per configuration were performed, taking configurations every 2 ps along 100 ns of the simulated trajectory.

3. RESULTS AND DISCUSSION

The marked amphipathic structure of lichenysin (Fig. 1) pointed towards a strong influence on the structural and functional properties of target phospholipid membranes. These effects could be well behind its physiological function, and thus an experimental study was conducted to establish the mechanism of permeabilization of biological and model membranes by this lipopeptide. The cmc of lichenysin (MW 1021.66) was determined in a previous work, giving a consistent value of 14.7 µM.27 This cmc was of the same order of magnitude to the 7.9 µM value published for the cmc of the related biosurfactant lipopeptide surfactin,53 for which a wide number of interesting chemical properties and potential applications has been described.9 It is important to remark that most of the interesting membrane actions of lichenysin reported below took place at concentrations well below its cmc, indicating that they were not due to its membrane solubilization activity.

3.1. Lichenysin-induced permeabilization of red blood cells Biosurfactants behave in the same way as chemical surfactants in the sense that they can penetrate biological membranes, and alter their structure and function. Interaction of a biosurfactant with erythrocytes normally leads to hemolysis,15,54,55 which can be due to either permeabilization of the membrane, through formation of 'pores' or zones of increased permeability to polar solutes, or to the disruption (solubilization) of the membrane. These effects usually occur at various ranges of biosurfactant concentrations, which depend on the chemical structure of the molecule and its


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physiochemical characteristics, the cmc being of particular relevance. Biosurfactants present very interesting potential applications, for example as drug vehicles in different formulations or in cosmetics, therefore the characterization of the hemolytic activity is of great importance. To the date, various representative compounds have been investigated. These include lipopeptides, like surfactin56,57 and iturin,15,58 as well as glycolipids like the rhamnolipids produced by Burkholderia pseudomallei]59 and Pseudomonas aeruginosa55, or Rhodococcus sp. trehalose lipids54. Figure 2 shows experiments of hemolysis of human erythrocytes induced by lichenysin, as determined by release of hemoglobin. Panel A shows the kinetics of hemolysis at various lichenysin concentrations, falling mostly below its cmc. Upon addition of the lipopeptide hemoglobin released from red cells in a time-dependent manner, up to completion. The rate of the process increased with lichenysin concentration in a non-linear manner, since an abrupt jump around the cmc could be observed. Panel B shows the percentage of hemolysis as a function of lichenysin concentration upon 24 min incubation. Hemolysis varied with lichenysin concentration in a sigmoidal manner since, as commented above for the initial rate, it could be seen that the extent of hemolysis also increased sharply around 4-5 µM, well below the cmc, to reach a maximum around 12 µM. The lichenysin-induced hemolysis curves shown in Fig. 2 are indicative of a slow process, which takes place even at rather low concentrations of the lipopeptide (4 µM), well below its cmc. These curves differ from those shown before, for instance for trehalose lipid biosurfactants54 which displayed a lag period in the range of minutes in the hemoglobin release curves, suggesting that the proper accommodation of the glycolipid biosurfactant prior to hemolysis was a slow process. The absence of this lag in the curves of lichenysin indicates that insertion of the lipopeptide in the target membrane is rather fast. However, since experimental data were collected every 4 min, we cannot discard the presence of a lag period shorter than this value, although the shape of the curves suggests its absence. Comparison of erythrocyte K+ release kinetics to hemoglobin release kinetics sheds light on the molecular mechanism causing hemolysis. For this purpose lichenysininduced K+ leakage from human red blood cells was continuously monitored in a separate assay (Fig. 3), under the same conditions as described above for release of hemoglobin. The main observation was that K+ release preceded the leakage of


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hemoglobin resulting in, for instance, a K+ leakage of ca. 45% of the maximum after 4 min, whereas hemoglobin leakage was still 15% of maximum. This was a solid indication to discard the lytic mechanism of hemolysis, pointing to the osmotic lysis.

3.2. Osmotic protection of hemolysis The different kinetic behavior of K+ and hemoglobin leakage suggested a colloidosmotic mechanism of hemolysis. Thus, an osmotic protection experiment was conducted (Fig. 4), to provide additional evidence which allowed confirming this mechanism. In this experiment osmotic protectants of different sizes were used, which also allowed determination of the size of the membrane ‘pore’ generated by the biosurfactant. Figure 4 shows the percentage of hemolysis vs. the diameter of the osmotic protectant used in each case. It was found that hemolysis could be impeded by molecules with a size larger than that of PEG 3350. This finding was independent of the concentration of lichenysin, and allowed to conclude that the pore size was close 34 Å (the diameter of PEG 3350). Two general mechanisms have been proposed for the hemolytic activity of surfactants.60 Hemolysis may occur, on the first hand, by membrane solubilization, i.e. the

direct disruption of

membrane structure,

which takes place

at high

surfactant/membrane ratios. On the second hand, hemolysis may be the consequence of local increase of membrane permeabilization to polar solutes of small size, which takes place usually at low surfactant concentrations and leads to the so-called osmotic lysis. Altogether these events constitute the well known colloid-osmotic mechanism. Colloidosmotic hemolysis is a well established mechanism61 that has been described in a large number of experimental works, including the effect of a wide variety of compounds with very different chemical structures such as ethanol,62 chlorodinitrobenzene,63 bile salts,64 trehalose lipid biosurfactant,65 methacrylate copolymers,66 or human IgG,67 as a few representative examples. The faster release of K+ vs. hemoglobin (Fig. 3) upon addition of lichenysin, was a first indication that lichenysin-induced hemolysis did not occur through membrane solubilization. Most likely it suggested that lichenysin induced early formation of permeability regions in the erythrocyte membrane, allowing K+ release, which gave rise to osmotic changes across the membrane, leading to hemolysis through a colloidosmotic mechanism. If a colloid-osmotic mechanism is operative, addition of the


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appropriate concentration of an impermeant solute of a certain size to the outer solution should impede hemolysis. The explanation is as follows: the balance of the intracellular osmotic pressure generated by hemoglobin with an external solute which cannot pass through the ‘pore’ will protect the red blood cell. Thus, the osmotic protection experiment carried out (Fig. 4), confirmed that a colloid-osmotic mechanism was taking place in our system. A pore diameter of ca. 34 Å (corresponding to PEG 3350) was obtained, which should allow the flux of small solutes like K+ ions (ionic radius 1.33 Å), as we have observed, but not hemoglobin. It is possible to speculate on the structure of these ‘pores’ in the erythrocyte membrane, based on the experimental results and on the light of the molecular dynamics data discussed below. We propose that the leaky 'pores' might be formed by clusters

of

lichenysin

molecules

surrounded

by

phospholipids,

which

are

physicochemically feasible given its strong amphiphilic nature. These clusters in the target red blood cell membrane could result in a local increase of the permeability of the membrane to solutes of small size (like K+), similar to the lichenysin-induced release of CF in phosphatidylcholine model vesicles (see below). Altogether, these events will result in a pore-type behavior. Such type of domains have been also described for Rhodococcus sp. trehalose lipid biosurfactants.65,68

3.3. Lichenysin-induced leakage of vesicle contents

The release of vesicle contents, in model phospholipid vesicles, was monitored by using the fluorescent probe CF entrapped into POPC LUV. Figure 5 shows curves of leakage of CF upon addition of lichenysin (increasing concentrations covering a range below and above the cmc) to POPC LUV suspensions, as a function of time. A general observation was that leakage was a slow process, with an initial lag period of 10-20 s observed for all lichenysin concentrations, which did not depend on the concentration of the lipopeptide. The initial rate of leakage, obtained at the onset of CF leakage, increased concomitantly with the concentration of lichenysin, but it can be observed that leakage was not completed within the 5 min time range shown in this figure. Therefore longer incubation times were checked (Fig. 6). It can be seen that leakage continued increasing at longer incubation times (30 and 60 min) for all lichenysin concentrations, and was essentially complete, even for the lowest ones (2 and 4 µM), after 2 h. The finding that leakage was such a slow process was an initial indication that membrane


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solubilization was not taking place under these conditions.69 Nevertheless this was further confirmed by monitoring sample turbidity which did not change in these samples (results not shown), indicating that we were below the solubilization ratio. Similar results were obtained using other lower and higher POPC LUV concentrations (not shown). The cmc of lichenysin27 is close to that reported for other biosurfactants of glycolipidic nature, like rhamnolipids,55 or lipopeptides.57 Our results

show that

lichenysin induces the release of low molecular weight solutes, like CF, from liposomes at concentrations below that value (Fig. 5), in a slow process which does not cause membrane disintegration or solubilization. Although the hemolysis and liposome experiments shown here cannot be directly compared since they were carried under somehow different experimental conditions due to the particular characteristics of each assay, it is possible to observe, by looking at the data shown in Figure 3 (hemolysis) as compared to Figures 5 and 6 (liposome leakage), that both processes took place and were essentially completed within the same time scale. This was a solid support for the operation of the 'pore' mechanism in both systems.

3.4.

Membrane

lipid

composition

affects

lichenysin-induced

membrane

permeabilization

To get additional information on the molecular mechanism of membrane permeabilization, the lipid composition of the target bilayer was changed. Figure 7 shows the dependence of extent of CF leakage 30 s after addition of lichenysin as a function of lichenysin concentration, for LUV of various lipid compositions. The extent of leakage showed a tendency to increase with the lipopeptide concentration within this range of concentration. Membrane lipid composition was observed to strongly influence CF leakage extent. We checked the incorporation of cholesterol, POPE or DPPC into the POPC membrane, at 25ºC, which resulted in a variety of effects. The effect of cholesterol was particularly interesting since it was concentration-dependent. Thus, at a high ratio (POPC/cholesterol 1:1) the extent of leakage decreased as compared to that of pure POPC, whereas at lower ratios (POPC/cholesterol 2:1) the effect of cholesterol was to enhance the extent of leakage. POPE was found to decrease the leakage extent at the 1:1 ratio shown in the figure, and also at other lower and higher ratios (not shown). Finally, inclusion of DPPC, which is in the gel phase at 25ºC, resulted in a considerable


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increase of the extent of CF leakage as compared to pure POPC, at all lichenysin concentrations studied. Summarizing, it was found that DPPC and cholesterol (at low ratios) enhanced leakage, whereas cholesterol (at high ratios) and POPE reduced leakage. The effect of POPE could be explained by the molecular shape theory of lipids. When considering the molecular shape of lichenysin, it has to be taking into account that it is formed by a large hydrophilic headgroup, constituted mainly by the amino acid ring, and a hydrophobic group of small size, i.e. the acyl chain. Accordingly lichenysin should be considered as a molecule with inverted-cone geometry, conferring positive curvature to its target membranes,70 which results in structural destabilization. The negative curvature introduced by POPE counteracts that of lichenysin, resulting in protection. Similar results have been described for other biosurfactants such as the related lipopeptide surfactin from B. subtilis,53 and P. aeruginosa dirhamnolipids.55 The observed effect of cholesterol was more complex. It is well known that cholesterol has a strong influence on the structural and functional properties of membranes. Incorporation of 40-50% cholesterol in phosphatidylcholine bilayers resulted in a large reduction of passive permeation to ions and CF,71 contents leakage induced by various toxicants of environmental interest,72 and detergent-induced leakage.73 Cholesterol incorporated in bilayers affects phospholipid lipid chain order which modifies the mechanical properties of the membrane.73,74 We have observed that incorporation of cholesterol at high ratios protects the membrane towards lichenysininduced permeabilization, which could be due to an increase in acyl chain motional order caused by the sterol,70,75 increasing lipid packing,76 hampering the adequate insertion of lichenysin, and consequently impeding formation of leaking ‘pores’. On the other hand, we have also consistently found that lower ratios of cholesterol (0.33 molar ratio) enhance lichenysin-induced leakage, and this effect of cholesterol could be of particular importance to explain the differential action of lichenysin observed on mammalian vs. bacterial membranes. It has been reported that lichenysin displays toxicity towards spermatozoa, erythrocytes and Vero cells,22 all from eukaryotic organisms, whereas we have found a complete absence of effect of lichenysin towards a series of Gram positive and Gram negative bacteria (results not shown). It should be remarked that eukaryotic cell membranes contain a large amount of cholesterol, in contrast to bacterial membranes,74 therefore our results on model phospholipid membranes clearly support a role of cholesterol in modulating the membrane action of


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lichenysin. Cholesterol has been also shown to hamper membrane permeabilization by surfactins.53 Altogether, the effects of DPPC, POPE, and cholesterol, all at relatively high proportions, reflect a general influence of lichenysin on the physicochemical properties of the POPC bilayer, rather than a direct interaction of these lipids with the biosurfactant. Thus, it seems that lipid membrane composition plays a role in the target membrane selectivity of lichenysin.

3.5. Molecular dynamics simulations

The average area per lipid is a key parameter of phospholipid bilayers. We have used the area per lipid to monitor stability of the equilibrated bilayer of the simulated systems. The area per lipid (i.e. the box area/number of POPC molecules) in each monolayer gave a value of 0.642 ± 0.013 nm2 (not shown), averaging over 100 ns of the trajectory. The area per lipid for the simulated systems did not show any significant drift, indicating that the systems can be considered to be in equilibrium. On the experimental side, values of 0.66 nm2 (T=310K),77 0.65 nm2 (T=298K),78 0.64 nm2 (T=298K)79 and 0.63 nm2 (T=297K)80 have been reported for the area per lipid for POPC bilayers. Thus our value is in good agreement with experimental data as well as with previous MD studies.81-83 This concordance validates our molecular model. Figure 8 shows the atomic mass density distribution along the z-axis, which is normal to the membrane surface. A maximum for phospholipids is observed in the polar head region containing the phosphorous atom, while in the center of the bilayer a minimum is observed corresponding to the carbon tails. Lichenysin is located well distributed along the bilayer occupying the hydrophobic core of the membrane. Sodium ions can also penetrate inside the bilayer through the lichenysin molecules. The permeability of the POPC/lichenysin bilayer to a penetrative hydrophilic particle of water relates to the excess chemical potential of the particle along the bilayer normal. The chemical potential profiles are shown in Fig. 9 for a POPC membrane and a lichenysin-containing POPC membrane. As expected for a hydrophilic particle, the profiles show a maximum in the hydrophobic core of the bilayer, where the interactions between the water particle and the hydrophobic tails are weaker, and minima in the edge regions of the bilayers, where the phospholipid polar heads are located with the water layer. It can be clearly seen that water chemical potential profiles are significantly modified in the center of the POPC bilayer by the presence of lichenysin in the


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membrane. Therefore the presence of lichenysin in the membrane clearly increases the permeability of the membrane to hydrophilic molecules facilitating the flux of polar molecules across the lipid palisade. The particular structure of lychenysin, with a hydrophobic tail and apolar amino acid residues, like leucine and valine, enables the molecule to partition in a hydrophobic environment as it is the POPC bilayer. However, lichenysin is a negatively charged molecule due to the presence of an acidic amino acid residue, the aspartyl residue, and besides, it contains glutamine, a polar residue. Our experimental results show that the presence of lichenysin inside the membrane facilitates the flux of polar molecules like CF, water or K+ ions, and this observation is supported by our results on MD simulations showing that the lipopeptide enhances the flux of water and cations, such as Na+. The area per lipid is not significantly altered by the presence of lichenysin since it is mainly located inside the membrane and not in the polar head-groups, as can be seen in the mass density profile (Fig. 8), but a water molecule can permeate throughout the membrane with greater ease if lichenysin is present (Fig. 9), decreasing the energy barrier of the hydrophobic core of the membrane. After the molecular dynamics simulations, a snapshot of the simulated POPClichenysin membrane was obtained (Fig. 10), where some water molecules as well as some Na+ ions can be seen inside the bilayer, in the hydrophobic core of the membrane, which does not occur in pure POPC membranes. It is also important to emphasize that lichenysin is almost homogeneously distributed along the z-axis but not in the xy plane, since it tends to form aggregates inside the membrane along the normal to the membrane surface plane (z-axis). In this work trajectories of several hundred nanoseconds have been simulated, which is sufficient to study molecular interactions in lipid membranes. However, large conformational changes take place at larger time scales, i.e. from micro to seconds. It has been proposed that the membrane perturbing activity and bioactivity of fengycin, another lipopeptide biosurfactant, are caused by its aggregated form.84 The observed behavior of lichenysin in our simulations allows arguing in this same direction, with the formation of higher aggregates at larger time scales facilitating the flux of more complex molecules or even the generation of porelike structures in the membrane. These findings are compatible with a scenario in which lichenysin forms domains that might be considered as structural lipid defects in the target bilayer, which behave as size-selective ‘pores’, allowing CF leakage through them.


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4. CONCLUSION

Lichenysin, a biosurfactant secreted by B. licheniformis causes colloid-osmotic hemolysis of human red blood cells. The experimental results support lichenysininduced ‘pore’ formation, resulting from domains with increased permeability in the erythrocyte membrane. The kinetics of K+ and hemoglobin release correlate well with previous results on membrane permeabilization induced by other biosurfactants like trehalose lipids. This suggests that both biosurfactants share a similar mechanism, and indicates that the lipid constituent of the bilayer plays a central role in hemolysis. Addition of lichenysin to POPC membranes gives rise to its permeabilization to solutes of small size, like CF, without membrane solubilization, with characteristics compatible with the 'pore' mechanism as well. Cholesterol plays a central role in mediating the action of lichenysin on phospholipid membranes, which is of great importance given the absence of cholesterol in bacterial cell membranes, as compared to eukaryotic membranes. For applications concerning the administration of drugs or other compounds with biological activity, it is important to consider molecules with the capacity to traverse the lipid membrane, like lichenysin. The results shown in this work are useful to establish a molecular mechanism for the biomembranes-related biological actions of this lipopeptide biosurfactant secreted by B. licheniformis, as well as for the development of future applications.

ACKNOWLEDGEMENTS

This work was partially supported by project CTQ 2014 59632-R (Spain). J.R.C. thanks SENESCYT-ESPOL (Ecuador) for the financial support for a six month stay at the Department of Biochemistry and Molecular Biology-A of the University of Murcia to carry out this study.


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FIGURE LEGENDS

Figure 1. The chemical structure of Bacillus licheniformis lichenysin D used in this study, containing the sequence QLlVDlL linked to a β‑hydroxy-myristoyl group.

Figure 2. Lichenysin-induced hemolysis of human erythrocytes. (A) Time dependence of hemolysis. Erythrocytes were incubated at 37ºC at various lichenysin concentrations (numbers on the curves, µM), samples were collected at time intervals, and the percentage of hemoglobin released was determined. The data correspond to the average of three independent experiments ± SE (error bars). (B) The dependence of hemolysis on lichenysin concentration. The data correspond to those shown in panel A upon 24 min incubation.

Figure 3. Hemolysis vs. potassium release induced by lichenysin in human erythrocytes. Lichenysin (6 µM) was added at time zero from a 2 mM stock solution in DMSO, samples were collected at 4 min intervals and hemoglobin (open circles) and K+ (closed circles) were determined. Temperature was kept at 37ºC. The initial and final external K+ concentration is indicated on the curve. Data correspond to the average of three independent experiments ± SE (error bars).

Figure 4. Osmotic protection experiment. Lichenysin-induced hemolysis was determined under the same conditions as in Figure 2B, in the presence of osmotic protectants, namely sucrose, polyethylene glycol 400, 600, 1000, 3350, 6000 and 10000, from smaller to higher diameter. Lichenysin concentrations were 6 (circles), 9 (triangles) and 12 µM (squares). Data correspond to the average of three independent experiments ± SE (error bars).

Figure 5. Lichenysin-induced liposome contents release. Lichenysin was added, from a DMSO stock solution, at increasing concentrations (µM, numbers on the curves) to POPC LUV containing CF. Total POPC concentration was 25 µM, and temperature 25ºC. Data correspond to one representative experiment of three repetitions.


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Figure 6. Lichenysin-induced liposome contents release upon long time incubation. Experiments were carried out as described in Fig. 5 prolonging incubation up to 30, 60 and 120 min. Lichenysin concentrations were 2 (black), 4 (light grey) and 6-25 µM (dark grey). The data correspond to the average of three independent experiments ± SE (error bars).

Figure 7. Effect of lipid composition on the extent of lichenysin-induced liposome contents release. CF leakage experiments were carried out as explained in Fig. 5 with LUV of various compositions: POPC (black circles), POPC/cholesterol 2:1 (open circles), POPC/cholesterol 1:1 (open squares), POPC/POPE 1:1 (black squares), and POPC/DPPC 1:1 (closed triangles). Total phospholipid concentration was 25 µM. Data correspond to one representative experiment of three repetitions.

Figure 8. Atomic mass density distributions of the locations of POPC (solid line), lichenysin (dashed), and Na+ ions (dotted) along the bilayer normal, the z-axis, for POPC bilayers containing lichenysin. The mass density scale for Na+ ions is indicated in the right vertical axis.

Figure 9. Excess of chemical potential profiles of a hydrophilic water particle along the bilayer normal for a POPC membrane (circles), and a POPC membrane containing lichenysin (squares).

Figure 10. Snapshot of the POPC bilayer in the absence (panel A) and presence (panel B) of lichenysin. The water layer is seen in white and red sticks at top and bottom of the picture. Lichenysin molecules are shown in purple sticks, the carbon tails of POPC in black lines, the phosphorous atoms as brown spheres, the Na+ ions as blue spheres, and the water molecules inside the bilayer have been drawn as white and red spheres.


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Figure 1. The chemical structure of Bacillus licheniformis lichenysin D used in this study, containing the sequence QLlVDlL linked to a β-hydroxy-myristoyl group. 99x73mm (300 x 300 DPI)


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Figure 2. Lichenysin-induced hemolysis of human erythrocytes. (A) Time dependence of hemolysis. Erythrocytes were incubated at 37ºC at various lichenysin concentrations (numbers on the curves, µM), samples were collected at time intervals, and the percentage of hemoglobin released was determined. The data correspond to the average of three independent experiments ± SE (error bars). (B) The dependence of hemolysis on lichenysin concentration. The data correspond to those shown in panel A upon 24 min incubation. 209x296mm (300 x 300 DPI)


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Figure 3. Hemolysis vs. potassium release induced by lichenysin in human erythrocytes. Lichenysin (6 µM) was added at time zero from a 2 mM stock solution in DMSO, samples were collected at 4 min intervals and hemoglobin (open circles) and K+ (closed circles) were determined. Temperature was kept at 37ºC. The initial and final external K+ concentration is indicated on the curve. Data correspond to the average of three independent experiments ± SE (error bars). 209x296mm (300 x 300 DPI)


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Figure 4. Osmotic protection experiment. Lichenysin-induced hemolysis was determined under the same conditions as in Figure 2B, in the presence of osmotic protectants, namely sucrose, polyethylene glycol 400, 600, 1000, 3350, 6000 and 10000, from smaller to higher diameter. Lichenysin concentrations were 6 (circles), 9 (triangles) and 12 µM (squares). Data correspond to the average of three independent experiments ± SE (error bars). 209x296mm (300 x 300 DPI)


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Figure 5. Lichenysin-induced liposome contents release. Lichenysin was added, from a DMSO stock solution, at increasing concentrations (µM, numbers on the curves) to POPC LUV containing CF. Total POPC concentration was 25 µM, and temperature 25ºC. Data correspond to one representative experiment of three repetitions. 209x296mm (300 x 300 DPI)


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Figure 6. Lichenysin-induced liposome contents release upon long time incubation. Experiments were carried out as described in Fig. 5 prolonging incubation up to 30, 60 and 120 min. Lichenysin concentrations were 2 (black), 4 (light grey) and 6-25 µM (dark grey). The data correspond to the average of three independent experiments ± SE (error bars). 209x296mm (300 x 300 DPI)


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Figure 7. Effect of lipid composition on the extent of lichenysin-induced liposome contents release. CF leakage experiments were carried out as explained in Fig. 5 with LUV of various compositions: POPC (black circles), POPC/cholesterol 2:1 (open circles), POPC/cholesterol 1:1 (open squares), POPC/POPE 1:1 (black squares), and POPC/DPPC 1:1 (closed triangles). Total phospholipid concentration was 25 µM. Data correspond to one representative experiment of three repetitions. 209x296mm (150 x 150 DPI)


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Figure 8. Atomic mass density distributions of the locations of POPC (solid line), lichenysin (dashed), and Na+ ions (dotted) along the bilayer normal, the z-axis, for POPC bilayers containing lichenysin. The mass density scale for Na+ ions is indicated in the right vertical axis. 261x187mm (200 x 200 DPI)


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Figure 9. Excess of chemical potential profiles of a hydrophilic water particle along the bilayer normal for a POPC membrane (circles), and a POPC membrane containing lichenysin (squares). 307x215mm (200 x 200 DPI)


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Figure 10. Snapshot of the POPC bilayer in the absence (panel A) and presence (panel B) of lichenysin. The water layer is seen in white and red sticks at top and bottom of the picture. Lichenysin molecules are shown in purple sticks, the carbon tails of POPC in black lines, the phosphorous atoms as brown spheres, the Na+ ions as blue spheres, and the water molecules inside the bilayer have been drawn as white and red spheres. 621x323mm (72 x 72 DPI)


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Kinetic and Structural Aspects of the Permeabilization of Biological

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