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The Potent Antimalarial Peptide Cyclosporin A Induces the Aggregation and Permeabilization of Sphingomyelin-Rich Membranes Slim Azouzi,†,§ Sandrine Morandat,†,§ and Karim El Kirat*,‡,§ †
Laboratoire de Genie Enzymatique et Cellulaire, CNRS UMR 6022, 60205 Compiegne Cedex, France Laboratoire de Biomecanique et Bioingenierie, CNRS UMR 6600, 60205 Compiegne Cedex, France § Universite de Technologie de Compiegne, BP 20529, 60205 Compiegne Cedex, France ‡
bS Supporting Information ABSTRACT: Cyclosporin A (CsA) is a hydrophobic peptide drug produced by the fungus Tolypocladium inflatum. CsA is commonly used as an immunosuppressive drug, but it also has antimalarial activity. The immunosuppressive activity of CsA is clearly due to its association with specific proteins of immune cells such as cyclophilins. By contrast, the antimalarial properties of this peptide are completely independent of the association with a parasite’s cyclophilins. Because of its hydrophobicity, CsA may interact with biological membranes, which may participate in its therapeutic effect. Recently, we have shown a marked preference of CsA for insertion into sphingomyelin (SM) monolayers. In this article, we measure for the first time the ability of CsA to induce permeabilization and aggregation and to change the lipid order, especially in the presence of SM. Calcein-release experiments permitted us to show that CsA causes the leakage of the fluorescent probe from SM-rich liposomes by 40% and PC liposomes by 11%, suggesting a lipid-selective effect. Electron microscopy and dynamic light scattering experiments confirmed the different interaction of CsA with SM and PC vesicles: it formed much larger aggregates with SM than with PC. Our results taken together suggest that CsA could specifically weaken and aggregate SM-rich membranes, which could in turn explain why CsA is efficient in the treatment of malaria. Indeed, CsA could inhibit the development of Plasmodium by permeabilizing and aggregating the SM-rich membrane network formed by the parasite during its intraerythrocytic growth cycle.
’ INTRODUCTION Cyclosporin A (CsA), a cyclic undecapeptide produced by the fungus Tolypocladium inflatum, is frequently used after organ transplantation as an immunosuppressive agent.1 The general mechanism proposed for the immunosuppressive effect of CsA proceeds via an interaction with the cyclophilins and calcineurin of immune cells. Indeed, CsA first forms a complex with cyclophilin A, and then the complex binds calcineurin, a calcium/calmodulin-dependent phosphatase. This ternary complex inhibits the dephosphorylation of the nuclear transcription factor of immune-activated T cells (NF-AT), which prevents the production of proinflammatory molecules (TNF alpha and interleukin 2), thereby suppressing the rejection of transplanted organs.2,3 Furthermore, CsA is involved in other biological functions such as antimalarial, anti-inflammatory, and antiviral activities.3 Despite these benefits, the CsA drug can cause severe side effects such as nephrotoxicity, hepatotoxicity, and neurotoxicity.4,5 The parasite responsible for malaria disease belongs to the genus Plasmodium. During its intraerythrocytic life, the parasite grows by digesting the host’s hemoglobin while forming a membrane network inside infected red blood cells. These newly formed membrane structures correspond to the parasitophorous vacuolar membrane (PVM) from which the tubovesicular r 2011 American Chemical Society
network (TVN) buds.6 It is worth noting that these membranes are especially enriched in sphingomyelin (SM).7 In general, the treatment of malaria is based on the family of quinine compounds (quinine, chloroquine, and mefloquine) that inhibits the polymerization of hemin released from the digestion of the host’s hemoglobin in the vacuole of the parasite. As a result, hemin molecules remain free, and because of their high hydrophobicity, they massively penetrate the parasites’ membranes, finally leading to their death by lysis.8 By contrast, CsA does not inhibit the polymerization of hemin (unpublished data), so its antimalarial activity may proceed through a different mechanism. The parasite produces cyclophilin analogues that are able to bind CsA. However, the explanation of the antimalarial effect of CsA through the formation of this complex is still not clear. Besides the formation of these complexes, if we consider the high hydrophobicity of CsA, it should be able to interact with lipid membranes. Many studies have suggested that this property can play an important role in its biological activity. For example, LeGrue has shown that the CsA Received: August 14, 2010 Revised: June 17, 2011 Published: June 21, 2011 9465
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Langmuir binding affinity for lymphocytes and for phospholipids is of the same order (∼107 M).9 Another study by Niebylski and Petty examined the effects of clinically used concentrations of CsA on the order of lipids in lymphocyte membranes using fluorescent probe DPH (1,6-diphenyl-1,3,5-hexatriene).10 They concluded that CsA causes a rapid increase in membrane order (within 5 min). CsA was also found to provoke the depolarization of lymphocyte membranes, accompanied by the leakage of potassium ions and the intracellular increase in calcium concentration.11 This drug was also shown to be able to modify the organization of model lipid bilayers in a concentration-dependent manner in the region of fatty acyl chains proximal to the headgroup.12 Fluorescence microscopy studies of CsA in dipalmitoylphosphatidylcholine (DPPC) monolayers showed that the drug is localized at the fluid/gel boundaries in membranes.13 CsA was also found to decrease the main transition enthalpy and phase-transition temperature (Tm) of DPPC liposomes.14 In addition, deuterium nuclear magnetic resonance (2H-NMR) measurements have revealed that CsA increases the acyl chain order in DPPC liposomes heated to above their Tm (∼41 °C). With the same technique, Schote and co-workers suggested that CsA could be incorporated into liposomal membranes with an upper limit of one molecule associated with nine phospholipid molecules.15 Moreover, cholesterol is able to inhibit the interaction of CsA with model membranes.13,16 Other studies also suggested that CsA induces the aggregation of lecithin liposomes and decreases the size of liposomes as observed by electron microscopy.17,18 In addition, Fahr and co-workers showed that CsA can bind to model membranes, which modifies their permeability to ions.19 We have recently shown a marked preference of CsA with respect to its insertion into SM monolayers, but we did not assess the membranolytic activity of CsA.16 This previous work did not allow us to draw conclusions about changes in permeability, aggregation, or even lipid order. However, given the growing resistance of classical antimalarial treatments generally aiming at inhibiting hemin polymerization and given the marked enrichment in SM of the intraerythrocytic membrane network formed by the parasite, it is clear that the SM/CsA interaction could inspire new strategies in the design of efficient drugs against paludism. In this work, we have compared the effect of CsA on SM and PC lipid bilayers by using microscopy and fluorescence techniques. All of the experiments confirmed a different interaction of CsA with SM-rich lipid bilayers. Our results show that CsA preferentially damages SM membranes and induces their aggregation. Most importantly, we report the first evidence of a lipid-selective membranolytic effect of CsA. This study provides a preliminary explanation of how CsA might proceed in the treatment of malaria. Indeed, CsA could inhibit the development of Plasmodium by permeabilizing and aggregating the SM-rich membrane network formed by the parasite during its intraerythrocytic growth cycle.
’ MATERIALS AND METHODS Chemicals. Egg sphingomyelin (SM), 1,2-dipalmitoyl-sn-glycerol3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Chol), calcein, sepharose 4B, and cyclosporin A (CsA) from Tolypocladium inflatum (purity was higher than 95%; molecular weight 1202.6 g/mol) were purchased from Sigma (St. Louis, MO). Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) was from Molecular Probes (Eugene, Oregon). Other chemicals were purchased from Merck (Darmstadt, Germany). For all of the experiments, a fixed
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volume of 10 μL of CsA dissolved in ethanol was added to the vesicles whatever the final concentration. To ensure that the solvent has no effect in our experiments, controls were performed by adding 10 μL of ethanol alone to vesicles. The distilled water was purified with a Millipore filtering system (Bedford, MA) yielding ultrapure water with a resistivity of 18.2 MΩ cm. Preparation of Unilamellar Vesicles. Unilamellar vesicles were prepared by dissolving the lipids, either pure or at the desired molar ratios, in chloroform. When CsA had to be preincorporated into lipid membranes at known molar percentages, CsA in ethanol was added to chloroform solutions of lipids (which was the case each time the CsA concentration was expressed in mol %). The lipids (or lipids/CsA organic mixtures) were dried under a stream of nitrogen to obtain a film and then kept under high vacuum for 2 h. The dry film was then dispersed in Tris buffer (10 mM Tris-HCl, 150 mM NaCl at pH 7.4) to obtain multilamellar vesicles (MLVs). To prepare large unilamellar vesicles (LUVs), the MLVs were extruded 19 times through 200 nm nuclepore polycarbonate membrane filters (Avestin Inc. Ottawa, Canada) at 55 °C using a syringe-type extruder (Liposofast, Avestin Inc.). To obtain small unilamellar vesicles (SUVs), MLVs were sonicated to clarity (three cycles of 2 min 30 s) using a 500 W titanium probe sonicator (Fisher Bioblock Scientific, France; 33% of the maximal power; 13 mm probe diameter) while being kept in an ice bath. Then the liposomal suspension was filtered on 0.2 μm Acrodisc (Pall Life Sciences, USA) to remove titanium particles. Dynamic Light Scattering (DLS). The hydrodynamic diameter of the liposomes was determined by DLS using a nano-ZS (Malvern Zetasizer, Malvern Instruments Ltd., Worcestershire, U.K.) consisting of a photon correlator spectrometer equipped with a HeNe laser source (wavelength 633 nm). DLS experiments were performed at 21 °C with a dynamic scattering angle of 90°. The sizes reported correspond to the mean z average (dz), and the measurements were realized within 5 min after CsA addition. The mean diameters measured for the LUVs of DOPC/DPPC (1:1 mol/mol), DOPC/SM (1:1 mol/mol), DOPC/ SM/Chol (4:3:1 mol/mol/mol), and DOPC/SM/Chol (2:1:1 mol/ mol/mol) were 170 ( 2, 182 ( 5, 180 ( 6, and 174 ( 3 nm, respectively. It is worth noting that the two latter compositions mimic the lipid composition of the eukaryotes’ plasma membrane and of the lipid rafts, respectively.20 The mean diameters measured for the SUVs of DPPC and SM were 45 ( 2 and 67 ( 3 nm, respectively. Calcein Release. The ability of CsA to permeabilize liposomes was measured with the calcein release assay. LUVs encapsulating the fluorescent probe were prepared as described above except for the hydration of the lipid film that was done in Tris buffer containing 35 mM calcein. Then, free calcein was separated from encapsulated calcein by gel filtration on a sepharose 4B column equilibrated with Tris buffer. Lipids were quantified with the method developed by Stewart.21 The calcein-loaded liposomes were diluted to a final concentration of 10 μM lipids in Tris buffer. Fluorescence measurements were performed at 21 °C immediately after CsA addition (within 1 min, the time required to stabilize the fluorescence signal) with Varian Cary Eclipse fluorescence spectrophotometer (Victoria, Australia) using excitation and emission wavelengths of 490 and 520 nm, respectively. The percentage of calcein released due to the action of CsA was defined as [(Ft F0)/ (Ftot F0)] 100, where Ft is the calcein fluorescence intensity recorded immediately after the addition of CsA, F0 is the initial fluorescence, and Ftot is the maximum fluorescence of the sample after the complete lysis of the membrane with 2 vol % Triton X-100 (TX-100). Laurdan Fluorescence. We have used the Laurdan probe to measure the influence of CsA on the lipid phases and on the order of lipid chains. SUVs were prepared to a final Laurdan/lipid molar ratio of 1/110. The Laurdan fluorescence was measured at an excitation wavelength of 360 nm using the Varian Cary Eclipse fluorescence spectrophotometer (Victoria, Australia) equipped with a thermoregulated cell 9466
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Figure 1. Influence of CsA on liposomal suspensions. Pictures of (A) CsA alone or in mixtures with (B) LUVs of DOPC/DPPC 1:1 mol/mol or (C) DOPC/SM 1:1 mol/mol. CsA was added to a final concentration of 120 μM to 1 mM LUVs in Tris buffer, and photographs were taken at the times indicated. Solid white arrows point to floating aggregates, and dashed white arrows indicate the pellets in the different series after 24 h of incubation with CsA. holder. The generalized polarization (GP) was calculated using the following relation22 GP ¼
I 435 I 490 I 435 + I 490
where I435 and I490 are the emission intensities obtained at 435 and 490 nm, respectively. The Laurdan probe is a lipophilic dye that is able to insert within membranes with its naphthalene moiety located just below the headgroups of lipids, among their glycerol backbones, and the linear hydrophobic dodecanoyl tail of Laurdan is embedded within the acyl chains of the lipids. Light excitation of Laurdan molecules modifies their dipole moments, which leads to a reorientation of water molecules surrounding the dye’s naphthalene moiety embedded in the headgroups/hydrophobic core interface of lipid membranes. In the emission spectrum, such an orientation of water molecules with the Laurdan dipole leads to a red shift of the probe’s emission. This phenomenon is even more important with an increasing number of water molecules around the dye. Therefore, in lipid membranes, the Laurdan probe is sensitive to the number of water molecules present within the bilayer.
Finally, variations in membrane water content provoke red shifts in the Laurdan emission spectrum, which can be quantified by calculating the GP value. If the lipids are well-ordered, then water molecules will have less access to the Laurdan probes embedded in the membrane, thus resulting in a high value of GP. In membranes with a lower order, the GP will decrease as a result of the greater access of water to the Laurdan probe inside the lipid bilayer.23 Transmission Electron Microscopy (TEM). CsA was added to a final concentration of 120 μM to an optical cell containing 1 mM DOPC/DPPC 1:1 or DOPC/SM 1:1 mol/mol LUVs. Pictures of the mixtures in the optical cells were taken at different times during 24 h. Then, aliquots of the pellet and supernatant were sampled for observation by TEM. Five microliters of the different samples were deposited on a carboncoated copper grid that was freshly cleaned for 5 min by UV ozone treatment (FHR UVOH 150 LAB, Germany). The excess liquid was carefully removed with absorbent paper. The samples were then stained with a solution of 2% uranyl acetate. TEM images were recorded at room temperature and under a vacuum of 105 Pa using a JEOL 1200 EX TEM (Tokyo, Japan) equipped with a high-resolution AMT camera. 9467
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Figure 2. TEM images of the membrane aggregates formed by CsA. Control TEM images are presented for (A) DOPC/DPPC 1:1 and (B) DOPC/SM 1:1 mol/mol LUVs. TEM images of (CE) aliquots sampled from the pellet and (FH) the supernatant for (C, F) CsA alone, (A, D, G) CsA mixed with DOPC/DPPC LUVs, and (B, E, H) CsA mixed with DOPC/SM LUVs. The scale bar is 500 nm for all of the images. The arrows point to remarkable structures. (See the text for details.)
Atomic Force Microscopy. Supported lipid bilayers (SLBs) were prepared in the presence of CsA and according to the vesicle fusion protocol.24 First, we have prepared SUVs with different lipids/CsA ratios at a 0.4 mM final concentration in Tris buffer containing 3 mM CaCl2. Afterwards, 150 μL of SUVs was deposited on freshly cleaved mica glued to steel sample disks (Agar Scientific, England) using Epotek 377 (Polytec, France). Samples were left for 6 h at 4 °C to accumulate SUVs on the mica surface. Samples were then rinsed with 3 mL of Tris buffer and held at 60 °C for 60 min to induce the fusion of liposomes adsorbed on mica. The SLBs were investigated using a commercial AFM (NanoScope III MultiMode AFM, Veeco Metrology LLC, Santa Barbara, CA) equipped with a 125 125 5 μm3 scanner (J scanner). Topographic images were recorded at 21 °C in contact mode using oxide-sharpened microfabricated Si3N4 cantilevers (Microlevers, Veeco Metrology LLC, Santa Barbara, CA) with a spring constant of 0.01 N/m (manufacturer specified), with minimal applied force (
