LDL uptake by Leishmania amazonensis: Involvement of membrane lipid microdomains

Experimental Parasitology 130 (2012) 330–340

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LDL uptake by Leishmania amazonensis: Involvement of membrane lipid microdomains Nuccia N.T. De Cicco a, Miria G. Pereira d, José R. Corrêa b, Valter V. Andrade-Neto c, Felipe B. Saraiva a, Alessandra C. Chagas-Lima a,f, Katia C. Gondim a,f, Eduardo C. Torres-Santos c, Evelize Folly e,f, Elvira M. Saraiva h, Narcisa L. Cunha-e-Silva d, Maurilio J. Soares g, Georgia C. Atella a,f,⇑ a

Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21.941-902, Brazil Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF 70.910-900, Brazil Laboratório de Bioquímica de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ 21.045-900, Brazil d Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21.941-902, Brazil e Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ 24.020-141, Brazil f Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21.941-902, Brazil g Instituto Carlos Chagas, Fundação Oswaldo Cruz, Curitiba, PR 81.350-010, Brazil h Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21.941-902, Brazil b c

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Article history: Received 11 May 2011 Received in revised form 10 February 2012 Accepted 13 February 2012 Available online 21 February 2012 Keywords: Leishmania LDL uptake Cholesterol uptake Lipid microdomains Lipid rafts

a b s t r a c t Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL1 particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-b-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Flagellate protozoa of the Trypanosomatidae family (Euglenozoa: Kinetoplastea) are obligatory parasitic organisms with a single flagellum and a small kinetoplast. Trypanosomatids parasitize all classes of vertebrates, as well as some invertebrates and plants, and are important in medicine because many species are pathogenic to humans (Maslov et al., 2001). The life cycle of digenetic parasites of the genus Leishmania Ross 1903 includes motile ⇑ Corresponding author. Address: Instituto de Bioquímica Médica, CCS, UFRJ, Av. Carlos Chagas Filho, Bloco H, 2° Andar, Sala 30, Ilha do Fundão, 21.941-902 Rio de Janeiro, RJ, Brazil. Fax: +55 21 2270 8647. E-mail address: [email protected] (G.C. Atella). 1 Abbreviations: CHO, cholesterol; CHOE, cholesteryl ester; DRM, lipid microdomains or detergent-resistant membrane; LDL, low density lipoprotein; 125I-LDL, LDL labeled with 125I; 3H-CHO-LDL, LDL with 3H-Cholesterol incorporated; TR-LDL, LDL with Texas-Red-Phosphatidylethanolamine associated. FITC-LDL, LDL with fluorescein 5’-isothyocianate associated; MBCD, methyl-b-cyclodextrin. 0014-4894/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2012.02.014

promastigotes in the alimentary tract of the insect vector (phlebotomine sandflies), and intracellular nonmotile amastigotes in mononuclear phagocytes of mammalian hosts. The species L. amazonensis belongs to the Leishmania (Leishmania) subgenus, which causes human cutaneous and mucocutaneous Leishmaniasis in the New World (Jhingran et al., 2008). There are four main strategies to control the transmission of parasites, based on vector biology, drug development, genetics and immunology. However, these strategies have been hampered by the lack of knowledge regarding the molecular aspects of parasite development in the host. Studies of lipid metabolism by independent groups demonstrated that trypanosomatids have incomplete de novo lipid synthesis (Korn et al., 1969). Therefore, they avidly take up lipids from the vertebrate bloodstream, presumably satisfying their requirements for growth and differentiation (Dixon et al., 1971, 1972). Furthermore, they depend on the presence of vertebrate plasma lipoproteins in the culture medium for rapid growth (Coppens et al., 1988, 1995; Soares and De Souza,

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1991). Many studies have shown that sterols are essential for the parasite viability (Urbina, 1997). Human low density lipoprotein (LDL) is a type of lipoprotein composed of a core of nonpolar lipids (triglycerides and cholesteryl esters) and a shell of polar phospholipids, cholesterol and apolipoproteins (Apo) (Davis, 1991). LDL is the major carrier of plasma cholesterol in humans (Gurr et al., 2002) and the main source of cholesterol for protozoa (Prioli et al., 1990; Coppens et al., 1991; Haughan et al., 1995; Morita et al., 2000; Labaied et al., 2011; Nishikawa et al., 2011). It has been shown that bloodstream forms of Trypanosoma brucei brucei acquire phospholipids and sterols through endocytosis and intracellular processing of LDL from the vertebrate host (Coppens et al., 1995). In this species, the LDL particles are recognized at the cell surface by an LDL receptor that is essential for optimal growth of the bloodstream stage of the parasite by providing cholesterol and other lipids (Coppens et al., 1988, 1995). The LDL receptor binds cholesterol-rich lipoproteins that contain ApoB-100 and/or ApoE, but not ApoB-48, and mediates their endocytic uptake (Brown and Goldstein, 1986). The main apolipoprotein on the LDL particle is ApoB-100, which determines the cellular uptake of lipoproteins by interacting with cell surface lipoprotein receptors. There are LDL receptors in nonmammalian species, such as Caenorhabditis elegans, chicken (vitellogenin receptor) and fruit fly (Y1 protein) (Willnow, 1999). In protozoa from the order Kinetoplastida, an LDL-binding protein that binds specifically and with a comparable affinity to LDL has been described in the bodonid Trypanoplasma borelli and in the trypanosomatids T. b. rhodesiense, T. equiperdum, T. vivax, T. congolense, L. donovani, Crithidia luciliae and Phytomonas characias (Bastin et al., 1996). Recently, Nagajyothi et al. (2011) demonstrated that the T. cruzi invasion is dependent of mammalian host LDL-receptor, however, no receptor for LDL was identified in these trypanosomatids. In 2003, Zeng et al. described oxidized LDL endocytosis through CD36, a scavenger receptor, in Chinese hamster ovarian cells and in C32 human melanoma cells. The endocytosis of LDL was localized to lipid raft domains independent of caveolin-1. Lipid rafts are plasma membrane microdomains enriched in cholesterol, glycosphingolipids and sphingolipids and are resistant to solubilization with non-ionic detergents at low temperatures (Galbiati et al., 2001). Because of this feature, lipid rafts are also known as detergent-resistant membranes (DRMs). A common type of glycosphingolipids found in lipid rafts is the ganglioside GM1, which contains one molecule of sialic acid and serves as a marker of rafts for binding subunit B of cholera toxin (Fujinaga et al., 2003). Lipid rafts have a distinct protein composition. Several classes of proteins, including caveolins, flotilins and stomatins, have been recently shown to structurally and functionally modify lipid rafts. Each of these proteins may be responsible for the formation of a distinct class of lipid raft (Galbiati et al., 2001). Many DRM proteins are linked to saturated acyl chains in two ways, either in the form of a glycosylphosphatidylinositol (GPI) anchor or through acylation with myristate or palmitate (Brown and London, 1998). DRMs play a role in a variety of processes such as vesicular trafficking, signal transduction and binding to antibodies, toxins, and pathogens (Simons and Ikonen, 1997). The disruption of lipid rafts stimulates apoptosis (Bang et al., 2005) and can inhibit raft-mediated endocytosis (van der Luit et al., 2002). There are many types of pathogens and toxins that utilize a raft pathway to invade host cells, for example, cholera toxin (Fujinaga et al., 2003), simian virus 40 (Anderson et al., 1996), human immunodeficiency virus (HIV) (Nguyen and Hildreth, 2000), pseudorabies virus, a type of alphaherpesvirus (Desplanques et al., 2008), Cryptosporidium parvum (Nelson et al., 2006), Escherichia coli and parasites such as Toxoplasma gondii and Plasmodium falciparum (Shin and Abraham, 2001a,b). In addition, Saint-Pierre-Chazalet et al. (2009)

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demonstrated that membrane sterols and lipid raft integrity are involved in the antileishmanial action of the drug miltefosine in L. donovani promastigotes. DRMs in parasites have been also implicated in invasion of mammalian host cells. Such mechanism has been described for L. braziliensis (Yoneyama et al. 2006). DRMs in parasites are also involved in endocytic pathways for important nutritional molecules, such as transferrin in Trypanosoma cruzi epimastigote forms (Corrêa et al., 2007; for review, see De Souza, 2007). In this work we investigated the capacity of LDL endocytosis by L. amazonensis and the involvement of a putative LDL receptor on its endocytosis. We also analyzed the involvement of L. amazonensis lipid membrane microdomains in LDL endocytosis. Methyl-bcyclodextrin (MBCD), which depletes cholesterol in membranes, was used to disrupt the lipid microdomains (Ilanguraman and Hoessli, 1998; Lambert et al. 2007). MBCD is strictly a surface-acting molecule and selectively extracts membrane cholesterol by including it in a central non-polar cavity of cyclic oligomers of glucopyranoside in alpha-1,4 glycosidic linkage (Pitha et al. 1988). Our data demonstrate that L. amazonenis is able to take up human LDL through DRM-mediated endocytosis. 2. Materials and methods 2.1. Parasites L. amazonensis (MHOM/BR/75/Josefa) promastigotes were cultured at 28 °C by weekly passages of log-phase parasites in M199 medium (Sigma–Aldrich Co, USA) supplemented with 25 mM of N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (HEPES) (Sigma–Aldrich Co, USA) and 10% of heat-inactivated fetal calf serum (Cultilab Ltda, Campinas, Brazil). Amastigotes from L. amazonensis (MHOM/77/LTB0016) lesions were isolated from infected Balb/c mice and cultivated at a parasite concentration of 5  105/mL in Schneider’s medium (Sigma– Aldrich Co, USA) at 26 °C, plus 1 M Hepes buffer and 10% of heatinactivated fetal calf serum (Cultilab Ltda, Campinas, Brazil). Following 3 days of incubation the recently transformed promastigotes were transferred to the same medium with a pH of 5.5, the same temperature, the same parasite concentration, a higher percentage of FCS (20%), and smaller amounts of antibiotics (60 UI/ mL penicillin and 60 mg/mL streptomycin). Following 6 days of incubation, the culture—now rich in metacyclic forms—was reincubated under the same conditions as above, but at a higher temperature (32 °C) (Bates, 1994). The resulting amastigote culture is maintained until the tenth passage at 10-day intervals. Metacyclic promastigotes were purified from culture of L. amazonensis (MHOM/BR/75/Josefa) stationary phase using a Ficoll gradient as previously described (Späth and Beverley, 2001). Metacyclics were obtained in the 10% ficoll layer and were characterized by its morphology (small and slender cell body, with a flagellum at least two times the size of parasite body), and by its typical swimming movement. The parasites were washed with phosphate buffered saline (PBS – 10 mM phosphate buffer pH 7.4, 150 mM NaCl) and resuspended in M199 for assays. For metabolic labeling, three-day-old promastigotes at the midlog phase of growth were used. For the binding assays (4 °C), the parasites were incubated on ice for three hours prior to the assay. 2.2. Methyl-b-cyclodextrin treatment To verify the involvement of lipid microdomains, L. amazonensis promastigotes, amastigotes and metacyclic forms were treated at different concentrations with methyl-b-cyclodextrin (MBCD) (Sigma–Aldrich Co, USA) in Schneider’s Insect medium or M199

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medium for 30 min at room temperature. After the treatment, the parasites were washed two times with PBS containing 1% sucrose and resuspended in various volumes of the same medium. 2.3. Lipoprotein purification The human plasma was courtesy of the Blood Bank Service of Hematology, University Hospital Clementino Fraga Filho of Federal University of Rio de Janeiro (Rio de Janeiro, RJ, Brazil), through agreement n.° 171/07, signed by the Ethics Committee for Researches from the same hospital. Low density lipoprotein (LDL) and high density lipoprotein (HDL) were purified from fresh human plasma as described by Chapman et al. (1981) with some modifications. Briefly, KBr was added to 15.0 mL of human cell-free plasma to adjust the density to 1.3 g/mL. The plasma was added to a centrifuge tube with 20.0 mL of saline solution (150 mM NaCl, 1 mM EDTA). This material was ultracentrifuged at 150,000g in a vertical angle Beckman RPVTi 50 rotor (Beckman Coulter Inc, Fullerton, CA, USA) at 4 °C for 18 h. The LDL and HDL fractions were localized and removed. In the LDL fractions, KBr was added again to the LDL fractions to adjust the density to 1.2 g/cm3, and the material was ultracentrifuged at 150,000g in the same rotor at 4 °C for 18 h. The purified lipoproteins were extensively dialyzed against PBS with 1 mM EDTA. Lipophorin (Lp) was purified from the hemolymph of the insect Rhodnius prolixus by a KBr ultracentrifugation gradient as described elsewhere (Gondim et al., 1989). The lipoprotein concentrations were quantified by the method described by Lowry et al. (1951) using 0.1% bovine serum albumin (BSA) as standard in the presence of 0.5% sodium dodecyl sulfate (SDS) (Markwell et al., 1978). For the uptake assays and culture, the LDL was sterilized by filtration with a 0.22 lm membrane (Millex-GV, Millipore S.A., Molsheim, France). The integrity of the sterile LDL was verified in a polyacrylamide (3–15%) slab. Gels were run under denaturing conditions with SDS (Laemmli, 1970) at a constant current of 100 V. 2.4. Fetal calf serum delipidation Lipid extraction of fetal calf serum (FCS) (Cultilab Ltda., Campinas, SP, Brazil) without protein precipitation was performed as described by Cham and Knowles (1976). Briefly, 5.0 mL of FCS, 10.0 mL of a mixture of di-isopropyl ether (DIPE) and n-butanol (60:40 v/v) and 5.0 mg of ethylenediamine tetraacetic acid (EDTA) were added to Falcon tubes in this order. The tubes were then fastened on a blood cell suspension rotator (Labquare Shaker, Lab Industries, Inc., Berkeley, CA, USA) to provide end-over-end rotation at 28–30 rpm for 30 min at room temperature. After extraction, the mixture was centrifuged at 2000 rpm for 2 min to separate the aqueous and organic phases. The aqueous phase was removed from the organic phase by careful suction with a needle and glass syringe. The remaining solvents were harvested with nitrogen gas (N2). The delipidated fetal calf serum (FCSd) was sterilized by filtration with a 0.22 lm membrane (Millex-GV, Millipore S.A., Molsheim, France). 2.5. Lipid analysis For lipid analysis, cells or serum were subjected to lipid extraction as described by Bligh and Dyer (1959) using methanol:chloroform:distilled water (2:1:0.8 v/v). The lipid extracts were analyzed by one-dimensional thin-layer chromatography (TLC) on Silica Gel 60 plates (E. Merck, Darmstadt, Germany) for neutral lipids using n-hexane:diethyl ether:acetic acid (60:40:1 v/v) (Vogel et al., 1962). Cholesterol, cholesteryl-oleate, glycerol-tryoleate, diolein,

oleoyl-glycerol and oleic acid (Sigma–Aldrich Co, USA) were used as standards. The lipids were visualized using a charring reagent (CuSO4) after heating at 200 °C for 20 min (Ruiz and Ochoa, 1997). After that, the chromatography plates were digitized. For the cholesterol metabolism assay, the lipids were visualized using iodine vapor. The cholesterol and cholesteryl ester spots were cut-off the silica gel and re-extracted, using methanol:chloroform:distilled water (2:1:0.8 v/v). The associated radioactivity was measurement by liquid scintillation. 2.6. Preparation of radioactively labeled LDL 125

I-labeled LDL (125I-LDL) was obtained by iodination with 125Isodium iodide (CNEN, São Paulo, Brazil) with 280 Ci/mg of protein using Iodo-gen (Sigma–Aldrich Co, USA) (100 g/mg of protein) following the manufacturer’s instructions. To remove the free iodide, the reaction mixture was passed through three Sephadex G-50 (Sigma–Aldrich Co, USA) spin columns (Penefsky, 1977). The specific radioactivity was determined by gamma counting. 3 H-CHO-LDL was obtained as described by Coppens et al. (1995). 3 H-cholesterol (PerkinElmer, Boston, MA, USA) stored in chloroform–methanol (2:1) was dried under a nitrogen stream, resolubilized in 30 lL of absolute ethanol and mixed with 12.0 mL of fresh human plasma. After incubation for 24 h at 37 °C, the LDL particles were purified as described above, and the specific radioactivity was determined by liquid scintillation counting. 2.7. Preparation of fluorescently labeled LDL Purified LDL was labeled with Texas-Red conjugated phosphatidylethanolamine (N-Texas RedÒ sulfonyl-1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt – Texas Red-PE) (Molecular Probes – Invitrogen, Carlsbad, CA) as described by Martin-Nizard et al. (1987) with slight modifications. Briefly, fluorescent lipid-coated glass beads (0.1 mg) were incubated with 1.0 mg of pure LDL. The mixture was gently stirred at room temperature for 40 min, and the glass beads were removed by centrifugation (5 min at 15,000g). Fluorescently labeled LDL-TR-PE was re-isolated by two Sephadex G-50 (Sigma–Aldrich Co, USA) spin columns (Penefsky, 1977). Purified LDL was also labeled with fluorescein 50 -isothyocianate (FITC) (Sigma–Aldrich Co, USA) following the instructions in the manual. LDL (1.0 mg) was incubated with 50 lL of NaHCO3 (1 M) and 50 lL of FITC (1 mg/mL) at room temperature for one hour with constant stirring. The mixture was then incubated with 15 lL of hydroxylamine phosphate (50 mM) for 30 min at room temperature. Finally, FITC-LDL was re-isolated by Sephadex G-50 (Sigma–Aldrich Co, USA) spin columns (Penefsky, 1977). For other assays, purified LDL was labeled with CholesterylBodipy (cholesteryl 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diazas-indacene-3-dodecanoate) (Molecular Probes - Invitrogen, Carlsbad, CA) at the same method described in item 2.6 of this section (Coppens et al., 1995). LDL labelling was confirmed by UV exposition of protein samples previously analyzed by electrophoresis. 2.8. L. amazonensis membrane preparation Plasma membrane-enriched vesicles were obtained from L. amazonensis promastigotes (107 cells/mL; 1.5–3.0 L) as previously described (Benaim et al., 1991) with some modifications. Cells were harvested, washed with cold PBS and centrifuged at 1500g for 10 min. After a final wash in a buffer containing 400 mM mannitol, 10 mM KCl, 2 mM EDTA, 3 ll/mL of protease inhibitor cocktail Set III, EDTA-free (Calbiochem, Merck KGaA, Germany), 1 mM phenylmethanesulphonyl fluoride (PMSF), and 20 mM HEPES/KOH pH 7.6, the cell pellet was mixed with glass beads

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(80–120 lm in diameter) at a ratio of 1:4 (wet wt/wt of beads) and disrupted by abrasion for 15 min in a mortar with pestle on an ice bath until 90% disruption was achieved as determined by examination under an optical microscope. Glass beads and large debris were removed by centrifugation at 1000g for 15 min at 4 °C (SA 600 rotor, Thermo Fisher Scientific Inc., Waltham, MA). The supernatant was subjected to three consecutive centrifugations at the same temperature: 5,000g for 20 min (SA 600 rotor), 16,000g for 30 min (SA 600 rotor,) and 105,000g for 1 h (70.1 Ti rotor, Beckman Coulter Inc, Fullerton, CA, USA). The resulting pellet was resuspended in 300 lL of buffer containing 10 mM Tris, 10 mM 3-(Nmorpholino) propanesulfonic acid (MOPS), 150 mM NaCl, 2 mM CaCl2, pH 7.4. The membrane preparation was separated into small aliquots and stored in liquid nitrogen until use. The protein concentration of the membrane preparation was quantified as described by Lowry et al. (1951) using 0.1% bovine serum albumin (BSA) as standard.

Reader Spectra Max M2 (Molecular Devices): excitation 596 nm and emission 615 nm.

2.9. Influence of LDL on parasite development

The membrane preparation was incubated at 28 °C for 90 min in the presence of 200.000 CPM of 125I-LDL (0.03 mg/mL) in binding buffer (10 mM Tris, 10 mM MOPS, 150 mM NaCl, 2 mM CaCl2, 2.0 mg/mL BSA, pH 7.4). To determine the binding specificity, an excess (2.0 mg/mL) of non-radioactive LDL or other lipoproteins [HDL and the insect lipoprotein, lipophorin (Lp)] was added. The incubation volume was 200 lL. Centrifugation binding assays were performed as described elsewhere (Hulme, 1992). After the incubation time, tubes were centrifuged at 14,000g for 5 min, and the supernatants were discarded. Pellets were washed twice with 1 mL of cold binding buffer, resuspended in 30 lL of the same buffer and counted in a gamma counter to determine the associated radioactivity. For each condition, a blank was measured in which no membrane was added. The radioactivity values obtained in the blanks were subtracted from the corresponding experimental samples.

Promastigote forms (107 cells/mL) were cultivated in the presence of normal fetal calf serum (FCS), delipidated FCS (FCSd) or FCSd with 10% sterile LDL (4.1 mg/mL). During seven days, 10 lL aliquots of culture were taken every 24 h. The cells were fixed with 4% formaldehyde in PBS and counted in a Neubauer chamber (Optik Labor Frisch., Balgach, Germany). Growth rates of parasites were measured relative to those of control cells incubated with normal FCS. 2.10. LDL uptake and cholesterol accumulation Promastigote forms (107 cells/mL) were incubated at 4 °C (binding assays) or at 28 °C (uptake assays) in the absence and presence of various concentrations of pure LDL. The incubation volume was 200 lL. After 24 h, cells were precipitated, and the supernatants were discarded. Cells were washed twice with 0.5 mL PBS containing 1% sucrose, lysed with chloroform and subjected to lipid extraction as described above. The organic phase was collected, evaporated under nitrogen and resuspended in 0.1 mL of chloroform. Total cholesterol was measured as described by Zlatkis et al. (1953) using commercial cholesterol (10 mg/mL, Sigma–Aldrich Co, USA) as standard. 2.11. LDL uptake and fluorimetric analyses Parasites (1  107 cells) were incubated for 30 min, 3 h or 24 h at 28 °C in the presence of 100 lg/mL of LDL coupled to Texas Red-PE or cholesteryl-Bodipy (Molecular Probes - Invitrogen, Carlsbad, CA), as described in 2.7 item. After, the parasites were washed twice in PBS pH 7.4 and fixed in 4% formaldehyde in PBS pH 7.4 for 20 min at room temperature. The cellular suspensions were adhered to 0.1% poly-L-lysine coated glass coverslips for 20 min and incubated with DAPI (5 lg/mL) in PBS for 5 min under light protection. Samples were mounted on 0.2 M n-propylgallate in glycerol:PBS (9:1). Images were acquired using appropriated filters in a Zeiss Axioplan epifluorescence microscope coupled to an Olympus X30 CCD camera, and were further processed using Adobe Photoshop CS2 (Adobe Systems, Inc.) and deconvoluted using Image J program (National Institutes of Health, Bethesda, MD). Cellular growth was measured by direct counting in a Neubauer chamber. Alternatively, the parasites (5  106 cells) was incubated at the same conditions described before, ressuspended in PBS pH 7.4 and the cellular suspensions were transferred to a black 96-well microplate. The Texas Red fluorescence was determined in a Microplate

2.12. Uptake of radioactively labeled LDL Promastigote, amastigote or metacyclic forms (107 cells/mL) were incubated at 4 °C (binding assays) or at 28 °C (uptake assays) with 200.000 CPM of 3H-CHO-LDL (0.2 mg/mL). Promastigote forms are also incubated in the presence of 200.000 CPM of 125ILDL (0.17 mg/mL). The incubation volume was 200 lL. At the indicated time, the cells were precipitated, and the supernatants were discarded. Cells were washed twice with 0.5 mL PBS containing 1% sucrose and resuspended in 30 ll of the same buffer. Cells were lysed, and the associated radioactivity was measured by liquid scintillation or gamma counting, respectively. 2.13. Centrifugation binding assay

2.14. Palmitic acid incorporation into cholesterol by L. amazonensis Promastigote forms (3  107 cells/mL) were incubated at 28 °C or at 4 °C in the presence of 10% FCSd and LDL (2,23mgmL). After 30 min 500.000 DPM of 3H-palmitic acid (palmitic acid [9,10-3H(N)], PerkinElmer, Boston, MA, USA) complexed with BSA was added. The incubation volume was 1 mL. After different times of incubation, cells were washed twice with 1 mL PBS containing 1% sucrose and resuspended in 100 lL of the same buffer. The cells were subjected to lipid extraction, and the lipids were analyzed by thin-layer chromatography and re-extraction as described above. Cholesteryl-ester associated radioactivity was measured by liquid scintillation counting, 2.15. Flow cytometry analysis Five samples with 1  107 promastigote forms of L. amazonensis each were incubated with 0.3 mg/mL of FITC-LDL at 28 °C or 4 °C. After three hours, the samples were washed three times with PBS pH 7.4 before analysis by flow cytometry. Data acquisition and analyses were performed using a FACSCalibur flow cytometer (Becton–Dickinson, Franklin Lakes, NJ, USA) equipped with Cell Quest software (Scripps Research Institute, La Jolla, CA, USA). 2.16. Statistical analysis All data are presented as the mean ± S.E.M. of the mean. The means were determined from three independent assays. Standard error bars are not shown where the error range is smaller than the symbol size. Statistical comparisons of means were evaluated using the Student’s T test (unpaired), the one-way and the

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two-way analysis of variance (ANOVA) followed by Dunnett tests and Bonferroni tests (paired). A p value 60.05 was considered significant.

3. Results 3.1. The effect of LDL on parasite multiplication and development In the vertebrate host, it is extremely likely that trypanosomatids require lipid sources, especially cholesterol, to complete their metabolism and membrane composition before internalization by macrophages and subsequent transformation. The main source of cholesterol for these parasites is LDL particles acquired by endocytosis (Dixon et al., 1971, 1972; Prioli et al., 1990; Coppens et al., 1991; Morita et al., 2000). We first investigated the role of LDL on L. amazonensis proliferation. Parasites were cultivated in delipidated fetal calf serum (FCSd) in the presence or absence of LDL (Fig. 1A). For this assay, the lipids from serum (Fig. 1B, lane A) were removed as described on Materials and Methods (Fig. 1B, lane B). The serum lipid reposition by addition of sterile LDL was confirmed by one-dimensional TLC (Fig. 1B, lane C). Basic medium supplemented with FCSd did not promote normal parasite proliferation (Fig. 1A), although the parasites had normal morphology and cell size (data not shown). On the contrary, the parasites grew normally when the basic medium was supplemented with 10% purified sterile LDL (Fig. 1A). The results indicated the significance of LDL lipids in promoting and sustaining parasite growth.

3.2. LDL uptake and cholesterol accumulation To investigate the in vitro uptake of LDL by L. amazonensis, parasites were incubated with 125I-LDL for different time periods. The incubation was stopped by removal of labeled lipoproteins, cells were then washed twice with PBS containing 1% sucrose and the radioactivity associated with the parasites was determined by gamma counting. Under these conditions, radioactivity from 125ILDL was transferred to the parasites in a time-dependent manner

(Fig. 2A). The amount of radioactivity transferred to L. amazonensis increased with time up to 24 h. L. amazonensis uptake of LDL led to cholesterol accumulation in the parasite. Incubation of cells with increasing concentrations of pure LDL (up to 4 mg/ml) did not indicate saturation of cholesterol accumulation (Fig. 2B). This resulted in an increase of the L. amazonensis cholesterol content to a level that is almost 150 mg/mg of cell protein. Flow cytometry analysis of cells previously incubated with FITC-labeled LDL showed that LDL uptake was impaired at 4 °C (Fig. 3B). The data were first plotted by population density, and all analyses were performed in the region corresponding to the highest cell density (Fig. 3A). The cells incubated at 4 °C showed no significant fluorescence, with a low number of labeled cells. On the other hand, the cells incubated at 28 °C were able to internalize FITC-LDL molecules (Fig. 3B). 3.3. Specific binding sites for LDL in L. amazonensis membranes Receptor-mediated endocytosis of human LDL was extensively reported in procyclic forms of T. brucei (Coppens et al., 1988, 1991, 1995). The presence of LDL-binding proteins was also suggested for others members of the Kinetoplastida order (Bastin et al., 1996). To confirm the presence of specific binding sites for LDL in L. amazonensis, a membrane preparation was obtained and incubated with 125I-LDL. Labeled LDL bound to the membranes in a specific manner, as this binding decreased in the presence of an excess of nonradioactive LDL (Fig. 4). It is noteworthy that the same effect was observed in the presence of an excess of HDL, but not the insect lipoprotein, lipophorin (Lp). This result indicates that LDL specifically binds to L. amazonensis, but that this same site seems to also bind HDL. 3.4. The LDL endocytosis in promastigotes from L. amazonensis To investigate the intracellular fate of lipids from LDL, we used the fluorescent lipid analogs Texas Red-PE or BODIPY- cholesteryl label to LDL. After 30 min of incubation, the TR-LDL was found in

Fig. 1. Influence of LDL on parasite multiplication and development. (A) Parasites at the log phase of growth (four days old) were transferred to new culture medium and cultivated in the presence of normal fetal calf serum (j FCS), delipidated fetal calf serum (N FCSd) or FCSd with 10% LDL (4.1 mg/mL) (.). Every 24 h, an aliquot was taken, the cells were fixed, and the cell growth (number of cells per milliliter in a suspension) was measured. Error bars represent mean ± S.E.M., n = 3. ⁄Statistically different (p < 0.05) and ⁄⁄Statistically different (p < 0.01) from FCSd values using one-way ANOVA followed by Dunnett’s multiple comparisons test. (B) One-dimensional thin-layer chromatography for neutral lipids of normal fetal calf serum (lane A), delipidated fetal calf serum (lane B) and delipidated fetal calf serum with 10% of sterile human LDL (lane C). PL, Phospholipids; MG, Monoacylglycerol; CHO, Cholesterol; FA, Free Fatty Acids; TG, Triacylglycerol; CHOE, Cholesteryl-esters; ND, Not determined.

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Fig. 2. 125I-LDL uptake and cholesterol accumulation by L. amazonensis. (A) The parasites (107 cells/mL) were incubated in serum-free medium with 200.000 CPM of 125I-LDL (0.12 mg/mL) for the indicated times at 28 °C. The cell-associated 125I-LDL radioactivity was measured by gamma counting and the results are expressed as ng of 125I-LDL/lg of cell protein. (B) The parasites (107 cells/mL) were incubated for 24 h at 4 °C (N) or 28 °C (j) in serum-free medium with the indicated concentration of LDL. Following incubation, the parasites were washed and lysed. The lipids were extracted and the amount of total cholesterol was determined.

Fig. 3. Flow cytometry analysis of L. amazonensis after incubation with FITC-LDL. Five samples of 1  107 cells each were incubated with 0.3 mg/mL of FITC-LDL at 28 °C or at 4 °C. After 3 h, the samples were washed three times with PBS before evaluation by flow cytometry. (A) Distribution of a representative assayed cell population according to cell size and granulosity. The R1 region represents a section with a very high density of morphologically similar, viable cells. All analyses were performed with cells from this region. (B) The y and x axis represent the number of events and log of fluorescence intensity (FL1), respectively. Black peak: cells incubated with FITC-LDL at 4 °C; grey peak: fluorescent cells after incubation with FITC-LDL at 28 °C.

tubular extensions from the anterior to the posterior region of the parasites (Fig. 5B–C). After 3 h of incubation, the same fluorescent tracer was localized at the anterior region of the cell, i.e., close to the flagellar pocket and in large compartments at the posterior region (Fig. 5E–F). The BODIPY-cholesteryl-LDL was useful to show that the lipid analog was distributed for the whole cell after 24 h of incubation (Fig. 6). The fluorimetric analysis was used to show the uptake rate of TR-LDL by promastigotes according to time (Fig. 7).

3.5. Cholesterol esterification by L. amazonensis

Fig. 4. Binding of 125I-LDL to L. amazonensis membranes. 125I-LDL (0.03 mg/mL) was incubated at 28 °C for 90 min with the membrane preparation (0.1 mg protein/mL) in the absence (total binding) or presence of an excess (2.0 mg/mL) of nonradioactive LDL, HDL or lipophorin (Lp). The binding of 125I-LDL to the membranes was determined, and the results are expressed as ng bound 125I-LDL/lg of membrane protein. Error bars represent mean ± S.E.M., n = 3. ⁄⁄Statistically different (p < 0.01) from total values using one-way ANOVA, followed by Dunnett’s multiple comparisons test.

The ability of L. amazonensis synthesize cholesteryl-ester (CHOE) using the LDL particle as source of cholesterol was studied. 107 cells/mL of L. amazonensis promastigotes were incubated in the presence of LDL with 3H-palmitic acid. After incubation the parasites were washed and subjected to lipid extraction followed by TLC. The lipids were eluted and the radioactivity determined by scintillation counting. As shown in Fig. 8, the parasites were able to convert cholesterol from LDL into cholesteryl ester using the

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Fig. 5. Texas Red-PE-LDL endocytosis in promastigotes of L. amazonensis. The parasites were treated with TR-LDL (100 lg/mL) for 30 min (A–C) or 3 h (D–F) at 28 °C. The tracer was localized mostly in tubular structures (white arrows) after 30 min of endocytosis from the anterior to the posterior end of the parasites (B, C). After 3 h of incubation, we could visualize the LDL-Texas Red in large structures (white arrowheads) and along the parasite body (E, F). DAPI was used to label the nucleus and the kinetoplast positions. (C, D) Overlay images of DIC and TR-LDL. Bars: 5 lm.

3

H-palmitic acid added to the culture medium. After 24 h, at 28 °C a significant amount of CHOE was formed, but at low temperature only a negligible amount of radioactive CHOE was synthesized (Fig. 8).

treated cells regarding parasite survival and membrane integrity (data not shown).

3.6. Involvement of membrane lipid microdomains in LDL endocytosis in L. amazonensis

Studies of lipid acquirement and metabolism by Protozoan parasites are in evolution yet. In 2008, Portugal and colleagues described the mammalian LDL-receptor influence on lipid composition of T. gondii parasite during the infection. The parasite acquires LDL from the host plasma for cholesterol use as a source to its own metabolism. Most recently, Nishikawa et al. (2011) demonstrated that T. gondii cells are not able to synthesize sterols and cholesterol and take them the host cell using LDL-endocytosis pathway which contribute to parasite replication. Differently of Trypanosomatids, other parasite protozoa, Plasmodium sp, cannot store or synthesize sterols, so it relies on permanent supply of this lipid. Recently, Labaied et al. (2011) illustrated that Plasmodium sp are capable to internalize cholesterol from LDL from hepatocytes cytosol. Lipid metabolism in Trypanosomatid protozoa has been widely studied in Trypanosoma brucei, whereas other organisms have been neglected. These parasites synthesize ergosterol but not cholesterol (Korn et al., 1969). Thus, they require exogenous lipid sources for their metabolism. Fatty acids or serum lipoproteins, especially mammalian LDL, are essential for optimal growth of T. brucei in axenic culture (Coppens et al., 1988, 1995). Accordingly, in

To study whether incubation with MBCD would affect endocytosis of LDL, cells (promastigote, amastigote and metacyclic forms) were pre-incubated for 30 min with 20 mM of MBCD at room temperature. The cells were next washed and incubated in the presence of 3H-CHO-LDL. The promastigote forms are also incubated with 125I-LDL. Following the 24 h incubation, the 3H-Cholesterol associated with the amastigote (Fig. 9A), metacyclic (Fig. 9B) and promastigote forms (Fig. 9C) was significantly reduced by this treatment. When parasites were treated with increasing concentrations of MBCD and incubated in the presence of 125I-LDL at 28 °C for 24 h (Fig. 9D), shows a dose-dependent inhibition of LDL uptake. Several parameters were followed upon cell treatment with MBCD. Such data were collected through the use of either light microscopy or transmission electron microscopy (to analyze the membrane integrity). The parasite survival was analyzed by growth curve and trypan-blue marker. We have also followed blue fluorescence of the cholesterol-Filipin III complex as described in Beknke et al. 1984. No major changes were detected in MBCD-

4. Discussion

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Fig. 8. Cholesterol metabolism by L. amazonensis. Parasites (3  107 cells/mL) were incubated at 4 °C and 28 °C in serum-free medium with 10% FCSd and sterile LDL (2.23 mg/mL). After 30 min, 500.000 DPM of 3H-palmitic acid complexed with BSA was added. After the indicated times, the cells were washed and the lipids were extracted. The lipid fraction was analyzed by TLC. The cholesteryl-ester (CHOE) spots were scraped and the lipid eluted from silica. The lipid-associated radioactivity was measured by liquid scintillation counting. The results are expressed as DPM associated to CHOE/3  107 cells. Error bars represent mean ± S.E.M., n = 3. ⁄⁄⁄ Statistically different (p < 0.001) from CHOE at 4 °C values using two-way ANOVA followed by Bonferroni test.

Fig. 6. BODIPY-cholesteryl-LDL endocytosis in promastigotes of L. amazonensis. The parasites were treated with BODIPY-cholesteryl-LDL (100 lg/mL) for 24 h at 28 °C. (A) Overlay images of DIC and DAPI. DAPI (blue) was used to show the nucleus and kinetoplast positions. (B) After the period of the incubation the fluorescent tracer was found at the anterior and posterior regions of the parasites, indicating that there was uptake and redistribution of the ligant. Bars: 5 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. Endocytosis rate of Texas Red-PE-LDL by fluorimetric analysis. The promastigotes were treated with TR-LDL (100 lg/mL) for 30 min, 3 h and 24 h at 28 °C in M199 medium. Fluorimetric analysis using TR-LDL reveals that the tracer uptake is directly dependent on time. All the data are a result of three independent experiments in triplicate. Error bars represent mean ± S.E.M., n = 3. ⁄/⁄⁄Statistically different (p < 0.05/p < 0.01) from 30 min point values using one-way ANOVA. The fluorescence intensity was expressed in arbitrary units (A.U.).

Leishmania spp the cholesterol present in the cells is reported to be of exogenous origin and to arise by uptake from the medium, where it is supplied as a constituent in the foetal calf serum (Haughan et al., 1995; Ginger et al., 1999). Our data provide evidence that L. amazonensis promastigote, amastigote and the infective metacyclic forms are able to internalize human LDL as a source of cholesterol and that this process involves the participation of detergent-resistant membrane lipid microdomains. Elucidation for the roles of key nutrients present in the serum is essential for understanding the proliferation of Leishmania species. To analyse the importance of lipids and LDL from the culture medium for parasite survival, promastigote forms were cultivated in the presence of delipidated fetal calf serum. We observed a decline in the multiplication of the parasites in such condition (Fig. 1A). This decrease was reversed when LDL was added to the culture medium, showing that LDL-associated lipids are required by the parasites. Other studies have demonstrated the importance of serum lipids for growth and multiplication of different parasites and corroborate our observations. Thus, cultivation of T. brucei in cholesterol-free medium reduced the growth rate by 50% and cholesterol uptake by the parasites increased 40% after 24 h of growth in delipidated medium (Coppens et al., 1987). In addition, in T. brucei LDL or HDL isolated from different vertebrate sera were required to allow parasite multiplication in vitro under axenic culture conditions (Black and Vandeweerd, 1989). The dependency on exogenous lipids for growth and the presence of lipid uptake mechanisms in T. brucei has been documented by different approaches, although other trypanosomatid species remain to be deeply investigated. In the genus Leishmania, the amastigote forms are most studied. Leishmania amastigote forms are able to take up molecules from receptor-mediated endocytosis by rat bone marrow-derived macrophages (Rabinovitch et al., 1985). Russell and colleagues (1992) demonstrated the presence of different proteins in parasitophorous vacuoles (PV). Interestingly, transferrin, a protein that utilizes a receptor-mediated endocytosis pathway, was not found in vacuoles. Some years later, Schaible et al. (1999) described that small anionic and large molecules are delivered to parasites in PV through a pathway blocked by organic anion transporters and autophagy inhibitors.

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Fig. 9. Effect of methyl-b-cyclodextrin on LDL endocytosis by L. amazonensis. Parasites were pre-treated with the indicated concentrations of methyl-b-cyclodextrin (MBCD) for 30 min at room temperature. The untreated and pre-treated parasites (107 cells/mL) were then incubated at 4 °C or 28 °C in serum-free medium with 200.000 CPM of radioactively labeled LDL. After 24 h, the cells were washed and lysed, and the cell-associated radioactivity was measured by liquid scintillation (3H) or gamma counting (125I). The results are expressed as ng of labeled–LDL/lg of cell protein. (A–C) L. amazonensis (A) amastigote, (B) metacyclic and (C) promastigote forms were incubated with 3 H-CHO-LDL (0.2 mg/mL). Error bars represent mean ± S.E.M., n = 3. ⁄/⁄⁄Statistically different (p < 0.05/p < 0.01) from 28 °C control values using Student’s T test. (D) L. amazonensis promastigotes were incubated with 125I-LDL (0.15 mg/mL). Error bars represent mean ± S.E.M., n = 3. ⁄⁄Statistically different (p < 0.01) from control values using one-way ANOVA followed by Dunnett’s multiple comparisons test.

However, there is no evidence that the Leishmania amastigote forms in PV acquired lipids or lipoproteins from macrophage cytoplasm. Our results demonstrate that amastigote, promastigote and metacyclic forms of L. amazonensis are able to acquire and internalize LDL from culture medium (Fig. 2A and 6A–D). For promastigote forms, the LDL uptake was directly proportional to its concentration in the incubation medium (Fig. 2B). Endocytosis of LDL was also confirmed by flow cytometry (Fig. 3). In membrane binding assays, we observed that LDL and HDL competed for binding sites but not the insect lipoprotein, lipophorin (Fig. 4).The lipophorin receptor is a member of the LDL receptors superfamily. Likewise receptors capable of endocytosis of lipophorin endocytosis have been suggested in Trypanosoma rangeli (Folly et al., 2003) and Plasmodium gallinaceum (Atella et al., 2009). However, lipophorin was unable to compete with LDL for binding in the membrane of L. amazonensis, suggesting that the interaction between host lipoproteins and parasite receptor occurs through high specificity. Endocytosis was also studied by fluorescence images (Figs. 5 and 6) followed by fluorimetry (Fig. 7). The fluorescence images showed that the TR-LDL tracer was localized in tubular extensions after 30 min of endocytosis (Fig. 5A-C), resembling the multivesicular tubules first characterized in L. mexicana (Ilgoutz et al. 1999).

These compartments represent the site of accumulation of the protein and lipid that is ingested by the parasite (reviewed by De Souza et al., 2009). Moreover, the tracer was localized in large compartments along the parasite body after 3 h of endocytosis (Fig. 5D-F). The BODIPY-cholesteryl-LDL was found in all the parasite extension, indicating the BODIPY-cholesteryl uptake and distribution (Fig. 6). Further studies are been performed by our group using LDL gold particles aiming to determine the intracellular compartments involved in L. amazonensis lipid traffic. In T. brucei was suggested the presence of an acyl-CoA: cholesterol O-acyl transferase (ACAT) responsible for the esterification of cholesterol from LDL by the parasite (Coppens et al. 1995), which makes possible its accumulation in lipid bodies. Andrade-Neto and colleagues (2011) showed that the treatment of L.amazonensis with sterol biosynthesis inhibitors (SBIs) increase the LDL endocytosis, suggesting that LDL uptake is a compensatory mechanism in response to the presence of SBIs. Their results also demonstrated the exogenous cholesterol accumulation, indicating that there is a mechanism for the supply of cholesterol in the parasite citoplasm. In our results, when L. amazonensis was incubated with radioactive fatty acid in the presence of LDL, we were able to detect the radioactive cholesteryl ester (Fig. 8), suggesting that the existence of this enzyme is common to trypanosomatid family.

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Future enzyme assays and bioinformatic analysis should be performed to confirm the presence of suche gene in these parasites. The classic model of receptor-mediated endocytosis of macromolecules involves uptake through clathrin-coated pits, as exemplified by endocytosis via the LDL receptor or the transferrin receptor. More recent works have shown that this is just one of several pathways for endocytosis (Conner and Schmid, 2003; Zeng et al., 2003). It has been shown that endocytosis of oxidized LDL through CD36 occurs via a raft-mediated pathway and that caveolae are not necessary for the process (Zeng et al. 2003). More recently, Zhao et al. (2006), observed a macrophage model system showing constitutive cholesterol accumulation without the need for either LDL modification or LDL uptake mediated by receptors. The presence of detergent-resistant lipid microdomains (DRMs) in trypanosomatid membranes has been reported, which were purified and partially characterized in T. brucei (Nolan et al., 2000), T. cruzi (Corrêa et al. 2007) and L. braziliensis (Yoneyama et al. 2006). However, their enrollement as partners of LDL endocytosis by the parasites has not been pointed out. Besides, LDL endocytosis as mediated by DRMs has only been described in mammalian cells such as macrophages (Zeng et al. 2003) and fibroblasts (Storey et al. 2007). Thus, further studies are needed to elucidate the nature of the endocytic process that occurs in L. amazonensis responsible for the LDL uptake, in order to define whether this occurs through a classical clathrin-mediated endocytosis or if DRMs would be involved. Our results show that the LDL endocytosis was sensitive to MBCD treatment, which depletes cholesterol in membranes and disrupts lipid microdomains for all parasites forms (Fig. 9). These data suggest that the classic model of receptor-mediated endocytosis of LDL that involves protein uptake through clathrincoated pits may not occur in all the phases of the biological cycle, or during every metabolic event faced by Leishmania cells. In conclusion, the delineation of a DRMs pathway for endocytosis of LDL by L. amazonensis will provide the basis for future studies on detailed molecular mechanisms and regulatory molecules that control this process. To understand the relevance of DRMs for the parasite growth and infection may contribute in the future for a better knowledge of the interactions between parasites and their hosts. 5. Financial support This research was supported by grants from Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ). Acknowledgments We express our gratitude Dr. Juliany C. Rodrigues, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, for the Leishmania amazonensis donation and for starting our culture and to Lilian Soares da Cunha Gomes, Heloisa Coelho and Mileane S. Busch for excellent technical assistance. References Anderson, H.A., Chen, Y., Norkin, L.C., 1996. Bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae. Molecular Biology of the Cell 7, 1825–1834. Andrade-Neto, V.V., Cicco, N.N.T., Cunha-Júnior, E.F., Canto-Cavalheiro, M.M., Atella, G.C., Torres-Santos, E.C., 2011. The pharmacological inhibition of sterol biosynthesis in Leishmania is counteracted br enhancement of LDL endocytosis. Acta Tropica 119, 194–198. Atella, G.C., Bittencourt-Cunha, P.R., Nunes, R.D., Shahabuddin, M., Silva-Neto, M.A.C., 2009. The major insect lipoprotein is a lipid source to mosquito stages of malaria parasite. Acta Tropica 109, 159–162.

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