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Antimicrobial Agents and Chemotherapy, October 2008, p. 3573-3579, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00587-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of an ABCG-Like Transporter from the Protozoan Parasite Leishmania with a Role in Drug Resistance and Transbilayer Lipid Movement

Esther Castanys-Muñoz, José María Pérez-Victoria, Francisco Gamarro,^* and Santiago Castanys^*

Instituto de Parasitología y Biomedicina López-Neyra, CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n, 18100 Armilla, Granada, Spain

Received 5 May 2008/ Returned for modification 4 June 2008/ Accepted 11 July 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Leishmaniasis treatment is hampered by the increased appearance of treatment failure. ATP-binding cassette (ABC) transporters are usually involved in drug resistance both in tumor cells and in microorganisms. Here we report the characterization of an ABCG-like transporter, LiABCG6, localized mainly at the plasma membrane in Leishmania protozoan parasites. When overexpressed, this half-transporter confers significant resistance to the leishmanicidal agents miltefosine and sitamaquine. This resistance phenotype is mediated by a reduction in intracellular drug accumulation. LiABCG6 also reduces the accumulation of short-chain fluorescent phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. As a whole, these results suggest that LiABCG6 could be implicated in phospholipid trafficking and drug resistance.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
First-line treatment against visceral leishmaniasis is based on pentavalent antimonials, but their efficacy is compromised by the increased appearance of drug resistance (7, 9). Amphotericin B and miltefosine were recently recommended as alternative therapeutic drugs. Other drugs in clinical trials are paromomycin and the 8-aminoquinoline sitamaquine.

Experimental resistance to miltefosine due to inactivation of the miltefosine transport complex (26, 27), as well as to overexpression of ABC transporters (6, 28, 30), has been described recently. ABC transporters constitute one of the largest families of proteins described, with a broad variety of physiological functions and considerable medical and economical consequences. These proteins are highly evolutionarily conserved and ubiquitous; they are present from prokaryotes to humans (13). ABC transporters use energy from ATP hydrolysis to translocate their substrates (ions, heavy metals, carbohydrates, amino acids, antibiotics, anticancer drugs, proteins, phospholipids, steroids, or pigments) across the cell membrane. The essential structure of an ABC transporter consists of two highly conserved nucleotide binding domains (NBD), which bind and hydrolyze ATP, and two hydrophobic transmembrane domains (TMD), with six membrane-spanning helices. Eukaryotic ABC transporters can be organized either as full-size transporters (two NBD and two TMD) or as half-transporters, containing just one of each domain. Half-transporters are thought to require dimerization in order to assemble a functional protein.

Among the substrates of ABC transporters, many of the aminoquinolines used for malaria treatment (chloroquine, mefloquine, primaquine) can be found. MRP1 (ABCC1) transports both chloroquine and mefloquine (23, 38). P-glycoprotein (MDR1, ABCB1) interacts with mefloquine (3). The Plasmodium falciparum ABC pfmdr transports mefloquine and is involved in the phenomenon of drug resistance (34). On the other hand, many ABC proteins are implicated in the movement of phospholipids and cholesterol among organelles in mammalian and yeast cells (32). ABCA1 transports cholesterol and phosphatidylcholine (PC) to ApoA-I; ABCB4 transports PC into the bile (35). Similarly, members of the ABCG subfamily have been associated with lipid transport (18, 31, 35). ABCG transporters are half-transporters, with an NBD-TMD topology; therefore, dimerization is required for them to become functionally active. Mammalian ABCG1 mediates cholesterol, PC, and sphingomyelin efflux (17, 33), and it is thought to heterodimerize with ABCG4 (10). Although mammalian ABCG2 was described primarily as a multidrug transporter, it can also be involved in lipid transport processes. ABCG2 mediates the transport of unconjugated steroids and sulfated conjugates of bile acids and steroids (36). Mammalian ABCG5 and ABCG8, which are expressed to high levels in epithelial cells of the intestine and act as a heterodimer, have been associated with the efflux of phytosterols and cholesterol into bile (11). Recently, ABCG-like proteins required for the secretion of surface waxes have been described in Arabidopsis spp. (4).

The complete sequence of the Leishmania genome (14) has allowed the elaboration of a complete inventory of ABC transporters found in Leishmania spp.—42 transporters belonging to all subfamilies described in eukaryotes (A to H) (20). To date, only ABC transporters related to the ABCA, ABCB, ABCC, and ABCG subfamilies have been described for Leishmania spp. (2, 5, 6, 8, 24, 29). In this work, we report the characterization of the Leishmania infantum ABCG6 gene (LiABCG6), which encodes an ABCG-like transporter localized at the parasite plasma membrane (PM). LiABCG6 has phospholipids, alkyl-phospholipids, and aminoquinolines as potential substrates and seems to be responsible for their active outward transport from the cytoplasmic to the exoplasmic leaflet of the parasite PM. (While this work was in progress, the ABCG6 gene of Leishmania donovani was described. ABCG6 is involved in camptothecin resistance due to rapid efflux [5].)

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Materials. Miltefosine and perifosine were from Zentaris (Frankfurt, Germany). Edelfosine was purchased from Calbiochem (Darmstadt, Germany). Hexadecylphospho[1,2-ethylene-¹⁴C]choline ([¹⁴C]miltefosine; 1.33 MBq/mmol) was synthesized by Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom). Sitamaquine [6-methoxy-8-(6-diethylaminohexylamino)lepidine dihydrochloride] and [benzene ring-U-¹⁴C]sitamaquine ([¹⁴C]sitamaquine; 2.07 GBq/mmol) were kindly provided by GlaxoSmithKline (Greenford, United Kingdom). Chloroquine, mefloquine, primaquine, and camptothecin were purchased from Sigma-Aldrich (St. Louis, MO). The fluorescent analogues 1-palmitoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-sn-glycero-3-phosphocholine (NBD-PC), -phosphoethanolamine (NBD-PE), and -phosphoserine (NBD-PS) and 6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-hexanoyl-sphingosine-1-phosphocholine (NBD-sphingomyelin [NBD-SM]) were from Avanti Polar Lipids (Birmingham, AL).

Cell cultures. Promastigotes of L. infantum (strain MHOM/ES/1993/BCN-99) were grown in vitro at 28°C in modified RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 20% heat-inactivated fetal bovine serum (Invitrogen).

DNA constructs and transformation procedures. LiABCG6 (GenBank accession no. XM_001469659) was isolated from genomic DNA of L. infantum by PCR using sense (5'-GTCGCTCTGGACATACTTGC) and antisense (5'-CATTGGCAGAGAACATCTGC) primers. Nucleotide sequences were determined automatically as described previously (19). For homologous expression in parasites, the LiABCG6 gene was cloned into the Leishmania expression vectors pXG-GFP+2' and pXG-'GFP+, producing the GFP-LiABCG6 and LiABCG6-GFP constructs, with a green fluorescent protein (GFP) fusion at the NH₂ or COOH terminus, respectively (12). For the NH₂-tagged version, the LiABCG6 open reading frame was amplified by PCR using sense (5'-GAAGATCTCGATGTCTTCTCCAGCGCC; bearing the BglII site) and antisense (5'-CAGCGGCCGCTCACTTCCCCTCAGTGGACC; bearing the NotI site) primers. For the COOH terminally tagged version, the LiABCG6 open reading frame was amplified by PCR using sense (5'-CACCCGGGATGTCTTCTCCAGCGCCAC; bearing the SmaI site) and antisense (5'-CTAGATCTCTTCCCCTCAGTGGACCG; bearing the BglII site) primers.

To obtain a nontagged version of LiABCG6, the GFP-LiABCG6 construct was digested with BglII and NcoI in order to eliminate the GFP tag. The resulting plasmid was sequenced and renamed LiABCG6-pXG2.

Cell transformation and analysis. Promastigotes of L. infantum were transfected with the different constructs obtained and were selected for G-418 resistance as previously described (26). Parasite drug sensitivities were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT)-based assay as previously described (15). The 50% inhibitory concentration (IC₅₀) was defined as the drug concentration required for half-maximal inhibition of the cellular growth rate.

Fluorescence microscopy. For localization studies of LiABCG6 GFP-fused chimeras, live parasites were pelleted, washed three times in phosphate-buffered saline (PBS), and attached to poly-L-lysine-coated coverslips, and images were acquired using a Zeiss (Oberkoche, Germany) Axiophot microscope equipped with a SPOT camera (Diagnostic Instruments, Inc.). In order to differentiate the PM from intracellular organelles, parasites were incubated in hypotonic buffer (5 mM Tris-HCl, 100 µM phenylmethylsulfonyl fluoride [pH 7.4]) for 30 min on ice and gently homogenized, resulting in the formation of deflagellated ghosts that preserve the microtubular structure associated with the parasite PM (21). Expression of tagged LiABCG6 was verified by Western blot analysis using polyclonal anti-GFP antibodies (1:7,500) (Molecular Probes) and a horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (1:5,000).

Nucleic acid blotting. Total RNA was extracted from L. infantum promastigotes with Trizol reagent according to the manufacturer's instructions (Invitrogen); electrophoresis was performed on denaturing gels containing formaldehyde; and the RNAs were transferred to Hybond-N nylon membranes (Amersham) by standard methods. Filters were hybridized with [³²P]dCTP random-primed labeled probes prepared from gel-isolated DNA fragments by using the Perfectprep gel cleanup kit (Eppendorf). A specific probe was obtained after HindIII-BglII digestion of the LiABCG6 gene.

Analysis of fluorescent phospholipid uptake and endocytosis. Parasites (10⁷ ml^–1) were incubated in HPMI buffer (20 mM HEPES, 132 mM NaCl, 3.5 mM KCl, 0.5 mM MgCl₂, 5 mM glucose, 1 mM CaCl₂ [pH 7.4]) supplemented with 0.3% (wt/vol) bovine serum albumin (BSA) for 30 min at 28°C and were labeled with 10 µM NBD-PC, 10 µM NBD-PE, 10 µM NBD-SM, or 20 µM NBD-PS for 30 min at 28°C as described previously (6, 25). These NBD-lipid concentrations are nonsaturable and suitable for analysis of the accumulation level by flow cytometry. NBD-PS was applied at the highest concentration because its accumulation efficiency is lower than those of other NBD-lipids (25). HPMI buffer was supplemented with either 500 µM phenylmethylsulfonyl fluoride or 5 mM diisopropylfluorophosphate to block the catabolism of NBD-lipids (1). Parasites were washed twice in ice-cold PBS supplemented with 0.3% BSA and were resuspended in PBS for flow cytometry analysis using a Becton Dickinson FACScan (San Jose, CA) equipped with an argon laser operating at 488 nm. To compare the uptake of NBD-lipids with that of the endocytic marker FM4-64 (Molecular Probes), parasites (5 x 10⁶ ml^–1) were incubated for 30 min at 28°C with 5 µM FM4-64 in HPMI buffer plus 0.3% (wt/vol) BSA and then washed with cold PBS to remove the noninternalized probe, and the uptake was measured by flow cytometry as previously described (25).

Determination of radiolabeled miltefosine and sitamaquine accumulation. The internalization of [¹⁴C]miltefosine was measured as previously described (25). L. infantum promastigotes (2 x 10⁷) were incubated in culture medium with 0.09 µCi ml^–1 [¹⁴C]miltefosine (2.5 µM) at 28°C. At various times, 1-ml aliquots were removed and placed on ice. After a wash with PBS containing 10 mg ml^–1 BSA to allow the removal of the drug fraction bound to the outer leaflet of the PM, followed by a second PBS wash, the cell pellet was resuspended in 1% Triton X-100, and both the protein concentration and cell-associated radioactivity were determined. Under our experimental conditions, the process of miltefosine internalization is nonsaturable through 60 min of incubation (25).

For [¹⁴C]sitamaquine internalization, L. infantum promastigotes (2 x 10⁷) were incubated in Hanks balanced salt solution (137 mM NaCl, 5 mM KCl, 7 mM Na₂HPO₄, 6 mM glucose, 21 mM HEPES [pH 7.4]) with 0.3 µCi ml^–1 [¹⁴C]sitamaquine (5.4 µM) at 28°C for 60 min. Under these conditions the process of sitamaquine internalization is nonsaturable (F. Gamarro, S. Castanys, and J. M. Pérez-Victoria, unpublished data). Parasites were washed with ice-cold PBS and resuspended in 1% Triton X-100, and both the protein concentration and cell-associated radioactivity were determined.

Chloroquine accumulation and efflux. Chloroquine accumulation was determined by measuring the decrease in fluorescence intensity after the incubation with parasites. The fluorescence of 2 µM chloroquine in HPMI buffer (pH 8.5) was measured at 28°C. After stabilization of the fluorescence signal, parasites were added to a final concentration of 2 x 10⁷ ml^–1, and the decline in fluorescence was measured as a function of time (excitation wavelength, 343 nm; emission wavelength, 382 nm; bandwidth, 4 nm) using a SLM-AMINCO fluorescence spectrometer (AMINCO-Bowman, Urbana, IL).

To determine chloroquine efflux, promastigotes (2 x 10⁷ ml^–1) were incubated in HPMI buffer plus 50 µM chloroquine for 30 min at 28°C. After the incubation time, parasites were washed twice in cold PBS and resuspended in HPMI buffer (pH 8.5) at a final concentration of 2 x 10⁸ ml^–1. Two hundred microliters was added to thermostatized (28°C) quartz cuvettes previously loaded with 1.8 ml of HPMI buffer (pH 8.5), and the increase in fluorescence was measured as described above. After the efflux, parasites were pelleted, and the fluorescence in the supernatant was measured.

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Molecular characterization and expression analysis of LiABCG6. The present work focuses on the characterization of the ABCG-like gene from chromosome 36, named LiABCG6 according to the classification recently proposed (20). LiABCG6 codes for a 683-amino-acid protein with a predicted molecular mass of 74.4 kDa. A sequence database search using the FASTA algorithm showed the best match with the ABCG/white subfamily of transporters. LiABCG6 shares 21.2% and 24.1% amino acid identity with human ABCG1 and ABCG2, respectively, 23.8% with the Drosophila white gene product, and 25.7% with yeast ADP1. The highly conserved NBD shares 50% identity with those of human ABCG1 and ABCG2. Hydrophobicity plots of LiABCG6 showed the expected topology for an ABCG half-transporter with a hydrophilic region, bearing the highly conserved motifs of ABC transporters at the NH₂ terminus and a hydrophobic region (TMD) with six transmembrane segments at the COOH terminus.

Localization of LiABCG6 in Leishmania promastigotes. To ascertain the subcellular localization of LiABCG6, GFP chimeras were created. Expression was determined by Western blotting (Fig. 1A). During the chimera selection process, high levels of expression were achieved, even at the minimum concentration of selectable drug (25 µg/ml) (data not shown).

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FIG. 1. Localization of LiABCG6 in Leishmania parasites. (A) Western blot analysis of GFP-transfected parasites overexpressing GFP-LiABCG6 or LiABCG6-GFP. The blots were stained with an anti-GFP antibody (dilution, 1:7,500) and a horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (dilution, 1:5,000). The positions of the prestained markers are indicated on the left. (B) Fluorescence microscopy analysis of L. infantum promastigotes overexpressing LiABCG6 tagged with a GFP fusion at the NH2 (GFP-LiABCG6) or COOH (LiABCG6-GFP) terminus. (Upper panels) Differential interference contrast (DIC) images. (Middle panels) Fluorescence images showing GFP fluorescence predominantly at the PM in GFP-LiABCG6 parasites or at the multivesicular tubule in LiABCG6-GFP parasites. (Lower panels) Fluorescence images of deflagellated ghosts prepared by hypotonic shock. GFP fluorescence remains associated with the PM.

Localization studies performed on GFP-LiABCG6- and LiABCG6-GFP-transfected promastigotes by fluorescence microscopy showed different localizations of LiABCG6, depending on the end to which GFP was fused. In parasites transfected with GFP-LiABCG6, the protein was localized mainly at the PM (Fig. 1B). This was verified with "ghost" parasites, in which the protein could still be observed at the PM. However, parasites transfected with LiABCG6-GFP showed a different localization of LiABCG6: the protein was localized exclusively at the multivesicular tubule, considered a terminal lysosome (22) (Fig. 1B).

Fluorescent phospholipid uptake in parasites overexpressing LiABCG6. To elucidate the possible role of LiABCG6 in lipid transport, we investigated the internalization of fluorescent phospholipid analogues in parasites transfected with LiABCG6-pXG2. LiABCG6 overexpression was analyzed by Northern blotting (data not shown). The accumulation of NBD-PC, NBD-PE, and NBD-PS was significantly lower in parasites overexpressing LiABCG6 than in mock-transfected parasites (Fig. 2). No significant differences in size were observed between mock and LiABCG6 transfectants by flow cytometry analysis (n = 4) (data not shown). The ratios of accumulated NBD-lipids between mock-transfected and LiABCG6-overexpressing parasites were 1.80 ± 0.09 for NBD-PC, 1.58 ± 0.19 for NBD-PE, and 1.42 ± 0.02 for NBD-PS (n = 4; P < 0.005). No significant differences were observed for NBD-SM (ratio, 0.98 ± 0.03; n = 4). No differences in the uptake of the endocytic marker FM4-64 were observed between mock-transfected and LiABCG6-overexpressing parasites (data not shown). Taken together, these results suggest that LiABCG6 activity specifically affects the accumulation of NBD-lipids by enhanced outward transport across the PM.

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FIG. 2. Fluorescent phospholipid accumulation in Leishmania parasites. Parasites were incubated with the fluorescent phospholipid analogue NBD-PC, NBD-PE, NBD-PS, or NBD-SM for 30 min at 28°C. After a wash, cell-associated fluorescence was measured by flow cytometry. Representative histograms are shown for each NBD-lipid. The shaded histogram represents mock-transfected cells; the open histogram represents cells of LiABCG6-transfected parasites; and the dotted line represents unlabeled cells.

Drug sensitivity profiles of Leishmania overexpressing LiABCG6. Since several ABC transporters have been implicated in drug resistance in Leishmania, we analyzed whether overexpression of LiABCG6 could confer resistance to several drugs. Different unrelated leishmanicidal drugs were tested, including alkyl-phospholipids (miltefosine, edelfosine, and perifosine), azoles (ketoconazole), aminoquinolines (sitamaquine, chloroquine, mefloquine, and primaquine), and camptothecin (Fig. 3). Parasites overexpressing LiABCG6 were about twofold more resistant to miltefosine, edelfosine, and perifosine than mock-transfected parasites, pointing to alkyl-phospholipids as possible substrates for LiABCG6 (Table 1). Overexpression of LiABCG6 conferred resistance to the aminoquinolines sitamaquine, chloroquine, and (slightly) mefloquine, whereas it did not affect susceptibility to primaquine (Table 1). These results suggest that aminoquinolines are putative LiABCG6 substrates. LiABCG6 is also involved in camptothecin resistance, as previously reported for L. donovani ABCG6 (5). LiABCG6 did not confer resistance to ketoconazole. Promastigotes overexpressing GFP-LiABCG6 showed behavior similar to that of parasites overexpressing untagged LiABCG6. Therefore, tagging at the NH₂ terminus did not interfere with functionality, whereas a tag at the COOH terminus inactivated the protein functionality while also altering the correct localization (Table 1 and Fig. 1B).

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FIG. 3. Structures of miltefosine, camptothecin, and the aminoquinolines tested.

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TABLE 1. Drug resistance profile for L. infantum promastigotes overexpressing LiABCG6a

Miltefosine and sitamaquine accumulation in Leishmania promastigotes overexpressing LiABCG6. To corroborate that LiABCG6 would have alkyl-phospholipid analogues and aminoquinolines as potential substrates, the intracellular accumulation of [¹⁴C]miltefosine and [¹⁴C]sitamaquine was measured. Parasites overexpressing LiABCG6 accumulated 75% and 70% of the levels of miltefosine and sitamaquine accumulated by mock-transfected parasites, respectively (Fig. 4A and B). We consider that these reductions in drug accumulation are compatible with 2-fold increased IC₅₀s, comparable to those previously described for Leishmania ABCG4 (6).

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FIG. 4. Miltefosine and sitamaquine accumulation in Leishmania parasites. Mock-transfected (filled bars) and LiABCG6-overexpressing (shaded bars) promastigotes were incubated with [14C]miltefosine (A) or [14C]sitamaquine (B) as described in Materials and Methods. After 30 min and 60 min, respectively, parasites were chilled on ice and washed with cold PBS, which, in the case of [14C]miltefosine, contained 10 mg ml–1 BSA. Cell-associated radioactivity was quantified and normalized to the protein concentration. Data are expressed as the percentage of [14C]miltefosine or [14C]sitamaquine incorporated; the amount incorporated by the mock-transfected parasites is taken as 100%. Each bar represents the mean for four independent experiments performed in duplicate; error bars, standard deviations. Significant differences from results for mock-transfected parasites were determined by Student's test (*, P < 0.01 in panel A and P < 0.005 in panel B).

These results are consistent with enhanced outward transport of both [¹⁴C]miltefosine and [¹⁴C]sitamaquine across the PM, probably due to the efflux activity of LiABCG6.

Chloroquine accumulation and efflux. To further corroborate that chloroquine is a putative substrate for LiABCG6, a transport assay was developed. Chloroquine is a diprotic weak base, only slightly fluorescent at a pH below 7.5, but its quantum yield increases severalfold at a basic pH (Fig. 5A). We therefore hypothesized that it could be possible to measure real-time chloroquine transport as a change in the fluorescence of live parasites in a medium at pH 8.5 by continuously monitoring the process in a spectrofluorimeter. Drug accumulation would be measured as a decrease in chloroquine fluorescence, because the drug would enter a more acidic compartment. Drug efflux from cells that have previously internalized a dye would be measured as an increase in fluorescence due to the exit of the drug to the more basic external medium.

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FIG. 5. Chloroquine accumulation and efflux in Leishmania parasites. (A) Chloroquine fluorescence. The fluorescence of chloroquine at 5 µM was measured in HPMI buffer at different pHs, ranging from 11 to 4. Fluorescence was higher at basic pHs and decreased toward neutral and acidic pHs. (B) Chloroquine accumulation. Mock-transfected (dotted line) and LiABCG6-overexpressing (solid line) promastigotes were resuspended in HPMI buffer (pH 8.5). When parasites were added to HPMI buffer (pH 8.5) plus 2 µM chloroquine, a reduction in fluorescence was observed as a result of chloroquine transport from the basic external medium to neutral or, more probably, acidic intracellular compartments. The accumulation was slower and less in LiABCG6-transfected parasites. Results of one experiment representative of three independent experiments, each performed in duplicate, are shown. (C) Chloroquine efflux. Mock-transfected and LiABCG6-overexpressing parasites were incubated in HPMI buffer (pH 7.4) with 50 µM chloroquine. After 30 min, parasites were washed and resuspended in HPMI buffer (pH 8.5), and the fluorescence was measured over 75 min. Results for mock-transfected parasites were subtracted from those for LiABCG6-overexpressing parasites, and the net trace is shown from the point at which parasites were added to initiate drug transport. Results of one experiment representative of three independent experiments are shown. (Inset) Determination of fluorescence in the medium. After 75 min of chloroquine efflux measurement, parasites were pelleted, and fluorescence in the medium was determined. Filled bar, mock-transfected promastigotes; shaded bar, LiABCG6-overexpressing promastigotes. Data are means for three independent experiments; error bars, standard deviations.

In order to prove this, we first measured chloroquine fluorescence in HPMI buffer (pH 8.5). After several seconds, parasites were added, and a time-dependent decline in fluorescence could be observed, due to chloroquine accumulation in promastigotes, with a pH lower than that of the external medium. Chloroquine accumulation in parasites overexpressing LiABCG6 was slower and less than that in mock-transfected parasites (Fig. 5B), probably due to LiABCG6 activity at the PM.

Chloroquine efflux was measured by incubating the parasites in HPMI buffer, pH 7.4, with 50 µM chloroquine for 30 min at 28°C. Parasites were washed and resuspended in HPMI buffer, pH 8.5, and fluorescence was measured (Fig. 5C). A faster increase in fluorescence intensity from LiABCG6-overexpressing parasites than from mock-transfected parasites was observed, consistent with protein-mediated translocation of chloroquine to the medium, where chloroquine becomes fluorescent. To further corroborate this, parasites were pelleted after the efflux, and the fluorescence of the supernatant was measured. The fluorescence of the LiABCG6 supernatant was twice that of the mock-transfected parasite supernatant (Fig. 5C inset). We can conclude that this efflux activity correlated well with a fourfold increase in IC₅₀s for chloroquine in parasites overexpressing LiABCG6.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this report we provide evidence for the lipid and drug transport activity of the ABCG-like half-transporter LiABCG6 in the protozoan parasite L. infantum.

Parasites overexpressing LiABCG6 accumulated significantly lower levels of fluorescent phospholipid analogues of PC, PE, and PS than control cells. The reduction in phospholipid accumulation was equivalent to that observed for other ABC transporters from Leishmania (2, 24, 30). However, no accumulation differences were noticed in parasites labeled with the sphingolipid analogue or with the endocytic/exocytic marker FM4-64. Therefore, the most straightforward explanation for these results would be enhanced outward transport of these lipid analogues across the PM by LiABCG6.

Parasites overexpressing LiABCG6 were resistant to the alkyl-phosphocholine derivatives miltefosine and perifosine, as well as to the alkyl-glycerophospholipid edelfosine. In agreement with this, parasites overexpressing LiABCG6 showed significantly reduced levels of [¹⁴C]miltefosine accumulation. Members of the Leishmania ABCA family are implicated in phospholipid trafficking in Leishmania, though they do not confer resistance to alkyl-phospholipids (2, 24). However, the Leishmania ABCB1 transporter (LtrMDR1) and LiABCG4 reduce the accumulation of a PC fluorescent analogue and are implicated in resistance to miltefosine (6, 28, 30).

We had previously reported that overexpression of an ABCG4 protein from Leishmana infantum confers resistance to sitamaquine (6). Parasites overexpressing LiABCG6 show a different pattern of resistance to aminoquinolines that could be attributed to the chemical structure of these drugs (Fig. 3). Chloroquine and sitamaquine have a long-chain substituent ending in a tertiary amine, whereas the substituent in primaquine ends in a primary amine. Mefloquine, which evokes slight resistance in LiABCG6-overexpressing parasites, has a substituent with a secondary amine. Furthermore, LiABCG6 confers resistance to camptothecin, as previously reported for L. donovani ABCG6 (5). Aminoquinolines and camptothecin show a quinoline ring as common motifs. Interestingly, several studies have reported that most P-glycoprotein modulators share some common chemical features, such as aromatic ring structures and a tertiary or secondary amino group (37). However, further investigation might be required. These results were further corroborated by the fact that LiABCG6-transfected parasites showed significantly reduced levels of [¹⁴C]sitamaquine accumulation. Chloroquine is a diprotic weak base that tends to accumulate in acidic compartments (16). Its fluorescence at basic pHs has allowed us to develop a novel real-time transport assay, showing that parasites overexpressing LiABCG6 accumulate less chloroquine due to increased outward transport. Finally, the ABCG4 and ABCG6 half-transporters show similar spectra of activity (reference 6 and this work), suggesting that these transporters would act as heterodimers conferring increased resistance. Further coexpresssion and coinmunoprecipitation experiments with both transporters would be needed to confirm this hypothesis.

In summary, LiABCG6 is required for the outward transport of short-chain phospholipid analogues, alkyl-phospholipids, and aminoquinolines. These findings could be of future clinical relevance, since LiABCG6, together with other ABC transporters and the miltefosine protein transport complex, contributes to resistance phenotypes in Leishmania.

 
We are grateful to S. Beverley (Washington University, St. Louis, MO) for the Leishmania expression vectors pXG-GFP+2' and pXG-'GFP+, to GlaxoSmithKline (Greenford, United Kingdom) for providing the sitamaquine and ([¹⁴C]sitamaquine, and to Æterna Zentaris (Frankfurt, Germany) for providing the miltefosine and [¹⁴C]miltefosine used in this study. We are grateful to José Ignacio Manzano for technical assistance.

This work was supported by Spanish grant SAF2006-02093 (to F.G.), ISCIII-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET) RD06/0021/0002 (to F.G.), Plan Andaluz de Investigación (Cod. BIO130), and Spanish grant MSC-FIS PI040830 (to J.M.P-V.). E.C.-M. was the recipient of an FPU fellowship from the Ministerio de Educación y Ciencia.

 
* Corresponding author. Mailing address: Instituto de Parasitología y Biomedicina López-Neyra, CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n, 18100 Armilla, Granada, Spain. Fax: 34 958 181632. Phone for Santiago Castanys: 34 958 181666. E-mail: castanys{at}ipb.csic.es. Phone for Francisco Gamarro: 34 958 181667. E-mail: gamarro{at}ipb.csic.es

Published ahead of print on 21 July 2008.

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Antimicrobial Agents and Chemotherapy, October 2008, p. 3573-3579, Vol. 52, No. 10
0066-4804/08/$08.00+0     doi:10.1128/AAC.00587-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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