The Leishmania LABCG2 transporter has a key role in the redox metabolism of these protozoan parasites. Recently, the involvement of LABCG2 in virulence, autophagy and oxidative stress has been described. Null mutant parasites for LABCG2 present an increase in the intracellular levels of glutathione (GSH) and trypanothione [T(SH)2]. On the other hand, parasites overexpressing LABCG2 transporter export non-protein thiols to the extracellular medium. To explore if LABCG2 may mediate an active transport of non-protein thiols, the effect of these molecules on ATPase activity of LABCG2 as well as the ability of LABCG2 to transport them was determined using a baculovirus-Sf9 insect cell system. Our results indicate that all thiols tested [GSH, T(SH)2] as well as their oxidized forms GSSG and TS2 (trypanothione disulfide) stimulate LABCG2-ATPase basal activity. We have measured the transport of [3H]-GSH in inside-out Sf9 cell membrane vesicles expressing LABCG2-GFP (green fluorescence protein), finding that LABCG2 was able to mediate a rapid and concentration-dependent uptake of [3H]-GSH in the presence of ATP. Finally, we have analyzed the ability of different thiol species to compete for this uptake, T(SH)2 and TS2 being the best competitors. The IC50 value for [3H]-GSH uptake in the presence of increasing concentrations of T(SH)2 was less than 100 μM, highlighting the affinity of this thiol for LABCG2. These results provide the first direct evidence that LABCG2 is an ABC transporter of reduced and oxidized non-protein thiols in Leishmania, suggesting that this transporter can play a role in the redox metabolism and related processes in this protozoan parasite.
Leishmaniasis, one of the most neglected tropical diseases, is caused by various species of the trypanosomatid protozoan Leishmania. According to World Health Organization, 310 million people have a risk of infection worldwide and over 20 000 deaths occur annually through a variety of clinical presentations, ranging from cutaneous lesions to fatal visceral leishmaniasis.
One of the most peculiar characteristics of trypanosomatids is their unique redox metabolism based on trypanothione [T(SH)2], a thiol synthesized from two molecules of glutathione (GSH) that are covalently bridged by a spermidine (Spd) in two ATP-dependent reactions. In the first step, glutathionylspermidine (Gsp) is formed, which is then combined with the second GSH to yield T(SH)2. T(SH)2 is involved in a variety of functions in cell metabolism like detoxification of ketoaldehydes and xenobiotics, reduction of ribonucleotides, regulation of DNA replication in kinetoplastids and detoxification of heavy metals. T(SH)2 can be considered the principal molecule for detoxification pathways in Leishmania and other trypanosomatids, as it participates in several spontaneous or tryparedoxin-mediated reactions of the redox metabolism [1,2].
In mammals, redox reactions rely on GSH and thioredoxin  instead of T(SH)2. Currently, only some proteins have been described to transport GSH or GSH conjugates through biological membranes, including some ABC (ATP-binding cassette) transporters. ABC transporters represent one of the largest families of proteins present in prokaryotic and eukaryotic organisms . They are highly conserved from prokaryotic to humans and play a critical physiological role in transport of different compounds across cellular membranes . The general structure of ABC transporters consists of four domains. Two of them, the transmembrane domains (TMDs), are hydrophobic, containing multiple α-helices, and are involved in substrate recognition. The other two domains are the nucleotide-binding domains (NBDs), which include the highly conserved motifs Walker A and B, responsible for the ATP-binding and hydrolysis necessary to get the energy required for transport . Eukaryotic ABC transporters can codify for full-transporters or half-transporters, as in Leishmania LABCG2 that needs to homo-/heterodimerize to reconstitute ATP-binding sites and generate an active protein .
In mammals, some members of the ABCC subfamily like MRP1–MRP5 and CFTR are involved in GSH/oxidized GSH (GSSG, l-glutathione disulfide) efflux and co-transport with other compounds . Additionally, the Atm1-family ABC exporter from Novosphingobium aromaticivorans directly transports GSSG and plays a role in heavy metal detoxification, probably by export of metal–GSH complexes . Furthermore, GSSG stimulates ATP hydrolysis of ABCB10, whereas GSH inhibits ATP binding and hydrolysis . In addition, Atm3 (ABCB25) from Arabidopsis thaliana and its ortholog Atm1 from Saccharomyces cerevisiae transport GSSG but not GSH . In contrast, CydDC ABC transporter from Escherichia coli transports GSH but not GSSG . Human ABCG2 has also been proposed as a GSH transporter, although this point remains controversial [13,14].
Recently, we have reported that Leishmania major parasites overexpressing LABCG2 present a reduction in the accumulation of SbIII due to an increased drug efflux, most likely, as SbIII–thiol complexes . ABCI4 and MRPA (PGPA) of Leishmania have been proposed to carry out an active efflux and sequestration into vesicles of thiol-metal conjugates, respectively [16,17]. Also, internal thiol levels in L. major lines overexpressing LABCG2 are lower than the control line, probably because LABCG2 is able to export thiols to the extracellular medium . Additionally, null mutant parasites for LABCG2 present a significantly high accumulation of GSH and T(SH)2, supporting the hypothesis that LABCG2 is able to export GSH and T(SH)2 to the extracellular medium . Thus, in order to understand the involvement of this transporter in redox metabolism of Leishmania, we have determined the ability of LABCG2 to transport GSH using Sf9 cell membrane vesicles expressing LABCG2. Additionally, we have analyzed by competition assays the affinity of the LABCG2 transporter for different thiols including T(SH)2. To our knowledge, the present study shows LABCG2 as the first ABC transporter able to transport non-conjugated thiols in Leishmania.
Adenosine 5′-triphosphate (ATP) disodium salt hydrate, GSH, GSSG, S-methyl-glutahione (methyl-GS), phenylmethylsulfonyl fluoride (PMSF) and magnesium chloride hexahydrate (MgCl2) were purchased from Sigma–Aldrich (St. Louis, U.S.A.). T(SH)2 was kindly provided by Prof. Luise Krauth-Siegel (Heidelberg, Germany). [glycine-2-3H] GSH (45 Ci mmol−1) was purchased from PerkinElmer (Waltham, U.S.A.). Anti-GFP polyclonal antibody and polyclonal goat anti-rabbit immunoglobulin antibody were provided by Rockland Immunochemicals (Pottstown, U.S.A.). Protease inhibitors were provided by Thermo Scientific (Waltham, U.S.A.). All chemicals were of the highest quality available.
Sf9 insect cells were cultured at 27°C in TNM-FH insect medium supplemented with 10% fetal bovine serum (hiFBS) and penicillin (100 U/ml, Sigma–Aldrich, St. Louis, U.S.A.)–streptomycin (100 μg/ml; Sigma–Aldrich, St. Louis, U.S.A.).
Generation of recombinant baculovirus and expression in Sf9 insect cells
L. major (MHOM/JL/80/Friedlin) LABCG2 (GeneDB — L. major, Accession Code LmjF06.0090) was cloned in pVL1392 vector containing a TEV cleavage sequence, eight times tandemly repeated glycine–threonine–serine sequence, GFP, Flag and His10 from the C-terminus of LABCG2; this construction was named LABCG2-GFP. To obtain Sf9 cells expressing nonfunctional LABCG2 (LABCG2K/M), a mutation was introduced in the Walker A motif of NBDs using inverse PCR-based mutagenesis with the In-Fusion® HD Cloning Kit (Takara Bio, Inc., Kusatsu, Japan), replacing lysine 108 for methionine (K108M) ; this construction was named LABCG2K/M-GFP.
Recombinant baculovirus containing LABCG2-GFP or LABCG2K/M-GFP were generated by co-transfection of Sf9 cells with the mentioned constructions and the linear baculovirus tDNA (Oxford Expression Technologies, Oxford, U.K.) using the FuGeneHD Transfection Reagent (Promega, Madison, U.S.A.), according to the manufacturer's instructions. The generated virus were amplified and stored at 4°C in darkness.
Baculovirus infection and preparation of Sf9 membranes expressing LABCG2
Sf9 cells (1 × 108) at a cell growth density of 2 × 106 cells/ml were infected with recombinant baculovirus containing DNA of LABCG2-GFP or LABCG2K/M-GFP at a multiplicity of infection of 3. After 48 h post-infection, cells were harvested and membrane preparation was performed as described previously . Briefly, cells expressing the protein were centrifuged, washed and disrupted twice in a glass homogenizer in TMEP buffer (50 mM Tris–HCl, 50 mM mannitol, 2 mM EGTA–Tris (pH 7.0), PMSF and protease inhibitors). The supernatant was collected and centrifuged at 40 000 g for 60 min. The resulting pellet was once more homogenized and stored in individual aliquots at −80°C.
Western blot analysis
Protein samples were fractionated by SDS–PAGE under standard conditions and electrotransferred onto Immobilon-P membranes (Merck Millipore, Darmstadt, Germany). Immunodetection was performed by using a 1 : 5000 dilution of rabbit anti-GFP polyclonal antibody in phosphate-buffered saline plus 0.01% Tween 20 and 0.1% bovine serum albumin. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit immunoglobulin G (DAKO, Glostrup, Denmark) using a 1 : 5000 dilution. Signal was detected employing the ECL chemiluminescent substrate (Pierce Biotechnology, Waltham, U.S.A.).
ATPase activity measurement by colorimetric assay
The ATP hydrolytic activity of LABCG2-GFP and LABCG2K/M-GFP has been determined as described in ref. . Briefly, purified membranes (50 µg) were incubated in 160 µl of reaction buffer (50 mM MOPS–Tris (pH 7.0), 50 mM KCl and 0.5 mM EGTA–Tris (pH 7.0)) supplemented with 12.5 mM Mg-ATP for 30 min at 37°C. The reaction was stopped by adding 100 µl of 5% SDS. Inorganic phosphate derived from ATP hydrolysis was determined by colorimetric reaction using a Cary 100 UV-Visible Spectrophotometer (Varian, Palo Alto, U.S.A.). The stimulation of ATPase activity for GSH, GSSG, T(SH)2 and TS2 was studied at different concentrations (0.5, 1 and 5 mM).
[3H]-GSH transport assays in Sf9 membrane vesicles expressing LABCG2
The transport assays were performed as previously described with minor modifications . Before uptake assays, ATPase activity of membranes obtained from different extractions was tested, establishing a minimal stimulation by 0.5 mM GSH of 20 nmol Pi/min/mg membrane protein. Isolated Sf9 membrane vesicles (70 µg protein) were incubated in the presence of 4 mM ATP and [3H]-GSH at different concentrations (0.1, 0.2, 0.5 and 1 mM), in 30 µl transport mix (0.1 M MOPS (pH 7.0), 1 M KCl and 0.1 M MgCl2) at 37°C for 30 min (non-saturated uptake time point). For the time course assay, purified Sf9 membrane vesicles were incubated with 0.5 mM [3H]-GSH at different time points (1, 5, 10, 30 and 60 min) in the same conditions described above. For competition assays, 10-fold excess concentrations (5 mM) of GSH, GSSG, methyl-GS, T(SH)2 and trypanothione disulfide (TS2) were added at the beginning of the incubation with 0.5 mM [3H]-GSH for 30 min. Finally, T(SH)2 was added to the reaction at the indicated concentrations to determine the IC50 of [3H]-GSH uptake. To stop the reaction, 25 µl of washing buffer (0.1 M MOPS–Tris (pH 7.0) and 1 M KCl) were added and quickly filtered through hydrophilic polyvinylidene fluoride (PVDF) membranes of 0.45 µm pore size (Merck Millipore, Darmstadt, Germany). The filters were washed three times and collected. Radioactivity was subsequently measured in scintillation fluid LS 6500 (Beckman Coulter, Brea, U.S.A.). TS2 was obtained by oxidation of T(SH)2 with hydrogen peroxide as described .
Experiments were performed in triplicate in three independent experiments, and the data are presented as means ± SD. Statistical comparisons between groups were performed using Student's t-test. Differences were considered significant at a level of P < 0.05.
Results and discussion
Expression of L. major LABCG2 in Sf9 insect cells
Sf9 cells infected with recombinant baculovirus have been previously used to express different human ABC transporters, including Pgp , ABCG2 , ABCA1  and ABCA4 . Additionally, we have previously described the use of baculovirus-Sf9 insect cell system for expression of Leishmania tropica P-glycoprotein (Pgp) transporter .
For the following experiments, Sf9 membrane vesicles expressing LABCG2-GFP or the mutant LABCG2K/M-GFP were analyzed by western blot. Uninfected Sf9 cells membrane samples were used as control. Western blot assay using an anti-GFP antibody showed a unique band at ∼100 kDa that matched the predicted molecular mass for LABCG2-GFP and LABCG2K/M-GFP. The protein was absent in uninfected Sf9 membrane preparation, and no significant degradation of LABCG2-GFP and LABCG2K/M-GFP was detected (Figure 1).
Expression of LABCG2-GFP in Sf9 insect cells.
Stimulation of ATPase activity by non-protein thiols in Sf9 membrane vesicles expressing LABCG2
We have previously described that L. major parasites overexpressing LABCG2 present lower internal thiol levels, as well as a significant increase in non-protein thiol efflux, in comparison with a control line with basal levels of transporter . Additionally, null mutant parasites for LABCG2 present higher levels of GSH and T(SH)2 than the control line . However, we cannot discard the possibility that these null mutant parasites present also higher levels of GSSG and TS2, because the employed methodology involves the chemical reduction of all thiols . Therefore, we support the hypothesis that LABCG2 is able to export thiols to extracellular medium. To validate this proposal, Sf9 membrane vesicles expressing LABCG2-GFP and LABCG2K/M-GFP were obtained and ATPase activity was measured in the absence (basal ATPase activity) and presence of increasing GSH and T(SH)2 concentrations (0.5, 1 and 5 mM). Additionally, we decided to include in ATPase activity studies the oxidized forms GSSG and TS2. The results showed a significant stimulation of LABCG2-GFP basal ATPase activity for all thiols assayed in a concentration-dependent manner (Figure 2). As a result, GSSG produced a higher stimulation than GSH. It has been reported that MRP1 presents higher affinity for GSSG than for GSH . In addition, ATPase activity of Atm1 from yeast and Atm3 from plants was stimulated by GSSG but not by GSH . However, the greatest ATPase activity stimulation was generated by T(SH)2 and TS2 at 5.0 mM concentration, reaching 281 and 344 nmol Pi/min/mg membrane protein, respectively (Figure 2). Interestingly, the differences in ATPase activity stimulation between the oxidized thiols and their reduced forms were not as high as described for other ABC transporters . Taking into account that T(SH)2 is the equivalent of GSH in the redox metabolism of trypanosomatids, these results are consistent with the supposition that a transporter of thiols could be specially stimulated by T(SH)2 and TS2 in Leishmania. As expected, membrane vesicles expressing LABCG2K/M-GFP did not show ATPase activity stimulation (Figure 2), supporting the specificity of LABCG2 transporter for these substrates.
Effect of different thiols and disulfides on ATPase activity of LABCG2-GFP and LABCG2K/M-GFP.
L. major LABCG2 is able to transport GSH
As ATPase activity assays proved the interaction of LABCG2 with different thiols and disulfides, we examined the ability of LABCG2-GFP to transport [3H]-GSH into Sf9 cell membrane vesicles. As shown in Figure 3, time course of [3H]-GSH uptake in Sf9 membrane vesicles expressing LABCG2-GFP was examined, demonstrating a rapid ATP-dependent uptake of [3H]-GSH that reaches steady-state levels (∼10 500 pmol/mg) in ∼30 min.
Time course of [3H]-GSH uptake by membrane vesicles from Sf9 cells expressing LABCG2-GFP.
As mentioned, GSH is a tripeptide involved in many signalling pathways; it acts as a cellular antioxidant that plays a critical role in protecting cells from oxidative damage and toxicity of xenobiotic electrophiles or heavy metals, as well as in maintaining redox homeostasis [27,28]. Currently, there are only few proteins characterized for the ability to transport GSH across membranes. As previously reported, cells overexpressing human ABCC1 are able to export GSH/GSSG and X-thiol conjugates with a depletion of levels of these molecules due to stimulation of the transporter by verapamil . Human ABCG2 has also been proposed as a GSH transporter [13,14]. However, these results remain controversial due to recent studies, suggesting that human ABCG2 is unable to transport GSH . To date, no ABC transporters with the ability to transport non-conjugated thiols have been described in trypanosomatids. In light of this, LABCG2 can be considered the first ABC transporter of GSH in Leishmania and in other trypanosomatids, being most probably involved in maintaining the redox balance in these parasites, which is critical for proper function of cellular processes and in the protection against oxidative damage.
Additionally, LABCG2-GFP mediated ATP-dependent uptake of [3H]-GSH in a concentration-dependent manner, reaching a biochemical plateau from concentrations higher than 0.5 mM, while membrane vesicles expressing the mutant version of the transporter did not accumulate [3H]-GSH (Figure 4). This uptake suffered a deviation from the Michaelis–Menten kinetic and appeared to follow a sigmoidal curve, suggesting that this transporter could present two substrate-binding sites that interact in a co-operative manner for GSH transport. Other ABC transporters present sigmoidal kinetics in the presence of different compounds. Among others, ABCC2 (MRP2) that mediated transport of the probe substrate estradiol-17β-glucuronide also exhibits a sigmoidal curve that suggests multiple substrate-binding sites with co-operative interactions .
Transport of [3H]-GSH into Sf9 cell membrane vesicles expressing LABCG2-GFP.
Substrate affinity of Leishmania LABCG2
To investigate the affinity of LABCG2-GFP for different thiols, [3H]-GSH uptake into Sf9 cell membrane vesicles containing this transporter was analyzed in the presence of 10-fold excess (5 mM) of GSH, GSSG, T(SH)2, TS2 and methyl-GS. The uptake of [3H]-GSH was notably decreased by all thiol derivatives tested (Figure 5). As expected on the basis of ATPase results obtained, T(SH)2 and TS2 produced the strongest competition, with 95% of the uptake abolished, indicating a significantly high affinity of LABCG2-GFP for these substrates. In addition, GSSG and methyl-GS inhibited more than 80% of [3H]-GSH uptake (Figure 5). On the other hand, Gsp (a compound not included in the study) constituted by one molecule of GSH and one molecule of Spd could be transported or interact with LABCG2.
Effect of different thiol derivatives in the uptake of [3H]-GSH into Sf9 cell membrane vesicles expressing LABCG2-GFP.
In view of the noticeable affinity of the LABCG2-GFP transporter for T(SH)2, the IC50 value for [3H]-GSH uptake was obtained in the presence of increasing concentrations of T(SH)2 (Figure 6). The uptake of [3H]-GSH was reduced dramatically at low concentrations of T(SH)2, showing an IC50 less than 100 μM (Figure 6), thus supporting that LABCG2 preferentially binds this substrate with a high degree of affinity.
Determination of IC50 of T(SH)2 against GSH uptake.
Recently, we have reported that null mutant parasites for LABCG2 accumulate T(SH)2 and GSH . In accordance with these results, transport studies have demonstrated that Leishmania LABCG2 is able to interact and mediate ATP-dependent transport of GSH, and probably T(SH)2 and their oxidized forms. As mentioned, redox metabolism is involved in several biological processes as protection of cells from oxidative damage. Additionally, a reduction in intracellular thiol levels mediated by ABCC1-dependent GSH efflux leads to a more oxidizing environment inside cells that triggers autophagy . It is well known that autophagy is an essential process involved in cellular differentiation and virulence in Leishmania . Besides, null mutant parasites for LABCG2 are defective in autophagy and metacyclogenesis . Therefore, taken together, these data suggest that LABCG2 could be involved in generating the oxidative conditions needed to prompt autophagy and the consequent cellular differentiation from non-infective to infective promastigotes, by extruding T(SH)2 and GSH to the extracellular medium depending on the intracellular balance between oxidized and reduced thiols. Additionally, the efflux of reduced thiols could help to maintain a reducing niche to counteract the oxidative stress inside macrophages. In fact, T(SH)2 is able to intercept nitric oxide and form a dinitrosyl–trypanothionyl iron complex harmless for parasites . As described, L. guyanensis amastigotes die inside BALB/c macrophages through apoptosis mediated by an increase in ROS (reactive oxygen species) levels due to infection . Thus, a decrease in ROS levels inside macrophages due to the action of parasite thiols could help to prevent the ROS-mediated apoptosis of host cells, protecting the niche of Leishmania intracellular forms. At the same time, thiols conjugated to nitric oxide inside parasites could be exported to the extracellular medium in order to help parasites to survive to oxidative stress. A similar idea has been suggested for human ABCG2 and its ability to efflux GSH in stem cells, for which the redox environment is decisive to trigger differentiation . Additionally, peroxynitrite produced into the macrophage's phagosome is a powerful oxidant and cytotoxic effector molecule that can oxidize lipids, proteins and DNA . Peroxynitrite can readily oxidize molecules such as thiols and metal centers . Kinetics studies of reactivity of different components of the tryparedoxin peroxidase antioxidant system in trypanosomes (trypanothione, cytosolic tryparedoxin peroxidases) with peroxynitrite show that the reaction rate of peroxynitrite with cytosolic peroxiredoxins is several orders of magnitude higher than with dihydrotrypanothione or reduced tryparedoxin . Afterwards, the oxidized peroxiredoxin is readily reduced by the tryparedoxin and trypanothione-coupled systems that restore the peroxiredoxin to the reduced thiol native state, with the consequent oxidation of T(SH)2 . The dynamic balance between intraphagosomal oxidant production and the activity of key antioxidant enzymes in the parasite at the onset of the infection critically determines parasite infectivity . In this way, T(SH)2 has a secondary protective key role against detoxification of peroxynitrite that facilitates the survival of parasites in the oxidative environment produced by activated macrophages in the first hours after infection . The ability of LABCG2 to transport T(SH)2 and TS2 could regulate the infection, protecting Leishmania parasites from the toxic effects of peroxynitrite and its secondary species.
In trypanosomatids, one of the most important mechanisms for detoxification of heavy metals involves the formation of thiol–metal complexes and subsequent extrusion outside cells by ABC transporters. For example, it has been shown that MRPA is able to export SbIII conjugated to trypanothione . For that purpose, T(SH)2 donates two hydrogen molecules in order to be conjugated with heavy metals in an oxidized and stable complex [SbT(S)2] . Thus, we have established that L. major parasites overexpressing LABCG2 present a lower accumulation of SbIII probably due to the efflux of metal–thiol complexes . Additionally, competition assays in this work have revealed that LABCG2 also interacts specifically with GSSG and TS2, as well as the conjugate methyl-GS. These results support the idea that LABCG2 is able to extrude X-thiol conjugates in order to protect parasites from the harmful action of xenobiotics.
In conclusion, the present work confirms the hypothesis that LABCG2 is an active thiol transporter and, consequently, the first ABC transporter of non-conjugated thiols found in Leishmania. These data provide new insights into the biological function of Leishmania LABCG2 transporter, revealing a determinant role in the redox metabolism of this parasite and related processes.
green fluorescent protein
reactive oxygen species
F.G. and S.C. designed the study and analyzed the results. A.P. and J.I.M. developed the experiments. Y.K. and K.U. participated in the generation of plasmid constructions, baculovirus generation and preliminary experiments. A.P., J.I.M. and F.G. wrote the manuscript. All the authors read and approved the final version of the manuscript.
This work was supported by the Spanish Grants SAF2015-68042-R (to S.C. and F.G.), SAF2012-34267 (to F.G.), by the Proyecto de Excelencia, Junta de Andalucia, Ref. CTS-7282 (to F.G.) and by FEDER funds from the EU to S.C. and F.G. A.P. was a student of the PhD program ‘Biochemistry and Molecular Biology’ of the University of Granada (Spain) and was supported by a fellowship for predoctoral contracts for PhD training from the Ministerio de Economia y Competitividad (in charge of Project SAF2012-34267).
We thank Luise Krauth-Siegel (Heidelberg, Germany) for providing T(SH)2 used throughout this research work and Ms Naoko Shiranaga for constructing pVL1392 vectors used in this work. Also, we thank András Váradi (Budapest, Hungary) for helpful advice concerning the heterologous expression in Sf9 cells, ATPase activity and transport in Sf9 vesicles.
The Authors declare that there are no competing interests associated with the manuscript.
These authors contributed equally to this work.