A New Type of High Affinity Folic Acid Transporter in the Protozoan Parasite Leishmania and Deletion of Its Gene in Methotrexate-resistant Cells

The protozoan parasite Leishmania is a folate auxotroph and thus depends on the uptake of folate from the environment to meet its folate requirement. We show here thatLeishmania contains several putative pteridine transporter genes. Some of these genes are deleted in methotrexate-resistantLeishmania cells where there is no measurable uptake of methotrexate. Transport studies suggest that Leishmania has more than one active folate transporter, and one of these, named FT5, corresponds to a very high affinity folate transporter (Km 84 nm). The uptake of both folate and methotrexate was impaired in an FT5 null mutant at low substrate concentrations (50 nm), although transport properties at higher concentrations (1000 nm) were not statistically different between wild-type and the FT5 null mutant. Modulation of the expression of FT5 also changes the susceptibility of Leishmania cells to methotrexate. These results have permitted the characterization of a novel class of folate transporters and suggest that the parasite Leishmania has several gene products possibly transporting folates and related molecules under varying conditions. The protozoan parasite Leishmania is a folate auxotroph and thus depends on the uptake of folate from the environment to meet its folate requirement. We show here thatLeishmania contains several putative pteridine transporter genes. Some of these genes are deleted in methotrexate-resistantLeishmania cells where there is no measurable uptake of methotrexate. Transport studies suggest that Leishmania has more than one active folate transporter, and one of these, named FT5, corresponds to a very high affinity folate transporter (Km 84 nm). The uptake of both folate and methotrexate was impaired in an FT5 null mutant at low substrate concentrations (50 nm), although transport properties at higher concentrations (1000 nm) were not statistically different between wild-type and the FT5 null mutant. Modulation of the expression of FT5 also changes the susceptibility of Leishmania cells to methotrexate. These results have permitted the characterization of a novel class of folate transporters and suggest that the parasite Leishmania has several gene products possibly transporting folates and related molecules under varying conditions. dihydrofolate reductase methotrexate open reading frame The protozoan parasite Leishmania is distributed worldwide and causes a variety of clinical symptoms ranging from self-healing cutaneous lesions to visceral infections that are usually fatal if left untreated (1Herwaldt B.L. Lancet. 1999; 354: 1191-1199Abstract Full Text Full Text PDF PubMed Scopus (1381) Google Scholar). The main chemotherapeutic regimen consists of pentavalent antimonials, but resistance to this class of antiquated drugs is now prevalent in several endemic areas (2Sundar S. Trop. Med. Int. Health. 2001; 6: 849-854Crossref PubMed Scopus (491) Google Scholar). New targets are urgently required. One metabolic pathway that has been exploited extensively for the development of drugs is the folate biosynthesis pathway. Folates are made of a pterin moiety conjugated topara-aminobenzoic acid and glutamic acid. Reduced folates are key cofactors in the biosynthesis of thymidylate, and inhibitors of this pathway act primarily at the level of the enzyme dihydrofolate reductase (DHFR),1 the enzyme responsible for supplying the cell with reduced folates (3Schweitzer B.I. Dicker A.P. Bertino J.R. FASEB J. 1990; 4: 2441-2452Crossref PubMed Scopus (311) Google Scholar). Antifolates have been used successfully as anticancer drugs (methotrexate) or in the treatment of bacterial (trimethoprim) or parasitic infections (pyrimethamine) (4Ouellette M. Leblanc E. Kundig C. Papadopoulou B. Adv. Exp. Med. Biol. 1998; 456: 99-113Crossref PubMed Scopus (10) Google Scholar). No effective antifolate chemotherapy has yet been established against infections with the protozoan parasite Leishmania. Nevertheless, many distinct features in the folate metabolism of this organism have been identified that could prove to be useful therapeutic targets (5Nare B. Luba J. Hardy L.W. Beverley S. Parasitology. 1997; 114 (suppl.): 101-110Crossref PubMed Google Scholar, 6Ouellette M. Drummelsmith J., El Fadili A. Kundig C. Richard D. Roy G. Int. J. Parasitol. 2002; 32: 385-398Crossref PubMed Scopus (79) Google Scholar). Our understanding of folate metabolism in Leishmania is derived mostly from work carried out on studies related to the mechanisms of resistance to the model antifolate drug methotrexate (MTX). Leishmania is sensitive to MTX, and step by step selection of mutants for MTX resistance has revealed a multiplicity of resistance mechanisms that have been permitted to pinpoint novel biochemical pathways. Amplification of the gene coding for the target DHFR (DHFR in Leishmania is a bifunctional enzyme where it is fused to thymidylate synthase) was shown to contribute to MTX resistance (7Coderre J.A. Beverley S.M. Schimke R.T. Santi D.V. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2132-2136Crossref PubMed Scopus (177) Google Scholar, 8Washtien W.L. Grumont R. Santi D.V. J. Biol. Chem. 1985; 260: 7809-7812Abstract Full Text PDF PubMed Google Scholar, 9Ellenberger T.E. Beverley S.M. J. Biol. Chem. 1987; 262: 13501-13506Abstract Full Text PDF PubMed Google Scholar, 10Arrebola R. Olmo A. Camacho A. Ruiz-Perez L.M. GonzalezPacanowska D. Mol. Biochem. Parasitol. 1996; 79: 129-133Crossref PubMed Scopus (1) Google Scholar, 11Kündig C. Leblanc E. Papadopoulou B. Ouellette M. Nucleic Acids Res. 1999; 27: 3653-3659Crossref PubMed Scopus (29) Google Scholar). A second locus, known as the H locus, was also found to be frequently amplified in MTX-resistant mutants. The gene present on this locus encodes for the pterin reductase PTR1 (12Papadopoulou B. Roy G. Ouellette M. EMBO J. 1992; 11: 3601-3608Crossref PubMed Scopus (187) Google Scholar, 13Callahan H.L. Beverley S.M. J. Biol. Chem. 1992; 267: 24165-24168Abstract Full Text PDF PubMed Google Scholar) that can reduce both pterins and folates and produce MTX resistance by bypassing the need for DHFR (14Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Crossref PubMed Scopus (152) Google Scholar, 15Nare B. Hardy L.W. Beverley S.M. J. Biol. Chem. 1997; 272: 13883-13891Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 16Wang J. Leblanc E. Chang C.F. Papadopoulou B. Bray T. Whiteley J.M. Lin S.X. Ouellette M. Arch. Biochem. Biophys. 1997; 342: 197-202Crossref PubMed Scopus (47) Google Scholar). This discovery further linked folate and pterin metabolism in Leishmania. Indeed, it is well established that pterins can have a folate sparing effect permitting Leishmania cells and related parasites to grow in folate-deficient medium (17Kidder G. Dutta B. J. Gen. Microbiol. 1958; 18: 621-638Crossref PubMed Scopus (94) Google Scholar, 18Trager W. J. Protozool. 1969; 16: 372-375Crossref PubMed Scopus (27) Google Scholar, 19Peixoto M.P. Beverley S.M. Antimicrob. Agents Chemother. 1987; 31: 1575-1578Crossref PubMed Google Scholar, 20Beck J.T. Ullman B. Mol. Biochem. Parasitol. 1990; 43: 221-230Crossref PubMed Scopus (42) Google Scholar, 21Papadopoulou B. Roy G. Mourad W. Leblanc E. Ouellette M. J. Biol. Chem. 1994; 269: 7310-7315Abstract Full Text PDF PubMed Google Scholar). It remains to be demonstrated, however, whether Leishmania can synthesize folates de novo, but Leishmania promastigotes in culture rely heavily on uptake systems for meeting their folate needs. A folate transport activity has been studied biochemically inLeishmania, but the gene and proteins involved have yet to be characterized at the molecular level. It consists of a saturable active transport system recognizing both folate and MTX withKm values ranging from 250 to 700 nmdepending on the species (22Ellenberger T.E. Beverley S.M. J. Biol. Chem. 1987; 262: 10053-10058Abstract Full Text PDF PubMed Google Scholar, 23Kaur K. Coons T. Emmett K. Ullman B. J. Biol. Chem. 1988; 263: 7020-7028Abstract Full Text PDF PubMed Google Scholar, 24Kündig C. Haimeur A. Légaré D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (90) Google Scholar). The transport activity is regulated according to cellular growth with maximum activity in the logarithmic phase (25Cunningham M.L. Beverley S.M. Mol. Biochem. Parasitol. 2001; 113: 199-213Crossref PubMed Scopus (81) Google Scholar). Another frequent mechanism of MTX resistance in Leishmaniais reduced accumulation of the drug (9Ellenberger T.E. Beverley S.M. J. Biol. Chem. 1987; 262: 13501-13506Abstract Full Text PDF PubMed Google Scholar, 23Kaur K. Coons T. Emmett K. Ullman B. J. Biol. Chem. 1988; 263: 7020-7028Abstract Full Text PDF PubMed Google Scholar, 26Papadopoulou B. Roy G. Ouellette M. Nucleic Acids Res. 1993; 21: 4305-4312Crossref PubMed Scopus (62) Google Scholar, 27Gamarro F. Chiquero M.J. Amador M.V. Legare D. Ouellette M. Castanys S. Biochem. Pharmacol. 1994; 47: 1939-1947Crossref PubMed Scopus (51) Google Scholar). This reduced uptake of MTX is paralleled by a marked decrease in folate uptake, suggesting that the expression of the common folate/MTX transporter is strongly down-regulated in MTX-resistant Leishmania. Given that Leishmania is a folate auxotroph, this brings up the question of how the resistant parasite can meet its folate requirements. An answer was found for Leishmania tarentolae in which the biopterin transporter BT1 (24Kündig C. Haimeur A. Légaré D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (90) Google Scholar, 28Lemley C. Yan S. Dole V.S. Madhubala R. Cunningham M.L. Beverley S.M. Myler P.J. Stuart K.D. Mol. Biochem. Parasitol. 1999; 104: 93-105Crossref PubMed Scopus (63) Google Scholar) was found to be overexpressed in all MTX-resistant cells with a markedly reduced folate uptake. Sufficient folates (but not MTX) could be transported through BT1 to meet the folate requirement of the cell (24Kündig C. Haimeur A. Légaré D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (90) Google Scholar). Here we show that BT1 is part of a large gene family and that some of these genes are consistently deleted in cells in which MTX uptake was abrogated. The molecular and biochemical analyses of one of these genes indicate that it corresponds to a high affinity folate transporter. The L. tarentolae cell line TarII WT has been described previously (29White T.C. Fase-Fowler F. van Luenen H. Calafat J. Borst P. J. Biol. Chem. 1988; 263: 16977-16983Abstract Full Text PDF PubMed Google Scholar). Leishmaniacells were grown in SDM-79 or M199 medium supplemented with 5 or 10% heat-inactivated fetal bovine serum, respectively, and 5 mg/ml hemin. The L. tarentolae MTX-resistant mutants TarII MTX1000.3 to TarII MTX1000.7 have been described previously (26Papadopoulou B. Roy G. Ouellette M. Nucleic Acids Res. 1993; 21: 4305-4312Crossref PubMed Scopus (62) Google Scholar). L. tarentolae promastigotes were transfected by as previously (12Papadopoulou B. Roy G. Ouellette M. EMBO J. 1992; 11: 3601-3608Crossref PubMed Scopus (187) Google Scholar). was and were J. T. A Scholar). The coding of the gene family were by The of are and The and in and are The were from a derived from an L. tarentolae (24Kündig C. Haimeur A. Légaré D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (90) Google with a that was from a a BT1 A the L. tarentolae was in the The was by which in the and at the of the This was expression B. Roy G. Ouellette M. Mol. Biochem. Parasitol. 1994; PubMed Scopus Google Scholar). The L. tarentolae FT5 gene was with a expression J. Papadopoulou B. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus (47) Google Scholar). A the L. tarentolae FT5 gene with of on of the gene was amplified by with and a and respectively, for This was An of the was made to of coding and by a expression derived from B. Roy G. Ouellette M. Mol. Biochem. Parasitol. 1994; PubMed Scopus Google Scholar). This was used for one FT5 by An L. tarentolae FT5 null mutant was by selection for of Beverley S.M. Mol. Biol. 1996; 16: PubMed Scopus Google Scholar) by the selection to and of the cell FT5 null mutants were characterized by and was on an of the was the and The here in the under the and Transport were as described previously (24Kündig C. Haimeur A. Légaré D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (90) Google Scholar). and were from Transport studies were carried out concentrations of to 5 to affinity The of was with Leishmania cell folate and MTX the uptake of cells on was Leishmania cells for MTX resistance have transport for MTX and of transport have been some the in transport consists of an of both MTX and folate (9Ellenberger T.E. Beverley S.M. J. Biol. Chem. 1987; 262: 13501-13506Abstract Full Text PDF PubMed Google Scholar, 26Papadopoulou B. Roy G. Ouellette M. Nucleic Acids Res. 1993; 21: 4305-4312Crossref PubMed Scopus (62) Google in class of mutant the uptake of MTX is not measurable K. Coons T. Emmett K. Ullman B. J. Biol. Chem. 1988; 263: 7020-7028Abstract Full Text PDF PubMed Google Scholar, 26Papadopoulou B. Roy G. Ouellette M. Nucleic Acids Res. 1993; 21: 4305-4312Crossref PubMed Scopus (62) Google Scholar). The L. mutant is a of the class of the mutant MTX has no measurable MTX uptake L. cells in which MTX uptake be we have shown that the biopterin transporter BT1 is overexpressed permitting a of folate to the cell (24Kündig C. Haimeur A. Légaré D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (90) Google Scholar). We that at high was to (24Kündig C. Haimeur A. Légaré D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (90) Google suggesting that the biopterin transporter is part of a We a to and we used a derived from this as a we could that this is part of a family Some more strongly than suggesting that several gene could This family is not present in L. tarentolae but also in all Leishmania species not BT1 can transport folates (24Kündig C. Haimeur A. Légaré D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (90) Google we that one of the shown in could to a folate transporter. The of L. tarentolae MTX-resistant mutants was to this the mutants MTX and MTX which have a in folate and MTX transport and not we not in the of of the gene family and in mutants L. tarentolae MTX MTX and MTX in which MTX uptake was impaired (26Papadopoulou B. Roy G. Ouellette M. Nucleic Acids Res. 1993; 21: 4305-4312Crossref PubMed Scopus (62) Google we the of a the of the and extensively the this to or if the of more than one gene of one gene to a decrease in the whether one of the deleted could to a folate transporter, we further characterized which contains a both the and that to be deleted in some of the MTX-resistant mutants The of the revealed open reading of in proteins of and These were to and and to the Leishmania BT1 A of has revealed that the of the genes were found and in the related parasite were found in T. One corresponds to the expression Mol. Biochem. Parasitol. 1994; PubMed Scopus Google and the of found on and of the T. and in to several have been Leishmania there are a of putative pteridine transporters and and found in and of the Leishmania were to the genes and that one of the is related to a of L. of and with and the is to FT5 and we have thus the for FT5 and have the for the gene derived from of L. The L. tarentolae gene to FT5 contains the that is in the MTX-resistant mutants TarII and The gene contains a and the of a in MTX-resistant mutants it is not whether this is of of or The family of transporter by FT5 is to putative as by the but are not related to of transport FT5 show with the reduced folate of J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) or proteins J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.M. J. PubMed Scopus Google Scholar, M. H. PubMed Scopus Google Scholar, E. J. Biol. Chem. Full Text PDF PubMed Google suggesting that if one of these gene products corresponds to a folate transporter, it a novel of transport whether or FT5 were of transporting we the genes in Leishmania expression and transfected tarentolae MTX in which folate and MTX uptake are impaired and The accumulation of and acid were thus in these the we could not in folate or MTX uptake in not a in folate and MTX transport The level of accumulation in the TarII FT5 is 10% that of wild-type suggesting that FT5 is not the folate transporter. The of this FT5 transporter for folate and methotrexate was a and found to be than the for folate and methotrexate uptake in cells This suggest that FT5 corresponds to a high affinity folate transporter. this we an L. tarentolae FT5 null of folate transport in L. of with a of substrate from to transfected of with a of substrate from to in a an FT5 null we made a to of the coding of FT5 by an expression This of on of the deleted FT5 gene for was in L. tarentolae wild-type in the of were by wild-type an FT5 is part of a gene are also The of the also a to and by the This is we have by one Leishmania is a and thus for a null need to be of can be by the of the drug (24Kündig C. Haimeur A. Légaré D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (90) Google Scholar, Beverley S.M. Mol. Biol. 1996; 16: PubMed Scopus Google Scholar). the of the and to the in a to high selection suggesting that the second was an at the of the wild-type FT5 to an FT5 suggesting that gene was with FT5 or that between these we carried out and that can between an of FT5 and a of the wild-type to a the deleted gene with the to a of the wild-type and to a the parasites as and the wild-type and deleted a was amplified in the FT5 mutant to of the of both FT5 and the of an FT5 null mutant. The of an FT5 null mutant has further studies on its in folate We have that folate the cells in the mutant. we used of folate or MTX, we a in the uptake of both but this was not statistically different from wild-type cells A and at a substrate of was a in the uptake of both folate and MTX by null mutant with wild-type cells and This further results suggesting that FT5 is a high affinity folate transporter The mutant TarII MTX is resistant to MTX and is with a marked in the uptake of the FT5 is deleted in this mutant and of FT5 in this mutant the transport of both folate and MTX MTX uptake is in the MTX we a in the MTX of the mutant. The FT5 was found to be more sensitive to MTX than the but the was not to wild-type suggesting that further mechanisms of resistance possibly transport have in this mutant. the of the FT5 null we could have a decrease in MTX cells were grown in a medium with a high of folates we could not a in MTX susceptibility between wild-type cells and the FT5 null mutants the of FT5 to be more under concentrations of folate we the wild-type cells and the FT5 null mutant in a medium in folates nm). the of MTX in Leishmaniacells is on the folate of the medium (5Nare B. Luba J. Hardy L.W. Beverley S. Parasitology. 1997; 114 (suppl.): 101-110Crossref PubMed Google B. Ouellette M. Adv. Exp. Med. Biol. 1993; PubMed Scopus Google and L. tarentolae wild-type cells were more sensitive to MTX in M199 than in SDM-79 and the FT5 null mutant was to MTX in M199 medium with an of prove that the effect was to a in folate we acid to M199 medium to the found in these of high folate we not a in MTX susceptibility between the wild-type cell and the FT5 null mutant a of genes involved in pterin and folate metabolism have been in Leishmania (5Nare B. Luba J. Hardy L.W. Beverley S. Parasitology. 1997; 114 (suppl.): 101-110Crossref PubMed Google Scholar, 6Ouellette M. Drummelsmith J., El Fadili A. Kundig C. Richard D. Roy G. Int. J. Parasitol. 2002; 32: 385-398Crossref PubMed Scopus (79) Google Scholar). The biopterin transporter BT1 was characterized with to both substrate affinity and (24Kündig C. Haimeur A. Légaré D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (90) Google Scholar) and its in parasite M.L. Beverley S.M. 2001; PubMed Scopus Google Scholar, G. Kundig C. M. Papadopoulou B. Ouellette M. Exp. Parasitol. 2001; PubMed Scopus Google Scholar). The BT1 gene for parasite B. Roy G. M. Kundig C. C. M. Ouellette M. 2002; PubMed Scopus Google Scholar). BT1 to be the biopterin transporter in Leishmania but can also transport folate although with a affinity than folate transporter (24Kündig C. Haimeur A. Légaré D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (90) Google Scholar). this work we have a novel large gene with a one transporting of could be in to open reading were found in L. and to L. and are also that are related to the folate transporter gene It is not known yet whether all these genes are active or if some to some of this gene family to be deleted in cells no measurable MTX uptake We that one of these genes could to a folate transporter. It is that with a decrease in MTX uptake, to have an of gene and in mutants TarII and the transport could be related to or to the of expression in one of the genes. It is also that the transport in these mutants was not related to this described gene whether one of the deleted genes in the mutant TarII corresponds to the folate transporter, we carried out gene and gene Our work on FT5 has shown that it corresponds to a high affinity and but low folate transporter. FT5 is not the folate transporter of and possibly related gene could to this transporter. We that at at a one to and the possibly to the main folate transporter. is in to this gene part of the Leishmaniais a pteridine auxotroph, and pterins as biopterin have been known to be growth M. Drummelsmith J., El Fadili A. Kundig C. Richard D. Roy G. Int. J. Parasitol. 2002; 32: 385-398Crossref PubMed Scopus (79) Google Scholar) although their remains One to be in parasite M.L. Beverley S.M. 2001; PubMed Scopus Google Scholar). to the of in it is that this parasite has a of in pteridine BT1 FT5 corresponds to a high affinity folate transporter, at one more transporter of acid is present as by the folate uptake in null mutant Leishmania between and and through of its the parasite a a high affinity folate transporter as and possibly the to have several more genes related to the Some of these products could have the to transport different of or genes are expression is at the parasite The of to the expression of genes the Leishmania The uptake of folate is known to be on the growth phase of the parasite (22Ellenberger T.E. Beverley S.M. J. Biol. Chem. 1987; 262: 10053-10058Abstract Full Text PDF PubMed Google Scholar, M.L. Beverley S.M. Mol. Biochem. Parasitol. 2001; 113: 199-213Crossref PubMed Scopus (81) Google Scholar). FT5 be the folate transporter characterized in to systems in 1999; Google Scholar, B. 1999; PubMed Scopus Google Scholar, Nucleic Acids Res. Mol. Biol. 2001; PubMed Google folate transport have been in D. R. Proc. R. Biol. Sci. PubMed Scopus Google Scholar) and in B. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.M. J. Biol. Chem. 1987; 262: Full Text PDF PubMed Google but the gene products responsible for these have not yet been in The FT5 gene is deleted in L. tarentolae mutants for MTX suggesting that it be in of this gene in the L. tarentolae mutant resistance MTX uptake cells to this We could have that a null to this was not in SDM-79 and under folate it to This is with FT5 a high affinity folate transporter. Indeed, in medium folates the cell of FT5, in medium the of FT5 is more MTX susceptibility was very by folate in Leishmania (5Nare B. Luba J. Hardy L.W. Beverley S. Parasitology. 1997; 114 (suppl.): 101-110Crossref PubMed Google Scholar, B. Ouellette M. Adv. Exp. Med. Biol. 1993; PubMed Scopus Google it that an FT5 null mutant more sensitive to MTX in a Indeed, folates the it as with The mutants were in the SDM-79 and thus the of be more in of MTX susceptibility We that FT5 was possibly with of the gene and the of both genes to the resistance Indeed, in to FT5 a of to an are deleted in Leishmania by M. E. D. Fase-Fowler F. Borst P. EMBO J. PubMed Scopus Google Scholar, K. Papadopoulou B. Ouellette M. Nucleic Acids Res. 1993; 21: PubMed Scopus Google and this could have between the of of the family that are we have described a novel gene that corresponds to a high affinity folate transporter. several related transporters are also present that could transport pteridine under a variety of is a parasite that is a pteridine auxotroph and has thus to a pathway of pteridine transporters and FT5 is part of a novel family of putative and the of the pteridine transporters family in Leishmania the to We for some of the and Leblanc for in and We Papadopoulou and of the and and El Fadili for useful on the

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