Article

• The EMBO Journal (1999) 18, 2342 - 2351
• doi:10.1093/emboj/18.9.2342

Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae

Christoph Kündig¹, Anass Haimeur¹, Danielle Légaré¹, Barbara Papadopoulou¹ and Marc Ouellette¹

1. Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada G1V 4G2

Correspondence to:

Marc Ouellette, E-mail: Marc.Ouellette@crchul.ulaval.ca

Received 5 October 1998; Accepted 10 March 1999; Revised 26 February 1999

• Keywords:

  • biopterin,
  • drug resistance,
  • folate transport,
  • Leishmania,
  • methotrexate

Introduction

Antifolates are inhibitors of the enzyme dihydrofolate reductase (DHFR), which supplies the cell with reduced folates which are essential cofactors used in many one-carbon donor reactions (Schweitzer et al., 1990; Kamen, 1997). Folates are made of three building blocks: a pterin moiety which is conjugated to para amino benzoic acid by dihydropteroate synthase (DHPS) and a glutamic acid which is conjugated to dihydropteroate by dihydrofolate synthase to produce dihydrofolate. Dihydrofolate is reduced to tetrahydrofolate by DHFR. Because the DHFR proteins of different organisms share little homology, this enzyme proved to be a valuable target for chemotherapeutic drugs. Various antifolates have been successfully used as anticancer drugs (methotrexate) or in the treatment of bacterial (trimethoprim) or of parasitic infections such as malaria or toxoplasmosis (pyrimethamine) (Schweitzer et al., 1990).

No successful 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 so far, which could prove useful therapeutic targets. By using methotrexate (MTX) as a model antifolate drug, several different resistance mechanisms were identified. Whereas some of them were similar to mechanisms found in cancer cells or bacteria, others turned out to be novel (Borst and Ouellette, 1995; Nare et al., 1997). Gene amplification as part of extrachromosomal elements is commonly seen in response to drug selection in Leishmania (Beverley, 1991; Papadopoulou et al., 1998). Amplification of the dhfr-ts gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) leads to overexpression of the main MTX target enzyme and has been observed in L.major in response to drug selection (Coderre et al., 1983; Ellenberger and Beverley, 1987b). In one case, a combination of overexpression with a point mutation within the L.major DHFR-TS was reported which resulted in a largely increased resistance level (Arrebola et al., 1994). Another locus that is often amplified in several Leishmania species selected for MTX resistance encodes for PTR1 (pterin reductase), an enzyme belonging to the family of short chain dehydrogenase/reductases (Callahan and Beverley, 1992; Papadopoulou et al., 1992). PTR1 is capable of reducing fully or partially oxidized pterins or folates (Bello et al., 1994; Wang et al., 1997). It is believed that overexpression of this enzyme confers MTX resistance by supplying the cell with a sufficient amount of reduced folates, thus by-passing the need for DHFR.

Besides the amplification of ptr1 and dhfr-ts genes, reduction of the drug uptake is the second main pathway by which Leishmania resist antifolates. Leishmania have long been believed to be auxotrophic for folates, and during in vitro growth the cells rely mainly on uptake of folates for growth. One common high affinity transporter for folate and MTX has been identified in Leishmania and related parasites and mutations within this gene lead to antifolate resistance (Dewes et al., 1986; Ellenberger and Beverley, 1987b; Kaur et al., 1988; Papadopoulou et al., 1993). These mutations are associated with a large variety of transport phenotypes ranging from a 2-fold decrease in folate/MTX transport to uptake levels below the detection limit. Mutant strains with no apparent measurable folate uptake are able to thrive under laboratory growth conditions. This suggests that Leishmania must be capable of de novo folate synthesis or that folates have alternative routes of entry. The conversion of radiolabeled biopterin (one of the building blocks of folic acid) into reduced folates has been demonstrated in L.donovani (Beck and Ullman, 1991) and is consistent with de novo synthesis. The exact mechanism of this conversion is unknown, but seems to differ from the conventional route via DHPS since incorporation of radiolabeled p-amino benzoic acid could not be detected in L.major (Kovacs et al., 1989), and several DHPS inhibitors are not active against Leishmania (Peixoto and Beverley, 1987; Kaur et al., 1988).

By transfecting a L.tarentolae gene bank into wild-type parasites and selecting for MTX resistance, we isolated a novel resistance gene coding for a high affinity membrane biopterin transporter, which also has low affinity for folic acid transport but does not transport MTX. Leishmania tarentolae cells resisting MTX by mutations in their common high affinity folate/MTX transporter showed an increase in the activity of their biopterin transporter.

Functional cloning of the novel MTX resistance gene orfG

Drug resistance genes in Leishmania are usually isolated by analyzing mutants selected for resistance by increasing drug concentrations (Borst and Ouellette, 1995). In order to isolate new resistance genes, we used functional cloning which was initially set up to study genes involved in lipophosphoglycan biosynthesis (Descoteaux et al., 1994). Wild-type L.tarentolae cells were transfected with a genomic cosmid bank and plated on MTX-containing plates (see Materials and methods). An identical cosmid called cMM4 was found in five of the transfectants obtained by functional cloning. Retransfection of the cosmid cMM4 into L.tarentolae TarII wild type (WT) restored the MTX resistance and this transfectant showed an increase of its EC₅₀ by 10-fold when compared with wild-type cells (Figure 1A). The level of resistance conferred by cMM4 differs from L.tarentolae cells transfected with cosmids containing either the dhfr-ts or ptr1 gene (Figure 1A), suggesting the presence of a novel MTX resistance gene on cosmid cMM4. The novelty of the resistance gene was confirmed by hybridization experiments since neither a ptr1 nor a dhfr-ts probe hybridized to cMM4 (not shown). The cosmid cMM4 was digested with either BglII, NheI or SpeI, and subcloned into the Leishmania expression vector pSPY-hyg (Papadopoulou et al., 1994b). After transfection in TarII WT, three different restriction fragments, a 6 kb BglII, a 6.8 kb SpeI and an 8.5 kb NheI fragment were associated with MTX resistance. A 2.3 kb BglII–NheI fragment was the smallest segment common to all three fragments (Figure 1B). Transfection of this fragment conferred a similar level of MTX resistance as the original cosmid cMM4 (Figure 1A).

Figure 1.

Functional cloning of a novel Leishmania MTX resistance gene. (A) Profile of MTX resistance of TarII WT () and transfected with dhfr-ts (), cMM4 (), the 2.3 kb BglII–NheI fragment of cMM4 () and ptr1 (). The cells were grown in SDM 79 medium supplemented with 5% heat-inactivated FBS. (B) Partial physical map of the orfG region of L.mexicana. Below the map, the restriction fragments associated with MTX resistance when transfected on a multicopy expression vector are depicted. B, BglII; N, NheI; S, SpeI. (C) Hydrophobicity plot of ORF G of L.mexicana (Kyte and Doolittle, 1982). Putative transmembrane segments are underlined and numbered. The sequence of the L.mexicana orfG gene can be found under the DDBJ/EMBL/GenBank accession No. AF078929. (D) Confocal laser scanning microscopy of L.tarentolae overexpressing the ORF G–GFP fusion protein, showing the membrane location of ORF G.

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DNA sequence analysis of the 2.3 kb BglII–NheI fragment (DDBJ/EMBL/GenBank accession No. AF078929) revealed an open reading frame (ORF) of 1893 bp with the TAG stop codon being part of the NheI site used for the subcloning of the gene. The ORF shared 88% identity with ORF G of L.donovani. ORF G was first described as part of LD1/CD1 amplicons spontaneously occurring in various Leishmania species (Myler et al., 1994). ORF G also shares considerable homology with ESAG10 of Trypanosoma brucei, a protein with unknown function encoded by an expression site-associated gene (Gottesdiener, 1994). The orfG gene was identified as a MTX resistance gene by functional cloning. It is possible that during selection we may have selected for a point mutation within the gene which is responsible for the observed resistance phenotype. To address this possibility, the orfG genes of L.mexicana, L.donovani and L.tarentolae were cloned in a Leishmania expression vector. Upon transfection, all these genes produced a similar level of MTX resistance as observed with the original cosmid cMM4 (not shown). These experiments indicated that the wild-type orfG genes from at least three different Leishmania species are able to confer antifolate resistance.

Hydrophobicity analysis of ORF G suggested the presence of 12 putative transmembrane segments (Figure 1C). Most of these transmembrane domains contain one or more hydrophilic amino acid residues that are predicted to form amphiphilic -helices or -strands, a structure that is typical for type IV integral membrane proteins (Singer, 1990). It has been suggested that members of this class of membrane proteins act as aqueous channels through the membrane and mediate specific transport of small hydrophilic molecules. To confirm the membrane location of ORF G, we constructed an ORF G–green fluorescent protein (ORF G–GFP) fusion. This fusion has the same activity as the intact ORF G since it confers the same level of MTX resistance (not shown) and it has the same pteridine transport properties (Figure 2). This suggests that the cellular location of the overexpressed ORF G–GFP is similar to that of the overexpressed ORF G. The localization of the ORF G–GFP fusion was studied by confocal microscopy (Figure 1D). Uniform staining of the plasma membrane was observed. In addition, the fusion protein was also detected within an intracellular compartment at the base of the flagellum that probably corresponds to the flagellar pocket.

Figure 2.

Accumulation of radiolabeled pteridines in Leishmania wild-type and MTX-resistant cells. The transport studies with TarII WT (), TarII transfected with orfG (), MTX 100.5 () and MTX 1000.6 () were carried out as described in Materials and methods. (A) [³H]MTX accumulation using 150 nM of the drug. (B) [³H]MTX accumulation using varying concentrations of the drug. (C) [³H]folate accumulation using 150 nM of substrate. (D) [³H]folate accumulation using varying concentrations of the substrate. (E) [³H]biopterin accumulation; TarII transfected with an orfG–GFP fusion (). Transport experiments with each individual cell line have been done at least three times and similar results were obtained consistently.

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Pteridine transport properties of MTX-resistant L.tarentolae and of an orfG transfectant

Computer analysis of ORF G suggested that it can specifically transport hydrophilic molecules. One possible way to increase the level of resistance would be an accelerated extrusion of the drug outside the cell (Borst and Ouellette, 1995). To test this possibility we measured the steady-state accumulation of [³H]MTX in wild-type and MTX mutant cells as well as in an orfG transfectant (Figure 2A). We have previously described two classes of L.tarentolae MTX/folate transporter mutants with a decreased uptake of 50 and >95%, respectively (Papadopoulou et al., 1993). As reported in the past, the mutant MTX 100.5 showed a 2-fold decrease in uptake while no MTX uptake could be measured in mutant MTX 1000.6 (Figure 2A). The accumulation of folic acid in wild-type and MTX mutant cells was very similar to the kinetics of MTX uptake although some folate uptake could be detected in MTX1000.6 (Figure 2C), further suggesting that Leishmania has a common folate/MTX transporter (Ellenberger and Beverley, 1987b). Overexpression of orfG led to no significant change in MTX accumulation compared with wild-type levels, indicating no involvement of ORF G in MTX export (Figure 2B). The sensitivity of Leishmania to antifolate drugs is heavily influenced by the concentration of exogenous folates (Beverley, 1991; Papadopoulou and Ouellette, 1993). Therefore, a selective increase of folate import compared with MTX uptake is another possible mechanism by which the putative transmembrane transporter ORF G could confer MTX resistance. Under standard conditions, the orfG transfectant showed no significant increase in folate transport compared with wild-type cells (Figure 2C). At higher concentrations of folate, however, ORF G effectively transported folic acid (Figure 2D). ORF G seems therefore to correspond to a low affinity folate transporter that can discriminate between MTX and folic acid (Figure 2B and D).

We also tested the possible involvement of ORF G in the transport of pterins using radioactive biopterin. Biopterin enables growth of Leishmania cells in a defined folate-deficient medium (Kaur et al., 1988; Beck and Ullman, 1990; Bello et al., 1994; Papadopoulou et al., 1994a). The uptake of biopterin was shown to be transport mediated (Beck and Ullman, 1990), and radiolabeled biopterin can be incorporated into reduced folate (Beck and Ullman, 1991). Wild-type L.tarentolae cells were shown to accumulate [³H]biopterin in a time-dependent manner (Figure 2E). Using standard conditions, under which no increase in folate transport could be detected (Figure 2C), the accumulation of [³H]biopterin was increased 10-fold in the orfG transfectant compared with wild-type cells and a similar increase was observed with the ORF G–GFP fusion (Figure 2E). MTX 100.5, a cell line with a 2-fold reduced MTX/folate uptake, accumulated biopterin at the same rate as wild-type cells. Interestingly, the cell line MTX 1000.6, without any detectable activity of its high affinity MTX/folate transporter, showed a biopterin accumulation that was several times greater than the wild-type level (Figure 2E).

The uptake of biopterin in L.tarentolae is probably mediated by an active transport mechanism as no accumulation could be measured when the cells were incubated on ice (Figure 3A). This was substantiated by the lack of biopterin accumulation in cells treated with the metabolic inhibitors sodium azide (20 mM) and 2,4-dinitrophenol (5 mM) (Figure 3A). Similar concentrations of inhibitors were shown to inhibit active folate uptake (Ellenberger and Beverley, 1987a) and active efflux of arsenite (Dey et al., 1994) in Leishmania. To characterize further the biopterin transport properties of ORF G, we measured the rate of uptake of biopterin in a wild-type cell and in an orfG transfectant while varying biopterin concentration. Wild-type cells exhibit uptake of biopterin with high affinity and an apparent K_m of 4.9 M (Figure 3B; Table I). A similarly high affinity biopterin with an apparent K_m of 4.7 M was observed for the orfG transfectant while its V_max was increased by >10-fold (Figure 3B; Table I). The increase in the rate of uptake (Table I) correlates well with the levels of the steady-state accumulation of biopterin observed in wild-type cells and orfG transfected cells (Figure 2E).

Figure 3.

Characterization of the biopterin transporter ORF G. (A) Biopterin transport (500 nM) in L.tarentolae wild-type cells () or incubated on ice (), or in the presence of 20 mM sodium azide () or of 5 mM 2,4-dinitrophenol (). (B) Lineweaver–Burk analysis of biopterin transport in TarII WT cells () or Tar II cells transfected with orfG (). Apparent K_m and V_max values found in Table I were determined from the intercepts of the x- and y-axes.

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Table 1: Biochemical characteristics of pteridine transport in L.tarentolae

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Wild-type Leishmania cells also have a high affinity folate transporter with a K_m value of 0.7 M for L.major (Ellenberger and Beverley, 1987a) and 0.23 M in L.donovani (Kaur et al., 1988). We have performed Lineweaver–Burk analysis of folate transport in L.tarentolae and found an apparent K_m of 0.26 M in wild-type cells (Table I). Similar kinetic parameters were observed for folate transport in L.tarentolae orfG transfectant (Table I), indicating that ORF G is not the high affinity folate transporter. Nonetheless, the L.tarentolae ORF G can transport folic acid (Figure 2D), at least when folate concentration is at 6 M. Attempts to characterize the ORF G-mediated folate transport activity in more detail were unsuccessful mainly for technical reasons (see Materials and methods). Although a precise K_m value could not be determined for folate transport mediated by ORF G, results indicated clearly that ORF G contributes only marginally, at least at low folate concentration, to folate transport (Figure 2D; Table I). Overall, our transport studies indicated that ORF G is a high affinity active biopterin transporter and a low affinity folate transporter, but does not transport the drug analog MTX (Figure 2B and D).

Phenotype of the L.tarentolae orfG knock-out mutant

To characterize further the role of ORF G in the pteridine metabolism of Leishmania, an orfG-null mutant was generated. The first allele was disrupted using an hygromycin phosphotransferase (hyg) expression cassette (Papadopoulou et al., 1994b). Southern blot analysis indicated that orfG is part of a 3.5 kb PstI fragment in wild-type cells (Figure 4A). After longer exposure we could detect several weaker fragments hybridizing to an orfG probe. One of these, which was not affected by the construction of orfG mutants, is visible in Figure 4A (marked by an asterisk). Analysis of the hygromycin-resistant cell pool showed the appearance of two additional fragments of 2.7 and 1.8 kb, which is consistent with the introduction of an additional PstI site within the hyg marker into orfG (Figure 4A). Leishmania is diploid and this makes it necessary to inactivate the second allele which can conveniently be done by loss of heterozygosity (Gueiros-Filho and Beverley, 1996). By increasing the selection pressure with hygromycin B, we observed an increase of the strength of the hybridization signal of the mutant fragments of 2.7 and 1.8 kb over the wild-type 3.5 kb fragment (Figure 4A, lane 3), suggesting that we have enriched for double disruptants within the cell pool. Cloning of this culture led to the isolation of the orfG-null mutant IIorfGYhygro.2, in which both alleles of orfG were disrupted by the hyg resistance marker (Figure 4A, lane 4).

Figure 4.

Analysis of a L.tarentolae orfG-null mutant. (A) Southern blot analysis of an orfG-null mutant. Total DNA was digested with PstI and hybridized to an intragenic orfG probe. Lane 1, TarII WT; lane 2, total population of orfGYhygro mutants; lane 3, orfGYhygro mutants selected with high levels of hygromycin B; lane 4, IIorfGYhygro.2, an orfG-null mutant. An orfG homologous gene is marked by an asterisk. A partial physical map of the orfG region of L.tarentolae wild-type and the orfG-null mutant is shown. Fragments obtained after PstI digestion are depicted below the map. P, PstI; C, Csp45I. (B) Measurement of biopterin accumulation. TarII WT (), IIorfGYhygro.2 (), IIorfGYhygro.2 transfected with orfG (). (C) MTX resistance of TarII WT (), IIorfGYhygro.2 () and IIorfGYhygro.2 transfected with orfG ().

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No biopterin accumulation could be measured in IIorfGYhygro.2 (Figure 4B), but by introducing an expression vector carrying the orfG wild-type gene into the orfG null mutant we were able to revert the transport phenotype of the mutant (Figure 4B). Inactivation of the high affinity biopterin transporter ORF G decreases the EC₅₀ to MTX by 4-fold compared with wild-type cells (Figure 4C). By overexpressing orfG on a plasmid in the orfG null mutant, we were able to reverse the hypersensitivity to MTX and produce a level of resistance which is close to that we have observed in wild-type cells overexpressing orfG (Figures 1A and 4C). An orfG null mutant of L.tarentolae is viable in culture and shows only a very small delay in its growth rate compared with wild-type cells. ORF G is therefore not essential for growth of L.tarentolae in culture medium, although it seems to be the only high affinity transporter for biopterin.

Overexpression of ORF G in folate transport mutants

The folate transport-deficient mutant MTX 1000.6 accumulates biopterin at a rate 3–5 times higher compared with wild-type cells (Figure 2C). As gene amplification is a common mechanism by which Leishmania survive drug challenge (Beverley, 1991; Papadopoulou et al., 1998), we tested whether amplification of orfG was responsible for increased biopterin transport in L.tarentolae MTX 1000.6 mutants. A 3.5 kb PstI fragment is recognized by an orfG probe in wild-type cells (Figure 5A, lane 1). The same probe also recognizes one copy of the orfG gene family (marked with an asterisk in Figure 5A). Gene amplification or DNA rearrangements could neither be detected in the mutant MTX 100.5 (Figure 5A, lane 2), nor in MTX 1000.4 or MTX 1000.5 (not shown), mutants in which folate/MTX transport was only reduced by 2-fold (Papadopoulou et al., 1993) (Figure 2A and C). However, novel non-amplified PstI fragments at a size of 4.8 kb hybridized to an orfG probe in mutants MTX 1000.6, but also in mutants MTX 1000.3 and MTX 1000.7 (Figure 5A, lanes 3–5). No measurable high affinity MTX/folate transport can be detected in the latter three mutants (Papadopoulou et al., 1993). The precise rearrangements may differ between the three mutants as the sizes of the rearranged fragments differ slightly. All three mutants showing rearrangement within the orfG region also demonstrated an increase in biopterin transport (Figure 5C). All mutants that resist MTX by a mutation in their high affinity MTX/folate transport are compensating by increasing the activity of the high affinity biopterin transporter.

Figure 5.

Overexpression of ORF G in MTX-transport mutants. (A) Southern blot analysis of total DNA of L.tarentolae TarII WT and MTX-transport mutants: total DNA was digested with PstI and hybridized with an intragenic orfG probe. Lane 1, TarII WT; lane 2, MTX 100.5; lane 3, MTX 1000.3; lane 4, MTX 1000.6; lane 5, MTX 1000.7. (B) Analysis of orfG RNA by Northern blot. Total RNA was hybridized to an intragenic orfG fragment of L.mexicana and re-hybridized to an -tubulin gene probe to monitor the amount of RNA layered in each lane. Lane 1, TarII WT; lane 2, MTX 1000.6; lane 3, TarII orfG transfectant. (C) Measurement of biopterin accumulation. TarII WT (), MTX 1000.3 (), MTX 1000.6 (), MTX 1000.7 ().

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The increase in biopterin transport in the mutants was not due to orfG gene amplification but to an increased steady-state accumulation of orfG RNA by 5-fold in MTX 1000.6 (Figure 5B, lane 2). In addition to the increased amount of RNA, we also noted that the orfG RNA in MTX 1000.6 (at least part of it) is larger than the wild-type RNA, which could possibly be a result of the described genomic rearrangement. The RNA of the transfectant also differs in size (Figure 5B, lane 3), but this is not surprising as only the protein-coding region of ORF G (without its own RNA maturation sequences) was cloned in a Leishmania expression vector. Overexpression of ORF G in MTX 1000.6 not only increases biopterin uptake but also augments uptake of folate when a high folate concentration was present in the transport assay (Figure 2D).

The increase of the steady-state orfG RNA level in MTX 1000.6 is commensurate with the high biopterin transport activity of this mutant. In one previous report, the RNA of orfG was shown to be increased following translocation of an orfG segment into the ribosomal locus (Lodes et al., 1995). To test whether this also occurred in the mutant MTX 1000.6, the chromosomes of wild-type and MTX 1000.6 cells were separated by TAFE and hybridized to an orfG and a ribosomal DNA probe. The orfG gene is part of a 2.1 Mb chromosome while ribosomal RNA genes are part of 1.5 and 1.8 Mb chromosomes. We could detect neither any gross gene rearrangements nor translocation of orfG into the ribosomal locus (not shown).

Isolation of a new MTX resistance gene by functional cloning

Leishmania often resists in vitro drug selection by amplifying specific portions of its genome as part of extrachromosomal elements (Beverley, 1991; Papadopoulou et al., 1998). Characterization of amplicons derived from MTX-resistant Leishmania led to the identification of dhfr-ts and ptr1 genes. The presence of repeated sequences flanking the resistance genes enhanced greatly the frequency with which gene amplification events are selected in Leishmania (Grondin et al., 1996). It is therefore likely that genes lacking such repeated flanking regions will not be amplified while selecting for stepwise increased resistance. To identify such genes, we introduced a Leishmania cosmid genomic library into wild-type cells and selected for genes which confer MTX resistance when overexpressed from a multicopy vector. The orfG gene was isolated in this way and constitutes a novel resistance gene. Functional cloning can therefore serve as a useful complementary approach to mutant analysis for the isolation of resistance genes. Using functional cloning, but screening for the ability of a L.donovani MTX-resistant mutant to thrive in folate-deficient medium supplemented with biopterin, the group of S.Beverley (Washington University, St Louis, MO) has independently isolated orfG and shown that it can transport biopterin (Moore and Beverley, Woods Hole Molecular Parasitology Meeting 1996, abstract 107, cited in Segovia and Ortiz, 1997).

ORF G is present in a chromosomal region called the LD1/CD1 locus (Stuart, 1991; Segovia and Ortiz, 1997). This region is present on a large 2.2 Mb L.donovani chromosome, but is also frequently amplified as part of linear or circular amplicons in several Leishmania species either cultured in the laboratory or isolated from the field. These linear or circular elements differ largely in size, ranging from 27 to >250 kb, but contain a 10 kb common region with four ORFs named ORF F, G, H and I (Myler et al., 1994). The role of these ORFs is unknown although sequence analysis indicated that ORF I contains a potential ATP/GTP binding motif, while ORF G has putative transmembrane domains and is highly similar to ESAG10 (Gottesdiener, 1994), an expression site-associated gene of unknown function present in the African trypanosomes. The membrane location of ORF G was confirmed by using a fully active ORF G–GFP fusion (Figure 1D). Fluorescence was also detected in an intracellular compartment at the flagellated end of the cell, which is likely to correspond to the flagellar pocket, a site where receptor-mediated endocytosis occurs (Webster and Russell, 1993). The generation of antibodies recognizing ORF G will be necessary to determine whether ORF G also localizes to that compartment in wild-type cells. The Leishmania integral glucose transporter Pro-1 was found to be located both in the plasma membrane and the flagellar pocket (Piper et al., 1995), indicating that dual location of a membrane protein is not without precedent in Leishmania.

Contrary to their host, Leishmania and other related parasites lack a de novo pathway for the biosynthesis of pterins. Several studies have shown that Leishmania requires pteridines (folates or pterins) for growth (Trager, 1969; Peixoto and Beverley, 1987; Scott et al., 1987; Kaur et al., 1988) but biopterin and other unconjugated pterins can clearly support the growth of Leishmania in folate-deficient medium (Beck and Ullman, 1990; Bello et al., 1994; Papadopoulou et al., 1994a). Pterins are important in folate metabolism in Leishmania (Figure 6) but also have other important as yet unidentified roles (Bello et al., 1994; Papadopoulou et al., 1994a), including one in oxidant resistance (Nare et al., 1997). The frequent amplification of LD1/CD1 in various Leishmania species may therefore be due to the presence of ORF G which can increase the uptake of pterins required in larger amounts under certain conditions, such as nutritional or environmental stresses, encountered throughout their life cycle. The high affinity biopterin transporter seems to be non-essential in L.tarentolae, as an orfG-null mutant with no measurable biopterin uptake (Figure 4B) could still grow in culture medium. The pterin requirements of this mutant are likely to be met by a pteridine hydrolyzing enzyme (Figure 6), which was described in Leishmania and other related parasites (Oe et al., 1983; Kaur et al., 1988; Ellenberger et al., 1989), and/or by other low affinity transporters that were not detected under our transport conditions.

Figure 6.

Model of the role of ORF G in MTX resistance of Leishmania. (A) The main route of entry of folate (and antifolates) into Leishmania wild-type cells is the high affinity folate transporter (FT), while pterins are taken up by the high affinity transporter ORF G. DHFR-TS reduces dihydrofolate to tetrahydrofolate. The main role of PTR1 is reduction of (bio)pterin but the enzyme can also reduce conjugated pterins. The arrows between the biopterin and folate pathways symbolize reported MTX hydrolase activity (Kaur et al., 1988; Ellenberger et al., 1989) and conversion of biopterin into tetrahydrofolate (Beck and Ullman, 1991). Gene amplification of ptr1 and dhfr-ts leads to antifolate resistance (Borst and Ouellette, 1995; Nare et al., 1997). (B) Mutations in FT confer high-level antifolate resistance but would decrease the intracellular concentration of folates. The lack of this high affinity folate uptake system is, however, compensated by overexpression of ORF G, which increases pterin uptake which presumably can be converted into folates. ORF G is also a low affinity folate transporter but does not transport MTX and this activity may also contribute to resistance, especially in cells grown under high folate concentration.

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ORF G confers MTX resistance and is overexpressed in folate transport mutants

Mutations in the common high affinity folate/MTX transporter are a frequent mechanism of resistance to MTX in Leishmania (Ellenberger and Beverley, 1987b; Kaur et al., 1988; Papadopoulou et al., 1993). Similarly, mutation in the reduced folate carrier is a common MTX resistance mechanism in cancer cells (reviewed in Gorlick et al., 1996; Kamen, 1997). We show here that in L.tarentolae MTX-resistant mutants in which there is no measurable high affinity folate/MTX uptake, the orfG gene is rearranged leading to an increased biopterin accumulation in the cell (Figure 5A and C). Remarkably, the overexpressed ORF G can also transport folic acid, albeit less efficiently, but it does not transport MTX (Figure 2). The folate requirements of these mutants may therefore be met by ORF G, provided that cells are grown in a folate-rich environment such as in SDM-79 medium. Recently, mutations in the mammalian reduced folate carrier were associated with increased affinity for folic acid and decreased affinity for MTX (Zhao et al., 1998), showing that pteridine transporters can also discriminate between the two closely related substrates folic acid and MTX. An increase in the intracellular concentration of biopterin mediated by overexpression of orfG is likely to translate into an increase in reduced folate synthesis (Figure 6) and should also contribute to the folate requirements of cells which have lost their high affinity folate transporter.

The ORF G was isolated by selecting for MTX resistance and indeed, L.tarentolae orfG transfectants and the orfG-null mutants are resistant or hypersensitive to MTX, respectively (Figure 4C). Resistance mediated by overexpression of ORF G is likely to be due to both increased biopterin uptake and to increased selective folate uptake. The SDM-79 medium used to grow our cells contains a high concentration of folates (13 M), which makes it possible that the selective increase in ORF G-mediated folate uptake contributes to resistance in the MTX-resistant cells that have lost their high affinity folate/MTX transporter. Indeed, at high folate concentration (6 M) we can observe folate transport in MTX 1000.6 whereas no transport can be measured when lower concentrations of folates are used (Figure 2D). Unpublished ongoing experiments clearly show, however, that the increased uptake of biopterin and its putative conversion into folic acid also contributes significantly to the MTX resistance phenotype. For example, orfG overexpression confers MTX resistance in media in which the folate concentration (<150 nM) is too low (Figure 2) to be transported significantly by ORF G (unpublished). Resistance in these cells is likely to be due to the increase in biopterin uptake and its subsequent conversion into folates. Overexpression of ORF G in a ptr1-null mutant which cannot synthesize reduced pterins does not lead to MTX resistance even when folic acid or biopterin are supplemented to the cells (unpublished). However, when dihydrobiopterin is supplemented to the same cells, we observed a >100-fold increase in MTX resistance (unpublished) which is probably due to an increase in the de novo biosynthesis of folates (Figure 6).

The increased activity of ORF G was not associated with gene amplification, but an excellent correlation was observed between the increased biopterin transport phenotype and a DNA rearrangement within the orfG locus (Figure 5A). In the mutant TarII MTX1000.6, we observed that the gene rearrangement was correlated with an increase in the steady-state level of the transcript (Figure 5B). The size of the transcript is different than in wild-type cells and may be a consequence of the rearrangement. Translocation of the orfG gene in the ribosomal locus has been demonstrated before, resulting in higher levels of orfG RNA (Lodes et al., 1995). Trypanosomatidae are capable of expressing their genes by an RNA polymerase I (Rudenko et al., 1991; Zomerdijk et al., 1991). The rearrangement observed in this study is different, however, from the one observed in the ribosomal locus. The increased expression of a gene correlating with gene rearrangement rather than gene amplification is likely to have occurred in at least one other drug-resistant mutant (Gamarro et al., 1994).

In conclusion, functional cloning has led to the isolation of a novel MTX resistance gene which was found to be involved in the resistance phenotype of drug resistant mutants. Interestingly, we showed that the loss of a high affinity transporter that is required to resist a drug analog (MTX) is compensated by the overexpression of another transporter. Transport-related mechanisms are frequent in drug-resistant organisms (Borst and Ouellette, 1995) and it is therefore possible that reduced uptake in these organisms is compensated by modulating the activity of other transporters.

Leishmania growth

The L.tarentolae cell line TarII WT has been described previously (White et al., 1988). Leishmania cells were grown in SDM-79 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 5 mg/ml of hemin (Brun and Schonenberger, 1979). MTX-resistant mutants of L.tarentolae and measurements of resistance levels have been described previously (Papadopoulou et al., 1993, 1994a). Leishmania tarentolae promastigotes were transfected by electroporation as reported previously (Papadopoulou et al., 1992).

DNA and RNA manipulations

Chromosomes in agarose blocks were resolved by transalternative field electrophoresis (TAFE) (Beckman) as described previously (Grondin et al., 1996). Total RNA was isolated using TRIzol (Gibco-BRL). Southern and Northern blotting, hybridization and washing conditions were performed following standard protocols (Sambrook et al., 1989). Probes containing coding sequences of orfG, -tubulin and 18S rRNA were obtained by PCR.

Functional cloning

A genomic cosmid library of partially Sau3AI-digested total DNA of L.mexicana in vector cL-Hyg (Ryan et al., 1993) was constructed following a detailed protocol (Descoteaux et al., 1994). After transfection of 10 g of the cosmid library DNA into L.tarentolae by electroporation (Bio-Rad Gene Pulser, voltage 0.45 kV and capacitance at 500 F), cells were propagated in SDM-79 medium overnight, grown for another 24 h in medium in which hygromycin B (Calbiochem) was added (200 g/ml), and then plated on SDM-79 agar plates containing 200 g/ml hygromycin B. After 10 days, the transfectants were pooled and multiplied in liquid SDM-79 medium containing 1 mg/ml of hygromycin B. After 24 h the cells were collected and replated on 600 M MTX-containing SDM plates. Several colonies grew on the MTX-containing plates, five of which were analyzed and found to contain an identical cosmid.

Generation of orfG expression vectors

The Leishmania expression vector pSLneo was constructed by insertion of a 2.5 kb BamHI–BamHI neo (neomycin phosphotransferase) expression cassette flanked by the -tubulin intergenic regions (Papadopoulou et al., 1992) into the single BamHI site of the vector pSL1180 (Pharmacia Biotech). pSLneo was used to overexpress orfG from a 2.3 kb BglII–NheI fragment of L.mexicana, a 2.3 kb BglII–NheI fragment of L.donovani and a 2.7 kb PstI–NheI fragment of L.tarentolae. To generate an ORF G–GFP fusion, pSLneo containing the 2.7 kb PstI–NheI orfG fragment of L.tarentolae was linearized with NheI, digested with Mung Bean nuclease to remove 5' extensions of DNA and ligated to a 0.7 kb blunt end HindIII–XbaI fragment of phGFP-S65T (Clontech Laboratories) containing the GFP gene. Correct in-frame fusion between orfG and GFP was confirmed by sequencing.

Construction of an orfG-null mutant

The 2.7 kb PstI–NheI L.tarentolae fragment containing orfG was subcloned into pSP72 (Promega) and a hygromycin B phosphotransferase expression cassette (hyg) derived from pSPY-hyg (Papadopoulou et al., 1994b) was introduced into the unique Csp45I site within orfG. This hyg-containing 3.3 kb BglII–NheI fragment was used to disrupt one chromosomal orfG allele by homologous recombination. An orfG-null mutant was obtained by selection for loss of heterozygosity (Gueiros-Filho and Beverley, 1996) by increasing the hygromycin B selection pressure to 600 g/ml and cloning of the cell pooL. Double knock-out mutants were identified by Southern blot analysis.

DNA sequence analysis

DNA sequencing was done on an Applied Biosystems 373 DNA automated sequencer. Analysis of the sequence was performed using the GCG software package (Genetics Computer Group, 1994) and DNA Strider™ 1.0. The nucleotide sequence reported here will appear in the DDBJ/EMBL/GenBank sequence database under the accession No. AF078929.

Pteridine transport experiments

Transport experiments were performed as described (Papadopoulou et al., 1993). Tritium-labeled pteridines {[³H]folate (14.6 Ci/mmol), [³H]MTX (23.6 Ci/mmol), [³H]biopterin (5.8 Ci/mmol)} were purchased from Movarek Biochemicals. Transport studies were carried out with varying concentrations of radioactive pteridines (50 nM to 50 M of biopterin, 50 nM to 20 M of folate) to determine apparent kinetic parameters (K_m and V_max) for folate and biopterin. The quantity of radioactivity incorporated was normalized with Leishmania cell numbers. We performed transport experiments with varying concentrations of the labeled pteridines. To measure biopterin transport in wild-type cells and in orfG transfectants, the accumulation of biopterin in an orfG-null mutant was subtracted, while for values of folate transport in wild-type cells and in orfG transfectants, uptake of cells incubated on ice have been subtracted. Data points used for the determination of kinetic parameters are the means of three experiments. Attempts to determine a K_m value for folate uptake mediated by ORF G failed due to insufficient sensitivity of the transport experiments. Significant folate uptake by ORF G is only detectable at high folate concentrations (>5 M), which made it necessary to dilute the radiolabeled folic acid solution with large amounts of unlabeled folate. This lowered the absolute counts of radioactivity and made the calculations very susceptible to small errors in pipetting, which resulted in very large standard deviations after compensating the data for dilution factors, thereby rendering it impossible to measure accurately a K_m value for folate for the L.tarentolae ORF G.

To measure the effect of metabolic poisons on biopterin transport in L.tarentolae, wild-type cells within logarithmic growth phase were washed twice and incubated for 30 min at room temperature in 20 mM sodium azide or 5 mM 2,4-dinitrophenol, respectively. After treatment, the cells were used directly in transport experiments. Cells that were washed after drug exposure showed no significant decrease in transport, indicating that the majority of cells were not irreversibly damaged. This was also confirmed by the fact that treated cells did not show any retardation in growth when cultured in SDM-79 medium.

Fluorescence microscopy

Leishmania tarentolae cells expressing the ORF G–GFP fusion protein were grown in SDM-79 to late log phase, washed in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde and resuspended in PBS at a density of 10⁷ cells/ml. Specimens were viewed on a Bio-Rad MRC-1024 confocal imaging system equipped with a Krypton-argon laser beam and mounted on a Nikon Diaphot-TMD. A 60 objective lens with a 1.4 numerical aperture was used. Confocal settings were as follows: 1 mW laser power, zoom 5, 1 s per scan Kalman filter and eight frames per image. The photomultiplier gain was set at maximum and the confocal aperture was adjusted for maximum resolution.

We thank S.Beverley (Washington University, St Louis, MO) for the cosmid cL-Hyg, C.Brochu of the Centre de Recherche en Infectiologie for plasmid pSLneo and Eric Pellerin (Hôtel Dieu, Québec) for help with the confocal microscope. This work was supported in part by the Natural Science and Engineering Research Council of Canada (NSERC) and by the Medical Research Council of Canada (MRC) to M.O. C.K. is a post-doctoral fellow of the Schweizerischer Nationalfonds, D.L. received a studentship from FCAR, B.P. is an MRC Scholar, and M.O. is an MRC Scientist and a Burroughs Wellcome Fund New Investigator in Molecular Parasitology.

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