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Originally published In Press as doi:10.1074/jbc.M400652200 on February 23, 2004

J. Biol. Chem., Vol. 279, Issue 18, 18575-18582, April 30, 2004
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Inactivation of the Leishmania tarentolae Pterin Transporter (BT1) and Reductase (PTR1) Genes Leads to Viable Parasites with Changes in Folate Metabolism and Hypersensitivity to the Antifolate Methotrexate*

Amal El Fadili, Christoph Kündig, Gaétan Roy, and Marc Ouellette{ddagger}

From the Centre de Recherche en Infectiologie du Centre de Recherche du Centre Hospitalier de l'Université Laval (CHUL) and Division de Microbiologie, Faculté de Médecine, Université Laval, Québec G1V 4G2, Canada

Received for publication, January 20, 2004 , and in revised form, February 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The protozoan parasite Leishmania is a folate and pterin auxotroph. The main biopterin transporter (BT1) and pterin reductase (PTR1) have already been characterized in Leishmania. In this study, we have succeeded in generating a BT1 and PTR1 null mutant in the same Leishmania tarentolae strain. These cells are viable with growth properties indistinguishable from wildtype cells. However, in response to the inactivation of BT1 and PTR1, at least one of the folate transporter genes was deleted, and the level of the folylpolyglutamate synthetase activity was increased, leading to increased polyglutamylation of both folate and methotrexate (MTX). Secondary events following gene inactivation should be considered when analyzing a phenotype in Leishmania. The BT1/PTR1 null mutant is hypersensitive to MTX, but in a step-by-step fashion, we could induce resistance to MTX in these cells. Several resistance mechanisms were found to co-exist including a reduced folate and MTX accumulation, demonstrating that cells with no measurable biopterin uptake but also greatly reduced folate uptake are viable, despite their auxotrophy for each of these substrates. The resistant cells have also amplified the gene coding for the MTX target dihydrofolate reductase. Finally, we found a marked reduction in MTX polyglutamylation in resistant cells. These studies further highlight the formidable ability of Leishmania cells to bypass the blockage of key metabolic pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The protozoan parasite Leishmania is responsible for a number of diseases with a wide range of clinical symptoms (1). Chemotherapy is currently the only effective way to control the infection, but the emergence of drug-resistant organisms, particularly against the first line antimony-containing drugs, is complicating the treatment (2). The discovery of new putative cellular targets is urgently required. Leishmania differs from its mammalian host in that it cannot synthesize pterins from GTP and differs from several microorganisms in that it cannot synthesize folates de novo. Indeed, it lacks enzymes necessary for the conjugation of the three building blocks (pterin, para aminobenzoic acid, and glutamate), leading to folates (reviewed in Ref. 3). This pterin and folate auxotrophy of Leishmania has resulted in a pterin (BT1) (4, 5) and a multiplicity of folate (FT)1 transporters (6, 7) that allow the transport of folate and pterin derivatives. These derivatives can be reduced into active molecules by the parasite dihydrofolate reductase-thymidylate synthetase (DHFR-TS) and by a novel pterin reductase PTR1 (3, 8). Leishmania does have an active folylpolyglutamate synthetase (FPGS) (9), permitting Leishmania to synthesize folylpolyglutamates that are essential for cellular retention (10). Reduced folates are one-carbon donors in various metabolic reactions including the synthesis of thymidine. Pterins are essential growth factors of Leishmania (11–14) and may play a number of other roles. Indeed, the almost completed Leishmania major genome project has revealed homologues to aromatic amino acid hydroxylases and to products involved in the biosynthesis of the molybdopterin cofactor (3). Finally, experimental evidence has been provided to link pterin levels with parasite metacyclogenesis (15).

Leishmania cells often resist the antifolate drug methotrexate (MTX) by reducing the accumulation of the drug (16–18). This can be achieved by gene deletion of some of the folate transporter FT genes (7). Since Leishmania is a folate auxotroph, this gene deletion event needs to be compensated for, and we have found that in Leishmania tarentolae strains, in which the main folate transporter is deleted, the biopterin transporter BT1, which can also transport some folates, is overexpressed (4). The BT1 gene has been inactivated in a number of species (4, 5, 15), and although cells can grow in culture flasks, they are less virulent in animal models (19), demonstrating an important role for pterin transport in parasite biology. The PTR1 gene has also been inactivated in a number of species, and these PTR1 null mutants were more sensitive to MTX (20, 21), had a decreased ability to reduce pterins, and recently were shown in animal models to be more virulent as low levels of reduced pterins induced metacyclogenesis (15).

Although pterins cannot serve for the de novo biosynthesis of folates, there are several interconnections between folate and pterin metabolism, and several Leishmania species can grow in folate-deficient medium provided that pterins are present (12, 14, 20). It has been suggested that pterins could have a folate-sparing effect, a phenomenon described 46 years ago in the related parasite Crithidia fasciculata (22), although the mechanism by which this is achieved is not understood. In an attempt to further increase our understanding of pterin metabolism in Leishmania, we succeeded, somewhat surprisingly, in generating mutants in which both the main pterin transporter (BT1) and the pterin reductase (PTR1) were disrupted by homologous recombination. Cells were viable, but some of their folate metabolism was changed. Since BT1 overexpression appears to compensate for mutations in the folate/MTX transporter in L. tarentolae MTX-resistant mutants (4), we assumed that selection of MTX resistance in a BT1/PTR1 null mutant would leave folate transport intact. Selection of the BT1/PTR1 cells for MTX resistance led to unexpected results that will be presented here.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Growth—The L. tarentolae cell line TarII WT and the TarII BT1/PTR1 null mutants were grown in SDM-79 (23) or M199 medium supplemented with 10% heat-inactivated fetal bovine serum and 5 µg/ml hemin. The BT1/PTR1 null mutant was selected for MTX resistance in a step-by-step fashion as reported previously (18) to lead to two mutants, MTX50.2 and MTX500.3, that were highly resistant to MTX. For HPLC analysis, cells were grown in M199 medium supplemented with 25 nM [3H]folic acid (25.7 Ci/mmol) or 25 nM [3H]MTX (26.62 Ci/mmol) (Moravek Biochemicals).

DNA Manipulations—Total DNA for Southern blot analysis was isolated using DNAzol reagent (Invitrogen). Southern blots, hybridization, and washing conditions were done following standard protocols (24). The PTR1, BT1, DHFR-TS, FPGS, neomycin phosphotransferase (NEO), hygromycin phosphotransferase (HYG), and FT probes were obtained by PCR. Wild-type L. tarentolae promastigotes were transfected by electroporation as reported previously (25). Selections were done with hygromycin B (>=100 µg/ml) and G 418 (>=40 µg/ml).

DNA Constructs—PTR1 inactivation was carried out using a construct described previously (20). The linearized 3.3-kb XhoI/XhoI fragment containing PTR1 interrupted by NEO was electroporated (25) in the TARII BT1 null mutant described previously in which the two BT1 alleles were inactivated by the HYG gene (4). Selection was initially with 40 µg/ml G418 (Invitrogen) for disrupting one PTR1 allele by homologous recombination. Disruption of the second PTR1 allele was obtained by selection for loss of heterozygosity by increasing the G418 selection pressure to 200 µg/ml and cloning of the cell pool. TARII BT1/PTR1 null mutants (TARII BT1/PTR1 KO) were characterized by Southern blot analysis using PCR probes for the BT1, PTR1, NEO, and HYG genes.

Enzymatic Assays in Crude Extracts—FPGS activity of Leishmania cells was assayed using extracts prepared essentially as described previously (9). L-[3H]glutamate (5 mCi/mmol) and 500 µM of either folic acid or MTX were added to the crude extracts of different strains. Separation of non-incorporated L-[3H]glutamate from the folylpolyglutamates or the MTX polyglutamates was done using a DEAE-cellulose column (Sigma).

Accumulation Studies—Transport experiments of folic acid, biopterin, and MTX were done as described previously (18). [3H]Folic acid (25.7 Ci/mmol), [3H]biopterin (6 Ci/mmol), and [3H]MTX (26.62 Ci/mmol) were purchased from Moravek Biochemicals, and transport studies were done using 200 nM substrate. Briefly, 1 x 106 cells were layered over 100 µl of dibutylphthalate (Sigma) and put in the presence of radioactive pteridine. Accumulation of radioactive substrates was stopped at various time points (0, 0.5, 2, 5, and 20 min) by centrifugation through the inert dibutylphthalate layer. Unincorporated substrates were removed by aspiration, cells were washed once in HEPES-NaCl buffer, and pellets were resuspended in scintillation liquid and counted. The amount of incorporated radioactivity was normalized with Leishmania cell number, and values of uptake in cells incubated on ice were subtracted.

HPLC Analysis—All HPLC reagents were obtained from U. S. Bioscience and were of HPLC grade. HPLC standards were purchased from Dr. Schirck's laboratory, Jona, Switzerland. The intracellular folylpolyglutamates in Leishmania wild-type cells and mutants were determined as described previously (9), and the extent of MTX polyglutamylation was assayed by HPLC using essentially the same procedure as described previously (26). Cells were incubated with 25 nM [3H]pteridines for 72 h.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of a BT1 and PTR1 Null Mutant of L. tarentolae—L. tarentolae cells becoming resistant to MTX by reducing folate/MTX uptake compensate by overexpressing the biopterin transporter BT1 gene (4). BT1 is a biopterin transporter that can also transport some folate. We reasoned that without BT1, we might select for mutations other than those affecting folate/MTX uptake. We first tried to select MTX-resistant mutants starting from a BT1 null mutant, but in all mutants studied, we found amplification of PTR1 (results not shown). We thus attempted to generate a BT1/PTR1 null mutant in the hope that after MTX selection, a novel resistant mutation could be found that would provide new insights into folate metabolism.

Leishmania cells disrupted either in PTR1 (15, 20, 21) or in BT1 (4, 5) have been described but not BT1/PTR1 null mutants. The L. tarentolae BT1 null mutant (4) is presented briefly in Fig. 1A. The single copy BT1 gene was interrupted by the HYG marker, and a BT1 HYG/HYG null mutant was obtained by loss of heterozygocity. Indeed, although the intact copy of BT1 is part of a 3.5-kb PstI-PstI fragment (Fig. 1B, lane 1), this fragment disappears in the BT1 null mutant and is replaced by two fragments of 2.7 and 1.8 kb, consistent with the integration of the HYG marker in the BT1 gene (Fig. 1, A and B, lane 2). In this BT1 null mutant, we introduced a NEO construct that would enable us to target the PTR1 gene (Fig. 1C). Transfection of the BT1 null mutant with this construct led to G418-resistant parasites that indeed had integrated the NEO construct at the level of one PTR1 allele (not shown). By increasing the concentration of the selective drug G418 and subsequent cloning of the cell pool, we were able to obtain by loss of heterozygocity a PTR1 null mutant in a BT1 null mutant background (Fig. 1). Indeed, although the intact PTR1 copy is part of an 2.3-kb XhoI-XhoI fragment (Fig. 1D, lane 1), this fragment increases to 3.2 kb in the null mutant and also hybridizes with a NEO probe (Fig. 1D, lane 2). Due to the central role of pterins in Leishmania growth, we were surprised that we could obtain a L. tarentolae BT1/PTR1 null mutant. Even more surprisingly, the genetic mutant had no measurable growth defect in the folate-rich (15 µM) medium SDM-79 (Fig. 2A) and had only a small growth defect in the low folate (20 nM) medium M199 (Fig. 2B). L. tarentolae cells disrupted in either PTR1 or BT1 were previously reported to be more sensitive to MTX, whereas cells transfected with PTR1 and BT1 were found to be resistant to MTX (4, 20). Indeed, the BT1 null mutant was four times more sensitive to MTX, whereas the PTR1 null mutant was 50 times more sensitive than a wild-type cell (Fig. 3A). The BT1/PTR1 null mutant was found to be 200 times more sensitive to MTX as compared with wild-type cells in SDM-79 medium (Fig. 3A). The roles of PTR1 and BT1 in MTX resistance were also confirmed by gene transfection. Transfectants overexpressing PTR1 become insensitive to MTX, whereas BT1 transfectants were ~10 times more resistant when grown in SDM-79 medium (Fig. 3B). The susceptibility of Leishmania cells is highly dependent on the folate concentration of the medium, and indeed, the EC50 of wild-type cells to MTX is 100 nM in M199 medium (as compared with ~25 µM in SDM-79), and the BT1/PTR1 null mutant was more sensitive to MTX with an EC50 of 15 nM (Fig. 3C). Cells in M199 medium overexpressing PTR1 were also highly resistant to MTX (result not shown), but surprisingly, BT1 overexpressing cells were not much more resistant to MTX as compared with wild-type cells in the M199 medium (Fig. 3C). We hypothesized that this discrepancy in BT1-mediated resistance between SDM-79 and M199 could be due to a difference in folate concentration. Indeed, when BT1 overexpressors were grown in M199 medium, but with the addition of folic acid to concentrations found in SDM-79, the parasites were MTX-resistant (Fig. 3C).



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  		FIG. 1.
Generation of a L. tarentolae BT1/PTR1 null mutant by gene targeting. A, a partial physical map of the L. tarentolae BT1 locus and the expected null mutant is shown. Only one of the two alleles is shown. The expected size of the fragments obtained after PstI digestion are depicted below the map. The BT1-containing genomic 3.5-Kb PstI-PstI restriction fragment now changes in two PstI-PstI restriction fragments of 2.7 and 1.8 kb upon the integration of the HYG gene that contains a new PstI site. B, Southern blot analysis of Leishmania cells. Total DNAs were digested with PstI and hybridized to BT1 and HYG probes independently. C, a partial physical map of the L. tarentolae PTR1 locus and its relevant restriction sites. Only one of the two alleles is shown. Upon integration of the NEO gene at the homologous locus, the genomic 2.3-kb XhoI-XhoI restriction fragment increases by the size of the NEO gene inserted. D, Southern blot analysis of PTR1 inactivation in the L. tarentolae BT1 null mutant. DNAs of wild-type cells and of a PTR1 mutant obtained by loss of heterozygocity were digested with XhoI, electrophoresed on an agarose gel, transferred, and hybridized with PTR1 and NEO probes. Lane 1, L. tarentolae wildtype (TarII WT); lane 2, L. tarentolae BT1/PTR1 null mutant.

 



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  		FIG. 2.
Growth properties of Leishmania cells. The growth of L. tarentolae TarII WT (•); of the BT1/PTR1 null mutant, TarII BT1/PTR1 KO ({circ}); or of the two BT1/PTR1 null mutant clones selected for high level methotrexate resistance TarII MTX 50.2 ({blacksquare}); and TarII MTX 500.3 ({square}) were measured in SDM-79 medium (A) or in M199 medium (B). Equal amounts of cells (5 x 106) were inoculated in 5 ml of medium and grown at 29 °C. Optical density at 600 nm was measured according to time. The average of triplicate independent measurements is shown.

 



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  		FIG. 3.
Methotrexate hypersensitivity of the L. tarentolae BT1/PTR1 null mutant. Leishmania cells were grown in SDM-79 medium (A and B) or M199 medium (± folic acid) (C), and their growth was measured at 68–72 h while varying the concentration of methotrexate. Cells that were studied include the wild-type cell TarII WT and several cells with genes inactivated or overexpressed. Cells in which PTR1 was the inactivated TarII PTR1 KO, in which BT1 was the inactivated TarII BT1 KO, cells in which both BT1 and PTR1 were the inactivated TarII BT1/PTR1 KO, or MTX-resistant cells derived from the latter, TarII MTX 50.2 and TarII MTX 500.3, were studied. Finally, the susceptibility of cells in which PTR1 TarII(PTR1NEO) or BT1 TarII(BT1NEO) were overexpressed as part of episomal plasmids was also studied. The average of a minimum of triplicate measurements is shown.

 
Biological Compensation Following Inactivation of the BT1 and PTR1 Genes of Leishmania—Based on the central role of pterins in Leishmania, it could be suggested that Leishmania cells would not thrive well in the absence of PTR1 and BT1, two proteins key in providing active pterins to the cell. The BT1/PTR1 mutant had minimal growth defects (Fig. 2), and cells have been propagated for over a year without problems. It is likely that several metabolic features are changed in this genetic mutant to compensate for the lack of BT1 and PTR1. As expected from a BT1 null mutant, there is no measurable biopterin uptake in the BT1/PTR1 null mutant (Fig. 4A). In L. tarentolae cells in which folate uptake is greatly impaired, we have observed an increased uptake of biopterin (4), and because there are several interconnections between folate and pterin metabolism, we tested here whether folate uptake was modulated in the BT1/PTR1 null mutant. Surprisingly, we found that both folate and MTX uptake were slightly but repeatedly decreased in the BT1/PTR1 null mutant (Fig. 4, B and C). Another aspect of folate metabolism that was recently studied in Leishmania cells concerned folate polyglutamylation, in which folates are found mostly as pentaglutamates (9, 27), whereas MTX is found predominantly in the form of triglutamates in L. tarentolae (26). We found that the glutamate chain lengths both of folate and most notably of MTX were increased significantly in the BT1/PTR1 null mutant as compared with wild-type cells (Tables I and II). The HPLC analysis of folates for the BT1/PTR1 null mutant led to the observation of other radioactive peaks consistent with polyglutamates longer than Glu6 (results not shown).



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  		FIG. 4.
Accumulation of radiolabeled pteridines in Leishmania cells. The transport studies in the L. tarentolae wild-type cell (•), TarII BT1/PTR1 null mutant ({circ}), and BT1/PTR1 null mutant cells selected for methotrexate resistance TarII MTX 50.2 ({blacksquare}) and TarII MTX 500.3 ({square}) were carried out as described under "Materials and Methods." Leishmania cells were grown in SDM-79, and pteridine uptake was measured using 200 nM substrate. The accumulated radioactivity was normalized with Leishmania cell numbers, and background uptake in cells incubated on ice was subtracted. A,[3H]biopterin accumulation; B, [3H]methotrexate accumulation; C, [3H]folate accumulation. The result of one experiment done in duplicate is shown, which has been repeated several times with similar results.

 


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  		TABLE I
Folylpolyglutamates in L. tarentolae cells

 


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  		TABLE II
Distribution of methotrexate polyglutamates in L. tarentolae cells.

 
The alteration in folate transport and polyglutamylation in the TarII BT1/PTR1 null mutant as compared with wild-type cells led us to test whether the copy number of genes coding for these activities was changed. Several genes are possibly implicated in folate transport (6, 7), and we used a probe recognizing several members of this gene family. A rearrangement of some folate transporter genes was observed in the BT1/PTR1 null mutant since a 2.5-kb PstI-PstI hybridizing fragment present in wild-type cells was absent in the genetic mutant (Fig. 5E, lanes 1 and 2). The copy number of FPGS was similar in the wild-type and null mutant (Fig. 5F, lanes 1 and 2), but FPGS activity was significantly higher in the null mutant as compared with wild-type cells (Fig. 6).



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  		FIG. 5.
Gene copy number determination of pterin/folate metabolic genes in Leishmania cells. Total DNAs were digested with the appropriate restriction enzymes XhoI (A, C, and D), PstI (B and E), or NcoI (F), electrophoresed and stained with EtBr (C), or transferred to nitrocellulose and hybridized with the probes indicated below the diagrams. Lane 1, TarII WT; lane 2, TarII BT1/PTR1 KO; lane 3, Tar IIMTX 50.2; and lane 4, TarII MTX 500.3. Molecular weights were deduced from the 1-kb ladder.

 



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  		FIG. 6.
Folylpolyglutamate synthetase enzymatic activity in Leishmania cells. The activity was measured from crude extracts of Leishmania cells grown in SDM-79 as detailed under "Materials and Methods." TarII WT (white bar), TarII BT1/PTR1 KO (gray bar), TarII MTX 50.2 (black bar), and TarII MTX 500.3 (hatched bar). The average of three independent experiments are shown.

 
Selection of MTX-resistant Mutants in the BT1/PTR1 Null Mutants—Despite the fact that BT1/PTR1 null mutant cells were hypersensitive to MTX, we were interested in generating MTX-resistant mutants in the hope of finding novel resistance genes. We succeeded in generating two highly MTX-resistant mutants TarII MTX 50.2 and TarII MTX 500.3 (Fig. 3). These highly resistant mutants had only a small growth defect as compared with their parent genetic mutant (Fig. 2). We first tested whether folate/MTX uptake was impaired in these mutants that had no active BT1. Against our expectations, these mutants exhibited a marked decrease in folate and MTX uptake (Fig. 4, B and C) with folate accumulating slightly more efficiently than MTX. Thus, Leishmania cells can survive without a biopterin transporter and with a much reduced folate transport activity. Both the BT1- and PTR1-disrupted loci remained intact upon MTX selection (Fig. 5, A and B). Leishmania cells often resist drugs by gene amplification (28, 29), and this can often be detected by a simple examination of ethidium bromide-stained gels (30). DNA amplification could easily be seen in the two independent mutants (Fig. 5C, lanes 3 and 4). This amplified DNA codes for DHFR-TS (Fig. 5D), the gene coding for the target of MTX but a locus usually not amplified in L. tarentolae selected for MTX resistance (31). The transport defect of the MTX-resistant mutant was due, particularly in mutant MTX 500.3 (Fig. 5E, lane 4), to a gross rearrangement and gene deletion in the folate transporter gene family. The FPGS gene loci remained intact in the mutant (Fig. 5F), but the distribution of MTX polyglutamates changed drastically (Table II). The distribution of folylpolyglutamates was decreased slightly in the mutants as compared with the parent strain with a decrease in Glu6 and Glu5 and an increase in Glu4 and Glu3. The distribution of MTX polyglutamates in the mutant, however, was greatly perturbed with a switch from Glu4 and Glu5 in the parent strain to the total absence of MTX polyglutamates in MTX 500.3 and only a small proportion of Glu2 with mostly MTX monoglutamates in MTX 50.2 (Table II). This drastic decrease in MTX polyglutamylation is not correlated with a decrease in FPGS activity (Fig. 6).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Pterins are important growth factors for Leishmania (11–14), and reduced pterins have been shown to be involved in parasite differentiation (15). We show here that cells without their main biopterin transporter and without their main pterin reductase are viable under the conditions tested (Fig. 2). The pterin requirement of the BT1/PTR1 null mutant must be achieved by other means. For example, the catabolism of folic acid could lead to a pterin moiety for the cell. This catabolism could, for example, take place via a pteridine hydrolyzing enzyme described in many species of parasites related to Leishmania (32). Alternatively, the genome of Leishmania seems to have several homologues of the folate/pterin transporters (7, 15), and alteration in the expression of one of these members could lead to sufficient biopterin accumulation to allow the cells to survive. In fact, we have observed something different where some of the FT members were deleted (Fig. 5E), and this or other defects led to a slightly decreased folate accumulation in the BT1/PTR1 null mutant (Fig. 4B). The reason for transporter gene deletion in a BT1/PTR1 null mutant is unclear. We could speculate that a pterin-containing compound could be transported by one of the FT gene family members and that the absence of its reduction (by PTR1) could lead to the build up of a compound detrimental to growth, thus explaining the deletion of some transporters. The deleted gene(s) do(es) not correspond to the main folate transporter, however (Fig. 4B). Further work will be required to try to test this speculative hypothesis, but in ongoing work, we sometimes find it difficult to overexpress some of the FT members in a wild-type background (7).2 It is possible also that in culture flasks, there is sufficient biopterin that the biopterin requirement of the cell is met by diffusion, a phenomenon that has been suggested to occur in Leishmania (4, 5). Since PTR1 is lacking in the same mutant, the reduction of pterins must also use another route. Since DHFR-TS cannot reduce pterins (33), it is likely that Leishmania has a second enzyme capable of reducing pterins, and evidence for this activity has been provided (21). Moreover, in Trypanosoma cruzi, a kinetoplastid parasite distantly related to Leishmania, two pteridine reductase enzymes have been characterized (34). In addition to these defects, the BT1/PTR1 null mutant had increased FPGS activity (Fig. 6), which leads to an increase in the level of long chain folylpolyglutamates (Table II). This increased polyglutamylation may lead to increased cellular retention of folates and thus compensate for the loss of some folate transporters resulting from gene rearrangements.

An important anticipated but not yet demonstrated observation of this study is that gene disruption in a protozoan parasite can lead to an alteration in the expression of several other genes. This change in gene expression is possibly essential for enabling growth, and this must always be taken into account when looking at a phenotype following a gene disruption event. We were able to detect some of these changes (deletion of some putative transporters, increased FPGS activity) because they are involved in the same pathways as the gene under study, but it is possible that the expression of other gene products will also be altered. DNA arrays or proteomic studies (35–37) could pinpoint at the global level the genes/proteins whose expression are modified following gene inactivation.

Although the BT1/PTR1 null mutant had no measurable growth defect, it had a clear phenotype of hypersensitivity to MTX, which appears to consist of the additive contributions of the disruptions of PTR1 and BT1. PTR1 is an established MTX resistance determinant (25, 38), although BT1 was isolated by functional cloning by selecting for MTX resistance (4). Amplification of BT1 has been observed in a number of Leishmania lines selected for MTX resistance (39).3 In contrast to SDM-79 medium (Fig. 3, A and B), we found that BT1 overexpressors were not more resistant to MTX in M199 medium (Fig. 3C). This may explain why BT1 was not isolated in an independent functional cloning experiment using MTX selection in M199 medium (40). We had hypothesized that BT1 may mediate MTX resistance by selectively transporting folate but not MTX in cells in which the folate transporters were deleted (4). Since folate concentration differs greatly between SDM-79 and M199, we tested whether the difference in resistance profiles mediated by BT1 is due to a difference in folate concentration between the media. This seems to be indeed the case (Fig. 3C). An increase in folate uptake mediated by BT1, measurable at high folate concentration (4), should confer MTX resistance as folate should compete with MTX for targets.

We selected for two BT1/PTR1 null mutants highly resistant to MTX. We thought that since resistance to MTX mediated by reduced activity of the folate/MTX transporter in L. tarentolae is compensated by overexpressing BT1, cells deleted for BT1 would not resist MTX by reducing the activity of the folate transporter. This was not the case, however, where a markedly decreased uptake of both folate and MTX was noted in MTX-resistant mutants (Fig. 4, B and C). These mutants have a small growth defect (Fig. 2) but less than one could have expected when both biopterin and folate uptake are decreased simultaneously (Fig. 4). Since Leishmania is likely not able to synthesize folate and pterins de novo, this suggests, at least in culture, that the small amount of pteridines taken up by these cells is sufficient to allow growth. Other mutations were also observed in these step-by-step selected resistant mutants, including amplification of the DHFR-TS gene (Fig. 5). Amplification of the DHFR-TS gene has been observed on several occasions in MTX-selected L. major (16, 41), but never in Leishmania donovani or L. tarentolae except when the PTR1 gene is deleted (31). We found previously that both the strength of the resistance gene and the environment of the genomic locus determine the efficiency with which a locus will be amplified in Leishmania (31).

In several Leishmania species, folates are found mainly as pentaglutamates (9, 27). In contrast, the glutamate chain length of MTX is much shorter, with few MTX polyglutamates in L. major and predominantly MTX triglutamates in L. tarentolae (26, 42). The difference between the two species is not due to a difference in FPGS activity but could be due to an {alpha}-MTX hydrolase that is highly active in L. major but not in L. tarentolae (17, 26, 42). Following the double inactivation of BT1 and PTR1, cells have increased FPGS activity (Fig. 6). This was observed for folate (Table I), but it was even more spectacular for MTX, in which the most abundant species became MTX-Glu5. In the MTX-resistant BT1/PTR1 null mutants, we found that MTX existed almost exclusively in the monoglutamate form (Table II), although long chain folylpolyglutamates were still present. Reduced polyglutamylation of MTX is a well established resistance mechanism in mammalian cells (reviewed in Ref. 43), and inactivation of a copy of FPGS in L. tarentolae can lead to MTX resistance (26). The absence of MTX polyglutamates in these mutants is not due to a decrease in FPGS activity (Fig. 6) and is not due to an increased activity of the MTX {alpha}-hydrolase (result not shown). A number of hypotheses can be advanced to try to explain why MTX is not polyglutamylated. The level of polyglutamylation in mammalian cells is controlled by the dual activities of FPGS and a {gamma}-glutamyl hydrolase (reviewed in Ref. 44). One could invoke a mutant form of {gamma}-glutamyl hydrolase that could preferentially depolyglutamylate MTX. Being less polyglutamylated, MTX would be expulsed more easily from cells. There is no evidence yet from the Leishmania genome project for the presence of a {gamma}-glutamyl hydrolase. Other enzymatic activities such as ferritin and glutamate carboxypeptidase could also control the level of MTX polyglutamates (45), although again, no evidence for these activities has been found in Leishmania. Alternatively, since the level of pteridine entering these MTX-resistant cells is low (Fig. 4), the little MTX that enters the cell may bind to the overexpressed DHFR-TS, which could then prevent MTX polyglutamylation. However, in another MTX mutant, TarII MTX 1000.6, we observed decreased MTX polyglutamates, but in this mutant, the DHFR-TS gene was not amplified (26). Further work will be required to pinpoint how exactly Leishmania manages to change the distribution of MTX polyglutamates, whereas keeping the distribution of folylpolyglutamates relatively untouched.

In this study, we have succeeded in generating a L. tarentolae BT1/PTR1 null mutant. These cells, under the conditions tested, are viable, but part of their folate metabolism is modified. The demonstration that the parasite responds to gene inactivation by altering various aspects of its metabolism has important consequences when one analyzes the phenotype of a genetic mutant. Despite the BT1/PTR1 null mutant being hypersensitive to MTX, we could still select for MTX-resistant mutants. This led to the demonstration that cells with greatly diminished folate and pterin uptake can grow and led to the most interesting observation of a drastic redistribution of MTX polyglutamates. Folate and pterin metabolism has a number of unique features as compared with their host cells that merit further studies to unravel putative new targets.


   FOOTNOTES
 
* This work was supported in part by the Canadian Institute of Health Research (to M. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Holds a Canada Research Chair in Antimicrobial Resistance and a Burroughs Wellcome Fund Scholar in Molecular Parasitology. To whom correspondence should be addressed: Centre de Recherche en Infectiologie, CHUQ, pavillon CHUL, 2705 boul. Laurier, Ste-Foy, Québec G1V 4G2, Canada. Tel.: 418-654-2705; Fax: 418-654-2715; E-mail: marc.ouellette{at}crchul.ulaval.ca.

1 The abbreviations used are: FT, folate transporter; MTX, methotrexate; DHFR-TS, dihydrofolate reductase-thymidylate synthetase; FPGS, folylpolyglutamate synthetase; NEO, neomycin phosphotransferase; HYG, hygromycin phosphotransferase; HPLC, high pressure liquid chromatography; WT, wild-type; KO, knock-out. Back

2 D. Richard and M. Ouellette, unpublished observations. Back

3 J. Drummelsmith and M. Ouellette, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank the members of the Ouellette laboratory for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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