Changes in Folate and Pterin Metabolism after Disruption of the Leishmania H Locus Short Chain Dehydrogenase Gene*

The short chain dehydrogenase gene Ztdh, which is derived from the H locus of Leishmania, confers high level resistance to the dihydrofolate reductase inhibitor methotrexate via a novel mechanism. Resistance is correlated with LTDH overproduction. High level resist- ance of Ztdh transfectants was observed even in poor media where reduced folates are essential for the syn- thesis of thymidylate precursors. The Ztdh transfectants were also capable of growing for several passages, at slightly reduced rates, in unsupplemented folate-defi-cient medium (fdDME-L), unlike the wild-type cells that could grow only in fdDMJ3-L if supplemented with vari- ous folates or pterins. An homozygous Ztdh mutant was obtained by gene targeting. This mutant became hyper- sensitive to methotrexate, but unlike wild-type cells, methotrexate toxicity could not be circumvented by the addition of thymidine. Although the homozygous mutant was capable of growing in fdDME-L supplemented with folate derivatives, it lost its ability to thrive in fdDME-L supplemented with pterins. Our results support the model in which LTDH is a key enzyme

Recently, conversion of biopterin to reduced folates has been demonstrated directly (Beck and Ullman, 1991). In Escherichia coli and other microorganisms, folates can be synthesized de novo from three main building blocks: a pterin derivative, paminobenzoic acid, and glutamate (reviewed in Brown and Williamson, 1987). The condensation of the pterin derivative to p-aminobenzoic acid is catalyzed by the enzyme dihydropteroate synthase. Leishmania are insensitive (or sensitive in a folate-independent concentration) to sulfonamides (Petrillo-Peixoto and Beverley, 1987), a class of selective dihydropteroate synthase inhibitors rendering it unlikely that pterins are converted to folates through the classical dihydropteroate synthase pathway, although a Leishmania sulfonamide-insensitive dihydropteroate synthase cannot be ruled out. The precise pathway responsible for the synthesis of folates from biopterin is therefore not known.
Resistance to the antifolate methotrexate (MTX),l a potent inhibitor of the enzyme dihydrofolate reductase (DHFR), is readily induced in Leishmania. Analysis of MTX resistance mechanisms has indicated several novel properties in the folate metabolism of Leishmania (reviewed in Ouellette and Papadopoulou, 1993). Three MTX resistance mechanisms have so far been described in Leishmania: amplification of the gene coding for the bifunctional DHFR-thymidylate synthetase (Coderre et al., 1983;Beverley et al., 1984); decreased uptake of the drug (Dewes et al., 1986;Ellenberger and Beverley, 1987b;Kaur et al., 1988;; and amplification of the H locus (Beverley et al., 1984;Hightower et al., 1988;Petrillo-Peixoto and Beverley, 1988;White et al., 1988;.
The gene responsible for MTX resistance on the H locus ( l t d h or hmtx') shows significant sequence identity with short chain dehydrogenases (Papadopoulou et al., 1992;Callahan and Beverley, 1992), a family of enzymes involved in a variety of oxidoreduction reactions (reviewed in Baker, 1991;Persson et al., 1991). The mechanism by which LTDH confers resistance is not known but it does not seem to involve MTX degradation (Ellenberger et al., 1989) or to impair MTX transport (Ellenberger and Beverley, 1987b;. We have proposed that LTDH might provide cells with reduced folates even if DHFR is blocked (Ouellette and Borst, 1991;Papadopoulou et al., 1992;. Either the Ztdh gene product uses the DHFR-thymidylate syn- The abbreviations used are: MTX, methotrexate; DHFR, dihydrofolate reductase; fdDME-L, folate deficient DME-L; kb, kilobase(s1. thetase substrate dihydrofolate, similar to the plasmid-encoded DHFR in trimethoprim-resistant bacteria (reviewed in Amyes and Towner, 1990), or it might be involved in the pathway responsible for the conversion of pterins to reduced folates. Interestingly, some pterin-metabolizing enzymes are part of the short chain dehydrogenase family (Ichinose et al., 1991;Park et al., 1991), and dihydropteridine reductase (Shahbaz et al., 1987;Dah1 et al., 1987;Lockyer et al., 1987) contains most signature sequences of short chain dehydrogenases (Whiteley et al., 1993). Using gene transfection and gene targeting experiments, we have further studied the role of LTDH in antifolate resistance and obtained evidence that the ltdh gene product is required in the metabolism of pterin derivatives. EXPERIMENTAL PROCEDURES Cell Lines and Culture-The Leishmania tarentolue cell line TarII wild-type and the transfectant TarII-pneo40.8, a TarII wild-type strain into which the ltdh gene was transfected, have been described previously (White et al., 1988;Papadopoulou et al., 1992). Cultures were grown routinely in SDM-79 medium (White et al., 1988), but when indicated they were also grown in M199 medium (Gibco-BRL) supplemented with 10% fetal calf serum, in the completely defined medium DME-L, or in the DME-L medium deficient in folates (fdDME-L) (Iovannisci and Ullman, 1983). Complementation studies were done with any of the following compounds, all purchased from Sigma: folic acid (10 p~), 6-biopterin (20 PM), 6,7-dimethyl-pterine (40 p~) , 6,7-dimethyl-5,6,7,84etrahydropterine (20 p~) , p-aminobenzoic acid (50 p~), thymidine (100 p~) , and dihydrofolate (20 p~) . Relative drug resistance values were obtained as previously described .
Gene Dansfection-Transfection of ltdh under the control of the expression cassette derived from pGEM3neo has been described previously (Papadopoulou et al., 1992). For the gene targeting experiments, the neomycin phosphotransferase gene (neo) and the hygromycin phosphotransferase gene (hyg) were derived from plasmids pSPYneo and pSPYhyg, respectively.2 These expression cassettes were cloned in the unique BamHI site of ltdh. Approximately 1 pg of the 2.3-kb XhoI-XhoI linearized ltdh fragment interrupted by neo or hyg, leaving 1.2 kb of homologous sequences on each side of the drug resistance gene, was electroporated in TarII wild-type cells under previously described conditions (Papadopoulou et al., 1992). Selections were with 40 pg/ml G418 (Gibco-BRL) or with 100 pg/ml hygromycin B (CalBiochem). Cells were cloned on SDM-79-1% agar plates containing the appropriate selective drug after each round of transfection before analysis by hybridization studies using It&-, neo-, and hyg-specific probes, all obtained by polymerase chain reaction.
Antibody and Western Blotting Procedure-A synthetic peptide corresponding to the last 12 amino acids of LTDH was custom synthesized (Hukabel Scientific). The peptide was coupled to albumin and injected into rabbits according to standard protocols (Harlow and Lane, 1988). After two boosts 1 week apart, the rabbits were bled and serum collected and used in Western blotting as described below. Leishmania cells were grown until late log phase, harvested by centrifugation, resuspended in 20 m M KCl, 20 m M Tris-HC1, pH 7.5,O.l m M EDTA, 1 m~ phenylmethylsulfonyl fluoride, and 6 m~ P-mercaptoethanol, and sonicated. An aliquot of each lysate corresponding to 50 pg of total protein was fractionated on 12.5% SDS-polyacrylamide gel electrophoresis, and proteins were electrotransferred to a nitrocellulose filter. The filters were saturated with a 3% Blotto solution (Harlow and Lane, 1988) and then incubated with a 1/5000 dilution of the antibody directed against the last 12 amino acids of LTDH or with non-immune sera as negative control for 90 min a t room temperature. Reaction was revealed with a goat antirabbit horseradish peroxidase-labeled antibody and chemiluminescence (ECL, Enhanced Chemiluminescence, Amersham).

RESULTS
Phenotype of ltdh Dansfectants-ltdh transfection leads to high level resistance to MTX (Papadopoulou et al., 1992;Callahan and Beverley, 1992). Increased resistance is correlated with an increase in ltdh mRNA (Papadopoulou et al., 1992). Here we show that ltdh overexpression is correlated with the overproduction of a 30-kDa protein corresponding to LTDH * B. Papadopoulou, G. Ray, and M. Ouellette, manuscript submitted for publication. (Fig. 1). The protein is barely visible in wild-type cells, but it is easily detectable in the ltdh transfectant TarII-pneo40.8. The size of the protein is in agreement with its expected molecular weight as deduced from sequence analysis.
According to our hypothesis, LTDH overproduction confers resistance to MTX because it is capable of alleviating the need for an active DHFR (Ouellette and Borst, 1991;Papadopoulou et al., 1992;. If this is the case, once overproduced, LTDH would confer resistance to all molecules acting as DHFR inhibitors in Leishmania. The ltdh transfectants were indeed shown to be highly resistant to MTX and to all other Leishmania DHFR inhibitors tested, including trimetrexate (Papadopoulou et al., 1992;Callahan and Beverley, 1992). DHFR inhibitors lead to thymidylate starvation and to cell death. In Leishmania. the action of these inhibitors can I " -21.5

FIG. 1. Overproduction of LTDH in transfectants.
Total protein lysates from TarII-pneo (vector alone) and the ltdh transfectant TarII-pneo40.8 were prepared as described under "Experimental Procedures," transferred to a nitrocellulose membrane for immunological detection. run on a 12.5 % SDS-polyacrylamide gel electrophoresis, and electro- The filter was incubated with a LTDH polyclonal antibody, raised against the last 12 amino acids of the protein, followed by incubation with a goat antirabbit horseradish peroxidase-antibody as described under "Experimental Procedures." Lane I , TarII-pneo40.8; lane 2, TarII-pneo. A filter was also reacted with preimmune serum, but no reaction was observed (not shown). The bands migrating above 45 kDa reacting with the antibody were also detected with the preimmune serum and served as internal standard to monitor the amount of protein layered in each lane. The molecular mass size standards (Bio-Rad) in kilodaltons are shown on the right.
The relative MTX resistance values were obtained by dividing the 50% growth inhibition value of the strain tested by the same value for the wild-type cells. A value of 1 indicates that the strain tested and the wild-type have the same sensitivity for MTX. tor alone; TarII-pneo40.8, a strain transfected with the fragment con-e TarII-pneo, a wild-type Leishmania strain transfected with the vectaining the ltdh gene.
ECS0 values for MTX in the different media are in parenthesis. be circumvented by adding thymidine but not biopterin (Table  I, see also Petrillo-Peixoto and Beverley, 1987;Kaur et al., 1988;Papadopoulou et al., 1992). Levels of MTX resistance mediated by ltdh have so far been measured in rich medium (Papadopoulou et al., 1992;Callahan and Beverley, 1992) where thymidylate precursors are present which reduce the need for reduced folates. If overproduction of LTDH can provide cells with sufficient reduced folates, it should also, in principle, be capable of conferring high level resistance to MTX even in poor medium, where reduced folates are absolutely necessary for the synthesis of thymidylate precursors. The level of resistance provided by ltdh transfectants was therefore measured in both rich and defined medium. The results indicated that poorer the medium in either folate or thymidylate precursors, the more Growth of Leishmcmia Ztdh transfectants in unsupplemented folate-deficient defined medium. The empty bur corresponds to TarII wild-type and the hatched one to the ltdh transfectant TarII-pneo40.8. Each bar corresponds to one passage. The first passage for both TarII wild-type and TarII-pneo40.8 reached stationary phase in 3 days. The wild-type cells stopped growing in fdDME-L after the second passage, but significant growth was still observed in the ltdh transfectant TarII-pneo40.8. At the eight passage, about 60% of the initial growth was observed after 9 days of growth for TarII-pneo40.8. A.
hdh 1 H sensitive the cells to MTX (Table I). The difference in MTX sensitivity of Leishmania cells in different media has been previously documented (Kaur et al., 1988). More importantly, the ltdh transfectant TarII-pneo40.8, in which LTDH is overproduced (Fig. l), was resistant to high level MTX in both rich and poor media (Table I). These results are in line with the proposed role of LTDH in reduced folate synthesis.
When wild-type cells were passaged from SDM-79 to the completely defined folate-deficient medium fdDME-L, they grew well for one passage (six to eight generations), probably using their stored reduced folate pools (see also Kaur et al., 1988). In some instances, two passages were necessary to observe a complete arrest of growth. TarII wild-type stopped growing when it was passaged for the second (or third) time in fdDME-L. However, significant growth was observed in TarII-pneo40.8 after the same number of passages (Fig. 2). The growth of TarII-pneo40.8 in unsupplemented fdDME-L was observed in three independent experiments. Its growth rate decreased as the number of passages increased, but growth nevertheless continued for several passages (Fig. 2) to eventually stop at the 10th passage. This suggests that overproduction of LTDH might contribute to the synthesis of a cofactor absent in fdDME-L that is most likely related to folate or pterin metabolism, as addition of any of these compounds can support the growth of wild-type cells in the same medium (see below). To examine the function of LTDH in folate or pterin metabolism, a Leishmania ltdh null mutant was generated by gene targeting.
Diploid Knockout of the ltdh Gene-In Kinetoplastidae, transfected DNA will integrate in the genome exclusively by homologous recombination (Cruz and Beverley, 1990;Lee and Van der Ploeg, 1990;Ten Asbroek et al., 1990;Eid and Sollner-Webb, 1991;Tobin and Wirth, 1992) rendering inactivation of any gene theoretically possible. Leishmania is diploid for most loci and, as no sexual crosses have yet been described, two successive rounds of targeting are necessary, which requires Generation of a Ztdh null m u t a n t by gene targeting. A, a schematic drawing of part of the H locus of L. turentolue with the ltdh gene and its relevant restriction sites (X, XhoI; B, BurnHI). Upon integration of the neo or hyg genes a t the homologous locus, the genomic 2.3-kb XhoI-XhoI restriction fragment would increase by the size of the resistance gene inserted. The segment with a thicker line represents the extent of the ltdh-neo and ltdh-hyg constructs. B, sequential inactivation of both ltdh alleles. DNAs of wild-type, of a single or a double ltdh mutant, were digested with XhoI, electrophoresed through an agarose gel, and hybridized with ltdh, neo, and hyg probes all obtained by polymerase chain reaction. Lane I , TarII wild-type; lune 2, TarII-ltdhYneo.1; lune 3, TarII-ltdhYneoYhyg. C, generation of a ltdh null mutant with a single construct. DNAs were processed as in B and hybridized to a ltdh or hyg probe. Lune I , TarII wild-type; lune 2, TarII-ltdhYhyg.2. two distinct selectable markers (Cruz et al., 1991;Lee and Van der Ploeg, 1991). Constructs containing the ltdh gene interrupted by either the neo or the hyg gene were made (Fig. 3-41.
The ltdh-neo construct was first introduced by electroporation in TarII wild-type. Cells resistant to G418 were obtained, and analysis of the clone TarII-ltdhYneo.1 was consistent with integration of the ltdh-neo construct in one allele of its homologous Ztdh locus. In the wild-type cell, the ltdh gene is present on a 2.3-kb XhoI-XhoI fragment (Fig. 3B, lane I). In TarII-ltdhYneo.1, one allele remained intact and the second's size increased by 0.9 kb with the insertion of neo (Fig. 3B, lane 2). The remaining intact allele of ltdh in TarII-ltdhYneo.1 was inactivated by a second round of targeting, in which the linearized ltdh-hyg construct (Fig. 3 A ) was introduced by electroporation. The analysis of one clone, named TarII-LtdhYneoYhyg is shown (Fig. 3B, lane 3 ) . In this strain, no more 2.3-kb XhoI-XhoI genomic fragments hybridized to a ltdh probe, even after overexposure of the autoradiogram (not shown). Indeed, the two Ztdh alleles were now migrating at a size consistent with the integration of the neo or hyg genes into the ltdh alleles (Fig. 3B, lane 3 1. The first ltdh allele of TarII wild-type was also inactivated with the ltdh-hyg construct (not shown). In one experiment using the same transfection and selection procedures, both genomic ltdh alleles were inactivated at once using only the ltdhhyg construct (Fig. 3C, lane 2). Indeed, in the hygromycinresistant clone TarII-ltdhYhyg.2, no more bands migrating with the genomic ltdh fragment were detected. This experiment was not repeated, but this result suggests that under appropriate conditions the rate of homologous recombination is sufficiently high to obtain homozygous mutants in a single round of targeting with one construct.
Phenotype ofthe ltdh Null Mutant-When cells overproduced LTDH, as in TarII-pneo40.8, they resisted to very high concentrations of antifolates (Fig. 4A). If LTDH contributes to the synthesis of reduced folates, it would be expected that inactivation of its corresponding gene would increase the sensitivity of such a strain to inhibitors of enzymes (like DHFR) involved also in the synthesis of reduced folates. The mutant TarII-ltdhYneo.1, containing one disrupted ltdh allele, was equally sensitive to MTX, a known Leishmania DHFR inhibitor (Coderre et al., 1983), as the wild-type cell. In contrast, the sensitivity of the ltdh null mutant TarII-ltdhYneoYhyg, measured in the rich medium SDM-79, increased by 50-fold (Fig.  4A), indicating that LTDH is probably not a target for MTX. The clone TarII-ltdhYhyg.2, where both alleles were inactivated at once by the hyg gene, also showed the same increased sensitivity to MTX (Fig. 4A), indicating that even when both alleles are inactivated in the same round of transfection, the resulting phenotype will be identical to a strain where alleles are inactivated sequentially. The transfection of the ltdh gene present on plasmid pneo40.8 (Papadopoulou et al., 1992) into TarII-ltdhYhyg.2 resulted in the cell line TarII-ltdhYhyg/ neo40.8. In this novel cell line, resistance to MTX was restored to the levels of wild-type cells transfected with the ltdh construct (Fig. 4A).
In rich medium, thymidine can circumvent the toxicity of MTX (Petrillo-Peixoto and Beverley, 1987;Kaur et a/., 1988;Papadopoulou et al., 1992), and this has been shown for TarII wild-type cells (Fig. 4B ). Leishmania mutants lacking a dhfr-ts gene were auxotrophic for thymidine (Cruz et al., 1991). These data suggest that, provided that thymidine is added, Leishmania does not require DHFR to grow in culture containing pterin derivatives. However, the addition of thymidine did not rescue TarII-LtdhYneoYhyg when challenged with MTX (Fig.  4B). This result indicates that reduced folates or pterin derivatives, provided by LTDH when DHFR is blocked, are absolutely required in an essential pathway different from that responsible for the synthesis of thymidylate precursors.
The possibility of growing cells in defined medium facilitated identification of the metabolic pathway inactivated once ltdh was disrupted. The strains TarII wild-type and the ltdh null mutants TarII-LtdhYneoYhyg and TarII-ltdhYhyg.2 were grown in fdDME-L, supplemented or not, to find the putative pathway in which LTDH is involved (Fig. 5). Supplementation of fdDME-L with folic acid or dihydrofolate resumed growth of TarII wild-type and of both ltdh null mutants (Fig. 5, A Z ) . Both TarII wild-type and TarII-LtdhYneoYhyg were passaged more than 20 times in fdDME-L supplemented with folic acid. The addition of biopterin and other pterin derivatives were capable of sustaining the growth of Leishmania major and Leishmania donouani in fdDME-L (Petrillo-Peixoto and Beverley, 1987;Kaur et al., 1988;Beck and Ullman, 19901, and this was also the case for L. tarentolae (Fig. 5A). The L. tarentolae TarII wild-type strain could be maintained indefinitely in fdDME-L supplemented with biopterin. The addition of biopterin or other pterin derivatives failed, however, to sustain the growth of TarII-LtdhYneoYhyg or TarII-ltdhYhyg.2 in fdDME-L (Fig. 5, B and C), suggesting that LTDH is involved in one of the steps where a pterin derivative is converted to reduced folates necessary for cell growth. When the null mutant TarII-ltdhYhyg.2 was transfected with the ltdh gene present on plasmid pneo40.8 the transfectant reverted to wild-type and was now capable to grow in defined medium supplemented with biopterin (Fig. 5 0 ) .
Thymidine alone cannot sustain the growth of wild-type cells  TarII-ZtdhYhyglneo40.8) ( D ) were passaged from SDM-79 to fdDME-L and grown for six to eight generations to decrease the pool of reduced folates and then repassaged in fdDME-L. The culture was supplemented with folate or pterin derivatives or molecules likely to be a substrate or an end product related to the synthesis of reduced folates at the concentrations indicated under "Experimental Procedures." Percentage of growth was measured between 72 and 120 h.
in fdDME-L (Fig. 5 A , see also Kaur et al., 1988), which has led to the suggestion that biopterin supplementation in fdDME-L not only contributes to thymidylate precursor synthesis, but has another, yet undefined, role (Beck and Ullman, 1990). This suggestion is supported in part by the incapacity of thymidine to reverse the effect of MTX in TarII-1tdhYneoYhyg (Fig. 4B).
Methotrexate-resistant L. donovani cells with a marked decrease in MTX accumulation were incapable of growth in fdDME-L supplemented with biopterin Ullman, 1990, 1991). Although the combination of thymidine and biopterin could resume the growth of the L. donouani MTX-resistant cells (Beck and Ullman, 1990), this combination, with and without p-aminobenzoic acid, could not permit the growth of TarII-1tdhYneoYhyg (Fig. 5B). These results indicate that different mutations with different phenotypes can affect the conversion of pterins in reduced folates. The disruption of the ltdh gene therefore has profound effects on folate and pterin metabolism.

DISCUSSION
Leishmania seems unable t o synthesize folates de nouo from p-aminobenzoic acid, a pterin derivative and glutamic acid (Petrillo-Peixoto and Beverley, 1987;Scott et al., 1987;Kaur et al., 1988). However, several pterin derivatives can sustain Leishmania wild-type growth in folate-deficient medium (Petrillo-Peixoto and Beverley, 1987;Kaur et al., 1988;Beck and Ullman, 1990; see also Fig. 5A 1. This finding indicates that Leishmania has the ability to synthesize folate derivatives from pterins, a suggestion recently confirmed by demonstrating L. donouani's ability to transform radioactive biopterin into tetrahydrofolates (Beck and Ullman, 1991). The pathway responsible for this conversion is not known. In the present study, evidence is provided that one step in this pathway requires the product of the short chain dehydrogenase gene ltdh. Cells can obtain reduced folates mainly from the reduction of dihydrofolate to tetrahydrofolate catalyzed by the enzyme DHFR. The Leishmania DHFR-thymidylate synthetase is a target for the antifolate MTX (Coderre et al., 1983). Under certain growth conditions, DHFR was dispensable because a Leishmania dhfr null mutant strain was viable, provided thymidine was added and biopterin present (Cruz et al., 1991). Similarly, the cytotoxic action of MTX in rich medium can be circumvented by the addition of thymidine (Fig. 4 B ; Petrillo-Peixoto and Beverley, 1987;Kaur et al., 1988;Papadopoulou et al., 1992). DHFR also seemed dispensable when LTDH was overproduced (Fig. 4A) even in poor medium where reduced folates are absolutely required ( Table I), suggesting that LTDH can provide sufficient levels of reduced folates to the cells even if DHFR is blocked. Reduced folates were found in an E. coli dhfr mutant (Hamm-Alvarez et al., 1990), indicating that alternative pathways for their synthesis exist also in other organisms. Ltdh transfectants, unlike wild-type, grew for several passages in unsupplemented fdDME-L medium (Fig. 2). This suggests that an essential molecule in the transfectant was overproduced when grown in rich medium and was therefore diluting out less rapidly with the number of passages in Of the diploid knockouts of genes so far described in Kinetoplastidae (Cruz et al., 1991;Curotto de LaFaille and Wirth, 1992;Chung et al.;, Cooper et al., 1993, few had a measurable or observable phenotype (Cruz et al., 1991;Cooper et al., 1993). The ltdh gene ofleishmania, first isolated because of its role in MTX resistance (Papadopoulou et al., 1992;Callahan and Beverley, 1992), has been inactivated by gene targeting fdDME-L. (Fig. 3B). Disruption of the two ltdh alleles with one single construct is also described (Fig. 3C). Under appropriate conditions, the production of homozygous mutant in embryonic stem cells with one construct has also been obtained (Moertensen et al., 1992). Both types of ltdh homozygous mutants were associated with a significant increase in MTX sensitivity and lost their ability to grow in minimal medium supplemented with biopterin. Functional complementation of the ltdh null mutant TarII-ZtdhYhyg.2 by transfection of the ltdh gene on an episomal vector is also described (Figs. 4 . 4 and 5 0 ) . The fact that folate but not pterin derivatives can support the growth of the null ltdh mutant (Fig. 5), in addition to the demonstration that biopterin can be converted into tetrahydrofolates in wild-type cells (Beck and Ullman, 1991), suggests that LTDH is involved in the metabolism of pterins, which leads to the synthesis of reduced folates. The hypersensitivity of TarII-ZtdhYneoYhyg to MTX (Fig. 4A) is also in line with a role for LTDH in reduced folate synthesis. How this is done is still unclear, as is the putative mechanism responsible for the condensation ofp-aminobenzoic acid to pterin necessary for folate synthesis. Radioactive p-aminobenzoic acid was reported to be transported inside Leishmania, but it was not incorporated into folates (Ellenberger and Beverley, 1987a;Beck and Ullman, 1991).
Analysis of ltdh homozygous mutant has also indicated that pterins or reduced folates do not serve exclusively for the synthesis of thymidylate precursors. In rich medium, the sole role of DHFR in wild-type Leishmania is to provide reduced folates for the synthesis of thymidylate precursors (Fig. 4B, Kaur et aZ., 1988;Cruz et al., 1991). Because thymidine could not support growth of wild-type cells in fdDME-L medium (Fig. 5 A ; Beck and Ullman, 1990) in contrast to folates or pterins, the latter two types of molecules were probably also serving in another essential role other than thymidylate synthesis. Folates were suggested to be a source of pterins (Beck and U11man, 1990). These pterin derivatives could be either obtained from folates by the action of a folate-MTX hydrolase activity present in Leishmania (Oe et al., 1984;Kaur et al., 1988;Ellenberger et al., 1989) or by the pathway involving LTDH. When DHFR is blocked in wild-type cells, this essential pterin cofactor is provided by LTDH, therefore, thymidine can rescue cells from MTX cytotoxicity. In cells overproducing LTDH, this pterin derivative is presumably converted to reduced folates at a n increased rate, hence permitting growth even in presence of saturating concentration of various DHFR inhibitors (Papadopoulou et aZ., 1992). However, in ltdh null mutant, the capacity for producing this essential cofactor is lost thus explaining why, when DHFR is blocked by MTX, cells will die whether thymidine is added or not (Fig. 4, A and B ) .
These studies present novel findings on folate metabolism and pterin utilization in Leishmania. Both thymidylate precursors and a pterin molecule are essential for growth. These two essential cofactors can be obtained from reduced folates generated by DHFR. Alternatively, the two molecules can also be obtained at sufficient levels when LTDH is overproduced. The identity and role of the essential pterin cofactor remains t o be determined. In mammalian cells, tetrahydrobiopterin is a cofactor of three aromatic amino acid oxidases, some of which are rate-limiting enzymes in the synthesis of biogenic amine neurotransmitters (reviewed in Blau et al., 1993). The role of such molecules in Leishmania is unknown. Evidence has been provided in the distantly related parasite Crithidia that pterins are involved in the synthesis of pyrimidines and of some amino acids (Kidder and Dewey, 1972;Kidder and Nolan, 1973). It is possible that a pterin derivative plays a similar role in Leishmania. Further work is required, however, to understand the role of this essential pterin derivative in Leishmania.