Characterization of the folylpolyglutamate synthetase gene and polyglutamylation of folates in the protozoan parasite Leishmania

Molecular & Biochemical Parasitology 124 (2002) 63 /71 www.parasitology-online.com

Characterization of the folylpolyglutamate synthetase gene and polyglutamylation of folates in the protozoan parasite Leishmania Amal El Fadili, Christoph Ku¨ndig, Marc Ouellette * Division de Microbiologie, Faculte´ de Me´decine, Centre de Recherche en Infectiologie du Centre de Recherche du CHUL, Universite´ Laval, 2705 boul. Laurier, Sainte Foy, Quebec, Canada G1V 4G2 Received 27 May 2002; received in revised form 1 August 2002; accepted 8 August 2002

Abstract Folates are polyglutamylated in most organisms by the enzyme folylpolyglutamate synthetase (FPGS). The Leishmania tarentolae FPGS gene was isolated. Its predicted product contains 538 amino acids and shows 33 and 30% identity with the human and yeast FPGS proteins, respectively. The level of folate polygtutamylation was studied in L. tarentolae promastigotes and in Leishmania infantum promastigotes and axenic amastigotes. In all species examined, folates were found predominantly as pentaglutamates, although monoglutamates were found in higher proportion in L. infantum axenic amastigote cells. Leishmania cells transfected with a FPGS containing plasmid (FPGS transfectant) exhibited a 6-fold increase in FPGS activity (32.7 pmol mg
1 h
1) compared with wild-type cells (4.7 pmol mg
1 h
1). HPLC analysis of the polyglutamylated forms of folates indicated a 2-fold increase of hexaglutamates in the FPGS transfectant compared with wild-type cells, while cells with one FPGS allele interrupted showed a higher proportion of short chain glutamates. The long-term accumulation of folates was greatly increased in the FPGS transfectant. Overall, this work indicates that FPGS activity is expressed in all forms of the parasite, and modulates the retention of folate, thereby possibly playing an important role in physiology. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Folylpolyglutamate synthetase; Leishmania ; FPGS transfectant; Folate metabolism

1. Introduction Reduced folates have important metabolic functions, notably in reactions requiring the transfer of one carbon groups, such as in the biosynthesis of the purine rings, the convervion of serine to glycine, the synthesis of methionine from homocysteine, the catabolism of histidine, and most importantly in the conversion of dUMP to dTMP [1,2]. Folates consist of a pterin ring, para aminobenzoic acid and glutamic acid. In most organisms which have been analyzed folates exist mainly as polyglutamate derivatives, with the polyglutamyl moiety linked to the g-carboxyl group of the side chain of folates [3,4]. Polyglutamylation of folates is carried out by the enzyme folylpolyglutamate synthetase (FPGS). The physiological importance of folylpolyglutamates is

* Corresponding author. Tel.: /1-418-654-2705; fax: /1-418-6542715 E-mail address: [email protected] (M. Ouellette).

now well established; they increase the cellular retention of folates, they are often better substrates or cofactors for some of the enzymes involved in folate metabolism, and finally, folylpolyglutamates are better accumulated in mitochondria where they are necessary for glycine biosynthesis [3,4]. Leishmania is a protozoan parasite distributed worldwide and responsible for a variety of clinical symptoms ranging from self-healing cutaneous lesions to visceral infections that can be fatal if untreated [5]. The mainstay in chemotherapy are pentavalent antimonial drugs, but resistance can be widespread in some parts of the world [6] and other chemotherapeutic alternatives are required. Currently, available antifolates are effective in the treatment of some protozoal infections such as malaria, but they are not effective against Leishmania [7,8] as they are poor inhibitors of the Leishmania dihydrofolate reductase [9]. Nonetheless, recent work has significantly increased our understanding of pterin and folate metabolism in Leishmania (reviewed in [2,7]),

0166-6851/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 0 2 ) 0 0 1 6 3 - 9

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which has led to the synthesis of novel antifolate inhibitors against Leishmania [10 /12]. Leishmania is auxotrophic for pterins and relies on folates from the environment to meet its folate requirements [7,13]. The Leishmania biopterin transporter BT1 [14,15] and several putative folate transporters have recently been identified in the genome of Leishmania [16]. One of these, FT5, was characterized in detail and found to correspond to a high affinity folate transporter [17]. Very few studies have dealt with folate polyglutamylation in Leishmania or in other parasites. In Plasmodium falciparum a bifunctional dihydrofolate synthetase /FPGS was identified by functional complementation of yeast and bacterial mutants [18]. In one study, it was found that folates are polyglutamylated in Leishmania major , with penta- and tetra-glutamates being the predominant forms, hence resembling folylpolyglutamates of mammalian cells [19]. In contrast, the antifolate methotrexate was not polyglutamylated in L. major [20]. Due to the importance of folate polyglutamylation in folate homeostasis and metabolism, we have cloned the Leishmania tarentolae FPGS gene and studied some of its properties.

2. Materials and methods 2.1. Strains and cultures The L . tarentolae cell line TarII WT [21] was grown in SDM-79 [22] or in M-199 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 5 mg ml
1 of hemin. The Leishmania donovani infantum promastigote line (MHOM/MA/67/ITMAP-263) was grown in MAA medium [23] at pH 6.5 at 25 8C while for the axenic amastigotes the cells were grown in MAA medium at pH 5.5 at 37 8C. For HPLC analysis, cells were grown in either supplemented M-199 or MAA medium with 25 nM [3H]-folic acid (20.2 Ci mmol
1) (Dr Shircks Laboratory, Jona, Switzerland). 2.2. Southern and Western blots DNA isolation, hybridization and washing conditions were done according to standard protocols [24]. The probe containing the coding sequence of FPGS was obtained by PCR. Expression of the FPGS/His6 fusion protein in Leishmania was measured by Western blotting using a polyclonal rabbit anti His6 antibody (Research Diagnosis Inc). Total soluble proteins of the Leishmania FPGS /His6 transfectants, of control cells were prepared by resuspending the cell pellet obtained from 60 ml of Leishmania cells in mid-log phase in 1.5 ml of protein suspension buffer (5 mM Imidazole, 0.5 M NaCl and 20 mM Tris /HCl pH 7.9). Cells were sonicated five times for 20 s, at 4 8C and centrifuged

at 16 000/g for 20 min. Electrophoresis of proteins, their transfer, antibody reaction and chemiluminescence detection were done as previously described [25]. 2.3. Cloning and sequencing of FPGS Sequence alignment of the yeast, human and Escherichia coli FPGS enzymes pinpointed conserved sequences from which oligonucleotides were synthesized (primer 1: 5?-GTCGAGGTSGGYMTBGGYGG, primer 2: 5?-GCCTCCCTTGAAGATGCCVSCCTT) (see Fig. 1) and used to amplify an internal FPGS fragment from L. tarentolae genomic DNA. The complete gene was isolated following screening of a L. tarentolae cosmid library [14]. DNA sequencing was done on an Applied Biosystems 377 DNA automated sequencer. Analysis of the sequence was performed using the Genetics Computer Group, 1994 (GCG) software package. The nucleotide sequence reported here appears in the DDBJ/EMBL/Gen Bank sequence database under the accession number AF284554. 2.4. DNA constructs The Leishmama expression vector pSLYNEO was constructed by insertion of a 900-bp Xba I-Eco RV neomycin phosphotransferase expression cassette downstream of a stretch of pyrimidines [26] into the Xba ISma I sites of the vector pSL1180 (Pharmacia Biotech), pSLYNEO was used to clone the L. tarentolae FPGS gene as part of a 2.7 kb Nco I fragment to lead to pSLYNEO /FPGS. Six histidine residues along with an Eco RI restriction site were added by PCR to the Cterminus of FPGS to yield an FPGS/His6 fusion that can be recognized by commercially available antibodies. In an attempt to disrupt one allele of the FPGS gene, the hygromycin B phosphotransferase expression cassette (HYG ) derived from pSPaHYGa vector (C. Dumas, unpublished) was introduced into the unique Bgl II site of FPGS (Fig. 2). pSPaHYGa was made by cloning the a tubulin intergenic sequence both upstream and downstream of the HYG marker in the vector pSP72 (Promega). The construct was linearized by Hin dIIIXba I in an attempt to interrupt one FPGS allele. Constructs were electroporated in Leishmania cells as described [27]. 2.5. FPGS enzymatic assays in crude extracts L. tarentolae or L. infantum cells (6 /108) in their logarithmic growth phase were washed twice and resuspended in a sonication buffer (22 mM Tris /HCl pH 8.3, 33 mM KCl, 27.5 mM MgCl2, 33 mM NaHCO3 and 47.5 mM b-mercaptoethanol) in a final volume of 0.4 ml. Cells were sonicated and centrifuged at 16 000/ g for 20 min at 4 8C. The supernatant was filtered using

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Fig. 1. Sequence comparison of the L. tarentolae FPGS. The amino acid sequences of various FPGS sequences were aligned using the GCG software package. Strictly conserved amino acids in all FPGS arc boxed in black while similar amino acids are boxed in gray. Dots within the sequence indicate gaps created to optimize the alignment. Amino acid residues of the different proteins are numbered at the left. The arrows represent the location of oligonucleotides used for amplification of a FPGS fragment from the Leishmania FPGS gene. The accession numbers are for L. tarentolae , AF284554; human, A46281; S. cerevisiae , Z75149; P. falciparum , AF161264; and E. coli , M32445.

a 0.45 mm filter. FPGS activity was determined essentially as described previously [28,29]. To 222 ml of crude extracts, 62.5 mM of L-[3H]-glutamate (5 mCi mmol
1, Dr Shircks laboratories) and 40 mM folic acid were added. Samples were prewarmed 10 min at 37 8C and

reactions were initiated by the addition of Na2ATP to 5 mM in a final volume of 0.25 ml. The samples were incubated for 2 h at 37 8C and the reactions were stopped by dilution with 0.75 ml of 93 mM sodium acetate, pH 5.2 and placing the tubes on ice. The

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Fig. 2. FPGS copy number in L. tarentolae wild-type cells, transfectants and mutants. (A) Schematic representation of the L . tarentolae FPGS locus and the disruption of one allele by the hygromycin phosphotransferase gene (HYG ). (B) Total DNA of Leishmania cells was isolated, digested with Nco I, electrophoresed, transferred and hybridized to a Leishmania FPGS probe obtained by PCR. Lanes 1 TarII WT: 2, TarII WT transfected with an FPGS episomyl construct; 3, TarII WT with one FPGS allele inactivated with the HYG marker. (C) Chromosomal location of FPGS as determined by hybridization to a TAFE blot where Leishmania chromosomes were resolved. Lane 1, TarII WT.

acidified mixture was applied to a DEAE-cellulose (Sigma) column for separation of polyglutamyl folate from unincorporated 3[H]-glutamate. Folylpolyglutamates were analyzed as described [28].

cells at 6, 24 and 48 h. Cells were washed in HEPES / NaCl and radioactivity was measured by liquid scintillation counter.

2.6. HPLC analysis of intracellular folylpolyglutamates

3. Results

PABAglun standards (n /l/6) were purchased from Dr Shircks laboratories. All HPLC reagents were obtained from US Bioscience and were of HPLC grade. Leishmania cells growing in either M199 or MAA medium were incubated in fresh medium containing 25 nM [3H]-folic acid (20.2 Ci mmol
1) for 72 h. About 6 /108 cells were washed twice with HEPES /NaCl buffer to remove extracellular labeled folic acid. The parasites were then resuspended in 200 ml of 20 mM potassium phosphate, pH 7 containing 5 mM b-mercaptoethanol. This suspension was boiled for 5 min and then cooled for 1 min on ice. Cellular debris were removed by a 5 min sedimentation at 16 000 /g . The resulting supernatants were collected and processed further. The cleavage of folylpolyglutamates to pABAglun was performed as described previously [30]. pABAglun from Leshmania were separated according to the glutamate chain length by HPLC using a Schimadzu HPLC system as previously described [31] on a Partisil 10 SAX anionic exchanger column. PABAglu1 to pABAglu6 were well resolved where unlabeled standards were monitored by their absorbance at 280 nm with retention times as follows: pABAglu, 7.5 min; pABAglu2, 17.5 min; pABAglu3, 26.6 min; pABAglu4, 31.5 min; pABAglu5 35.5 min and pABAglu6, 39.9 min.

3.1. Cloning and expression of the Leishmania FPGS gene

2.7. Uptake studies Cells were grown in M-199 media in the presence of 25 nM of 3[H]-folic acid. The long term accumulation of [3H]-folic acid was measured by pelleting aliquots of

Despite its key role in folate metabolism, folate polyglutamylation has been studied very little in protozoan parasites. As a first step towards increasing our understanding of folate polyglutamylation in Leishmania , we attempted to clone the L. tarentolae FPGS gene by PCR-homology cloning. Oligonucleotides corresponding to two conserved regions of FPGS genes from a number of organisms (see Fig. 1) were used in a PCR reaction to amplify the homologous gene from genomic DNA of L. tarentolae . A PCR product of 150 bp was sequenced and its putative open reading frame was found to show significant homology to other FPGS proteins. This fragment was used as a probe to screen a L. tarentolae genomic cosmid DNA library [14]. Two cosmids were isolated and a 2.7 kb Nco I-Nco I fragment was subcloned. Sequence analysis of this fragment revealed an open reading frame of 1614 bp encoding a putative protein of 538 amino acids exhibiting 30 and 33% identities to the yeast and human FPGS, respectively, and 24% identity with the recently described Plasmodium FPGS [18] (Fig. 1). The Leishmania FPGS is a single copy gene (Fig. 2B, lane 1 and results not shown) and is part of a large chromosome at around 2000 kb (Fig. 2C). To evaluate the role of the Leishmania FPGS enzyme in folate metabolism, the FPGS gene was cloned in a Leishmania expression vector. In one construct, the gene itself was subcloned while in a second, six histidine residues were added at the C-terminus of FPGS to

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create an FPGS/His6 fusion. Both constructs were transfected into L. tarentolae wild-type cells and transfectants were selected for G418 resistance. Southern blot analysis confirmed that the transfectants obtained had the correct plasmids and that the FPGS gene was present in high copy number (Fig. 2B, lane 2, and data not shown). Total protein extracts of the FPGS and the FPGS /His6 transfectants were reacted to an anti His6 antibody. A protein with an apparent size of 60 kDa was detected in the FPGS /His6 transfectant, but was absent in the FPGS transfectant control (Fig. 3). The size of the FPGS/His6 tagged version is commensurate with the expected theoretical size of the fusion protein. To verity if the expressed FPGS /His6 protein is active, we measured FPGS enzymatic activity in crude extracts from L. tarentolae wild-type, FPGS transfectant and FPGS /His6 transfectant cells. The crude extracts derived from both transfected strains show five to six times higher FPGS activity for the substrate folic acid compared with the wild-type crude extracts (Fig. 4). These results indicate that FPGS in the transfectants is overexpressed and active, and that the addition of His6 did not influence its function. 3.2. Polyglutamylation during the life cycle of Leishmania Few studies have looked at folate metabolism in Leishmania amastigotes. The availability of a L. infantum line that can be grown both as axenic promastigotes and amastigotes [23] is facilitating biochemical studies with amastigote parasites. The FPGS activity was measured in L. infantum amastigote and promastigote extracts and was found to be similar (Fig. 4). The distribution of folylpolyglutamates was also studied. Similarly to L. tarentolae (Table 1) and L. major [19], folates are mostly found as pentaglutamates in L. infantum promastigotes, although the levels of monoglutamates are higher (Table 1). In axenic amas-

Fig. 3. FPGS expression in Leishmania transfectants. Total proteins of Leishmania cells were prepared as described in Section 2, electrophoresed through a 10% SDS-PAGE gel, blotted to nitrocellulose membrane and reacted with a polyclonal antibody against His6-tag. 1, positive control of a bacterial integrase containing a His6-tag [43]; 2, L. tarentolae FPGS transfectant; 3, Leishmania FPGS /His6 transfectant.

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Fig. 4. In vitro measurements of FPGS activity in Leishmania cell extracts. The FPGS activity was measured as described under Section 2 from crude extracts of L. tarentolae wild-type cells, Leishmania FPGS transfectants and Leishmania donovani infantum promastigotes and amastigotes. An average of three independent measures is shown.

tigotes, the pentaglutamates also predominate but the proportion of monoglutamylated folates is even higher (Table 1). 3.3. Modulation of FPGS expression and folate polyglutamylation The pools of folylpolyglutamates in L. tarentolae wild-type cells and in the FPGS transfectant were studied using HPLC analysis. Folylpolyglutamates present in the extracts were cleaved to p -aminobenzolpolyglutamates. A representative HPLC spectrum can be found in Fig. 5. In L. tarentolae wild-type cells, the majority of folylpolyglutamates are found as the penta form (
/62%) followed by the tetra- (
/20%) and hexa(
/7%) glutamate forms (Table 1). In the FPGS transfectant with increased FPGS activity (Fig. 4), we noticed a shift of the polyglutamate forms with a significant increase in the hexaglutamates (Table 1). To test whether a decrease in the copy number of FPGS would also alter the levels of polyglutamylation we interrupted one FPGS allele with an hygromycin phosphotransferase marker (Fig. 2B, lane 3). The FPGS gene in Leishmania is single copy (Fig. 2B, and other results not shown) and a 2.7 kb Nco I-Nco I fragment hybridized to a FPGS probe. The integration of the HYG gene should leave one allele of FPGS intact as a 2.7 kb Nco I-NcoI fragment, while the insertion of the disruption cassette should lead to a 2.9 and 1.4 kb

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Table 1 Folylpolyglutamates in Leishmania cells Cell lines

L. tarentolae TarII WT TarIIFPGS TarII FPGS/HYG TarII FPGS/HYG/FPGS L . infantum promastigotes L. infantum amastogotes a

Glutamate chain length (%)a n l

n 2

n 3

n 4

n 5

n 6

6.391.8 3.092.2 2.390.2 1.890.1 20.090.4 32.092.5

1.392 0.790.3 1.691.1 0.690.1 3.891.2 1194.2

2.290.3 2.390.8 21.7910.4 2.990.4 4.591.6 9.097.5

20.391.8 16.790.7 47.696.2 21.394.9 18.791.3 17.192.4

62.092.5 60.690.4 24.6916.6 65.594.5 48.991.2 32.191.8

7.390.6 16.092.7 1.8590.8 7.890.9 4.290.1 3.290.5

Average of three independent measures9S.D.

with a shift towards tri- and tetraglutamates while the penta and hexaglutamates decreased significantly (Table 1). Reintroduction of an episomal copy of FPGS in the mutant restored the level of polyglutamylation found in wild-type cells (Table 1). Similar results were obtained while disrupting an FPGS allele with a NEO construct, but we were unable to generate an FPGS null mutant (A. El Fadili and M. Ouellette, unpublished observations). 3.4. Increased folate accumulation in FPGS transfectants In mammalian cells, increased polyglutamylation is often related to increased cellular retention of folates [4]. In our analysis, we noticed that there was systematically more radioactive folates in the FPGS transfectant suggesting that increased polyglutamylation augments folate accumulation. To prove this, we carried out longterm folate accumulation measurement in L. tarentolae cells. At 6 h incubation, there was already 4-fold more folate accumulated in the FPGS transfectant compared with wild-type cells (Fig. 6). This difference peaked at 24 h with close to ten times more folate accumulation in the

Fig. 5. HPLC analysis of folate polyglutamates. (A) A typical HPLC profile of pABAglun standards. (B) Analysis of radioactive 3[H]folylpolyglutamates as determined by HPLC separation of pABApolyglutamates in L. tarentolae wild-type cells.

FPGS-hybridizing Nco I-Nco I fragment (Fig. 2A). This was the pattern observed (Fig. 2B, lane 3) confirming the disruption of one allele. This FPGS haploid mutant, showed an increase in shorter chain folylpolyglutamate

Fig. 6. Steady-state accumulation of 3[H]-folic acid in Leishmania cells. The accumulation of 25 nM of 3[H]-folate was carried out in M199 medium and cells were pelleted at 6, 24 and 48 h. Results represent an average of three independent experiments.

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transfectant and three times more folate were accumulated in the FPGS transfectant compared with wild-type cells at 48 h (Fig. 6). No significant difference in long term folate accumulation was seen with the FPGS single allele mutant compared with wild-type cells.

4. Discussion In most cells, folates are found as polyglutamates [3,4]. Only one report dealt with folate polyglulamylation in Leishmania indicating that folates were polyglutamylated with the tetra- and pentaglutamate forms predominating in L. major [19]. In order to better understand the role of polyglutamylation of folates in Leishmania , we have cloned the Leishmania FPGS gene. Its predicted product is similar to other FPGS proteins (Fig. 1). The predominant folylpolyglutamates of wildtype L. tarentolae cells were found to be the Glu(4), and Glu(5), forms (Table 1), which is similar to what has been found in L. major [19]. The Glu(5) form also predominates in L. infantum promastigotes and amastigotes. It is worth noting that in L. infantum the level of monoglutamates was higher than in L. tarentolae and that the level of monoglutamate is even higher in amastigotes (Table 1). Since the FPGS activity is similar in L. infantum promastigotes, amastigotes and L. tarentolae (Fig. 4), differences in the level of monoglutamates is probably related to an increase in catabolism of folylpolyglutamates in L. infantum . Steady state levels of folate polyglutamates in mammalian cells are likely to depend on the balance of FPGS and of a gglutamyl hydrolase (GGH), a peptidase that cleaves the glutamate residues of folylpolyglutamates [32]. Novel folate catabolizing enzymes have also recently been isolated from mammalian cells. One corresponds to ferritin [33] and another to a glutamate carboxypeptidase II [34]. Although none of these activities have yet been reported in Leishmania , a species and stage-specific increase in the enzymatic activities of any of these gene product, could explain the observed increase in monoglutamates in L . infantum amastigotes. Modulation of the expression of FPGS influences the level of folylpolyglutamates. Overexpression of FPGS leads to a higher proportion of hexafolylpolyglutamates (Table 1). As a corollary, inactivation of one FPGS allele clearly reduces the levels of long chain folylpolyglutamates while proportions of shorter ones are increased significantly (Table 1). Attempts to generate a L. tarentolae FPGS null mutant by inactivating both alleles have failed so far (unpublished observations). Indeed, although it was possible to integrate both the NEO and HYG markers into the two FPGS alleles, one more intact FPGS allele was generated by the parasite (unpublished observation). The same type of polyploidy has occurred in other instances in Leishmania when

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attempting to disrupt the dihydrofolate reductase [35]; a cdc2 related kinase [36], and the trypanothione reductase [37] genes. These studies concluded that polyploidy occurred because under the conditions tested, the genes being inactivated were essential. Down regulation of FPGS using an antisense-based expression system in a human cell line led to inhibition of cellular proliferation [38]. The FPGS gene is essential in E. coli [39], while inactivation of the Saccharomyces cerevisiae FPGS gene leads to methionine auxotrophy [40]. Mutations in the FPGS gene of CHO cells led to strains auxotrophic for glycine, adenosine and thymidine [41]. In this study, supplementation of the medium with various combinations of these substrates did not help for selection of a Leishmania FPGS null mutant (data not shown). More work will be required to conclude whether FPGS is essential in Leishmania . An overexpression of FPGS increases the overall accumulation of folic acid in Leishmania cells (Fig. 6). In mammalian cells, there is a good correlation between increased polyglutamylation and increased cellular retention of folic acid (reviewed in [4,42]). The kinetics of accumulation in Leishmania appear to be complex, where both folate levels and the difference in accumulation between the FPGS transfectant and wild-type cells peaks at 24 h and then decreases afterwards. It is possible that at 24 h there are sufficient folates within the cells and a cellular process either increasing both polyglutamate catabolism and efflux or down regulating one of the folate transporters could explain the decrease in accumulation at 48 h. The kinetic of uptake of folic acid in L. major and Leishmania mexicana was also found to be complex [16]. In conclusion, Leishmania has a single copy FPGS gene whose gene product is closely related to other FPGS proteins. Folates are found predominantly as pentaglutamates in several Leishmania species and folates are also polyglutamylated in amastigotes although a higher proportion of monoglutamates was observed in this stage of the parasite. The level of folylpolyglutamates varies with the copy number of FPGS and increased FPGS activity is associated with increased folic acid accumulation. Folate polyglutamylation increases cellular accumulation of folates, which could be important in parasite physiology and possibly also in the future development of novel antifolate chemotherapeutic agents.

Acknowledgements We thank Nancy Messier for the gift of a sample of Int-His6. We thank Dr Jolyne Drummelsmith, Dr Danielle Le´gare´ and Dr Barbara Papadopoulou for critical reading of the manuscript. This work was supported in part by a grant from the Canadian

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A. El Fadili et al. / Molecular & Biochemical Parasitology 124 (2002) 63 /71

Institutes of Health Research (CIHR) to MO. C. Ku¨ndig was a post-doctoral fellow of the Schweizerischer Nationalfonds, and M. Ouellette is a CIHR Investigator and a Burroughs Wellcome Fund Scholar in Molecular Parasitology.

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