Dihydrofolate Reductase-thymidylate Synthase Research Articles

Characterization, crystallization and preliminary X-ray analysis of bifunctional dihydrofolate reductase-thymidylate synthase from Plasmodium falciparum.

The full-length pfdhfr-ts genes of the wild-type TM4/8.2 and the double mutant K1CB1 (C59R+S108N) from the genomic DNA of the corresponding Plasmodium falciparum parasite have been cloned into a modified pET(17b) plasmid and expressed in Escherichia coli BL21 (DE3) pLysS. Conditions for the expression and purification of the P. falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) have been established that yield approximately 1 mg of the soluble active enzyme per litre of culture. The purified enzymes have been crystallized using a modified microbatch method with PEG 4000 as the primary precipitating agent. X-ray diffraction data were collected to 2.50 and 2.64 A resolution under cryogenic conditions from single crystals of the two PfDHFR-TS proteins in complex with NADPH, dUMP and either Pyr30 or Pyr39. Preliminary X-ray analysis indicated that the crystals belong to the orthorhombic space group P2(1)2(1)2(1), with two molecules per asymmetric unit and approximately 52% solvent content (VM approximately 2.6 A3 Da-1). The use of a particular type of baby oil in the microbatch setup appeared to be beneficial to PfDHFR-TS crystallization and a preliminary comparison with another commonly used oil is described.

Toxoplasma gondii: generation of novel truncation mutations in the linker domain of dihydrofolate reductase–thymidylate synthase

Index Descriptors and Abbreviations: DHFR–TS/m2m3, high-level pyrimethamine resistant dihydrofolate reductase (EC 1.5.1.3)-thymidylate synthase (EC 2.1.1.45); FLAG, FLAG epitope; pyr R, high-level pyrimethamine resistance; SERA, serine repeat antigen

Genetic and metabolic analysis of folate salvage in the human malaria parasite Plasmodium falciparum.

Antifolate drugs that target the biosynthesis and processing of essential folate cofactors are widely used for treatment of chloroquine-resistant falciparum malaria. Salvage of pre-formed folate can strongly compromise the efficacy of these drugs in vitro and the availability of folate from the human host in natural infections also influences therapeutic outcomes. To investigate how different parasite lines respond to the presence of exogenous folate, we measured the effect of the latter on the susceptibility of parasites to sulfa-drug blockage of folate biosynthesis, utilising the parents and 22 progeny of the HB3-Dd2 genetic cross of Plasmodium falciparum, together with selected unrelated lines. Complete linkage of the folate utilisation phenotype was observed to a DNA sequence of 48.6 kb lying between nucleotide positions 738,489 and 787,058 of chromosome 4 and encompassing the dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene locus. Examination of the putative ORFs on this fragment upstream (3) and downstream (4) of dhfr-ts revealed no plausible candidate genes for folate processing. Similarly, a marked heterogeneity in the 5'-UTR regions of Dd2 and HB3, manifest as a directly repeated 256 bp sequence in the former, also did not correlate with the folate utilisation phenotype nor apparently influence levels of dhfr-ts transcripts or protein products. By contrast, the nature of the coding sequence of the dhfr domain appeared to play a direct role, with the single mutant (S108N) HB3-type utilising folic acid much less efficiently than other allelic variants. We also compared the processing of exogenous folic acid, folinic acid and p-aminobenzoic acid (pABA) in metabolic labelling studies of HB3 and Dd2. These support the view that DHFR is likely to have a low-level folate reductase activity as well as its normal function of reducing dihydrofolate to tetrahydrofolate, and that a significant hurdle in the utilisation of exogenous folic acid is the initial reduction of fully oxidised folic acid to dihydrofolate, an activity that the single mutant enzyme found in HB3 is postulated to perform particularly poorly. This would mirror earlier studies indicating that the DHFR activity of HB3 is also compromised relative to other variants.

Parasite-specific inserts in the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase of Plasmodium falciparum modulate catalytic activities and domain interactions.

Polyamine biosynthesis of the malaria parasite, Plasmodium falciparum, is regulated by a single, hinge-linked bifunctional PfAdoMetDC/ODC [ P. falciparum AdoMetDC (S-adenosylmethionine decarboxylase)/ODC (ornithine decarboxylase)] with a molecular mass of 330 kDa. The bifunctional nature of AdoMetDC/ODC is unique to Plasmodia and is shared by at least three species. The PfAdoMetDC/ODC contains four parasite-specific regions ranging in size from 39 to 274 residues. The significance of the parasite-specific inserts for activity and protein-protein interactions of the bifunctional protein was investigated by a single- and multiple-deletion strategy. Deletion of these inserts in the bifunctional protein diminished the corresponding enzyme activity and in some instances also decreased the activity of the neighbouring, non-mutated domain. Intermolecular interactions between AdoMetDC and ODC appear to be vital for optimal ODC activity. Similar results have been reported for the bifunctional P. falciparum dihydrofolate reductase-thymidylate synthase [Yuvaniyama, Chitnumsub, Kamchonwongpaisan, Vanichtanankul, Sirawaraporn, Taylor, Walkinshaw and Yuthavong (2003) Nat. Struct. Biol. 10, 357-365]. Co-incubation of the monofunctional, heterotetrameric approximately 150 kDa AdoMetDC domain with the monofunctional, homodimeric ODC domain (approximately 180 kDa) produced an active hybrid complex of 330 kDa. The hinge region is required for bifunctional complex formation and only indirectly for enzyme activities. Deletion of the smallest, most structured and conserved insert in the ODC domain had the biggest impact on the activities of both decarboxylases, homodimeric ODC arrangement and hybrid complex formation. The remaining large inserts are predicted to be non-globular regions located on the surface of these proteins. The large insert in AdoMetDC in contrast is not implicated in hybrid complex formation even though distinct interactions between this insert and the two domains are inferred from the effect of its removal on both catalytic activities. Interference with essential protein-protein interactions mediated by parasite-specific regions therefore appears to be a viable strategy to aid the design of selective inhibitors of polyamine metabolism of P. falciparum.

LINKAGE BETWEEN NUCLEAR AND MITOCHONDRIAL DNA SEQUENCES IN AVIAN MALARIA PARASITES: MULTIPLE CASES OF CRYPTIC SPECIATION?

Analyses of mitochondrial cytochrome b diversity among avian blood parasites of the genera Haemoproteus and Plasmodium suggest that there might be as many lineages of parasites as there are species of birds. This is in sharp contrast to the approximately 175 parasite species described by traditional methods based on morphology using light microscopy. Until now it has not been clear to what extent parasite mitochondrial DNA lineage diversity reflects intra- or interspecific variation. We have sequenced part of a fast-evolving nuclear gene, dihydrofolate reductase– thymidylate synthase (DHFR-TS), and demonstrate that most of the parasite mitochondrial DNA lineages are associated with unique gene copies at this locus. Although these parasite lineages sometimes coexist in the same host individual, they apparently do not recombine and could therefore be considered as functionally distinct evolutionary entities, with independent evolutionary potential. Studies examining parasite virulence and host immune systems must consider this remarkable diversity of avian malaria parasites.

Phylogenetic Classification of Protozoa Based on the Structure of the Linker Domain in the Bifunctional Enzyme, Dihydrofolate Reductase-Thymidylate Synthase

We have determined the crystal structure of dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Cryptosporidium hominis, revealing a unique linker domain containing an 11-residue alpha-helix that has extensive interactions with the opposite DHFR-TS monomer of the homodimeric enzyme. Analysis of the structure of DHFR-TS from C. hominis and of previously solved structures of DHFR-TS from Plasmodium falciparum and Leishmania major reveals that the linker domain primarily controls the relative orientation of the DHFR and TS domains. Using the tertiary structure of the linker domains, we have been able to place a number of protozoa in two distinct and dissimilar structural families corresponding to two evolutionary families and provide the first structural evidence validating the use of DHFR-TS as a tool of phylogenetic classification. Furthermore, the structure of C. hominis DHFR-TS calls into question surface electrostatic channeling as the universal means of dihydrofolate transport between TS and DHFR in the bifunctional enzyme.

An amino acid substitution in the Babesia bovis dihydrofolate reductase-thymidylate synthase gene is correlated to cross-resistance against pyrimethamine and WR99210.

The genomic locus and cDNA encoding Babesia bovis dihydrofolate reductase-thymidylate synthase (DHFR-TS) were cloned and sequenced. A single dhfr-ts gene, composed of four exons, encodes a 511 aa protein that is most closely related to Plasmodium falciparum DHFR-TS. The genomic locus is characterized by the presence of four other genes of which at least three are expressed during the erythrocytic cycle. Three of the genes were highly conserved in closely related Theileria species and for two of the genes and dhfr-ts, gene synteny was observed between B. bovis and Theileria parva, B. bovis in vitro cultures displaying approximately 10-20-fold decreased sensitivity towards the antimalarial drugs WR99210 and pyrimethamine were selected repeatedly after prolonged growth in presence of drugs. Five cultures examined in detail were shown to encode a DHFR-TS carrying amino acid substitution S125F. Three-dimensional-modelling, using the P. falciparum DHFR structure as a template, suggests that substitution S125F protrudes into the binding site of NADPH. The S125F mutant could be isolated by growth under pyrimethamine or WR99210 pressure conferring cross-resistance to both drugs. Although opposing selection for pyrimethamine or WR99210 resistance was reported recently using P. falciparum or P. vivax strains carrying wildtype dhfr, the results obtained here are reminiscent of a quadruple mutant of P. falciparum dhfr displaying strong resistance to pyrimethamine and 10-fold enhanced resistance against WR99210. Wildtype B. bovis DHFR carries three mutations present in this mutant possibly explaining the low sensitivity to pyrimethamine and the ease by which moderately WR99210 resistant mutants could be isolated.

Fitness effects of DHFR-TS mutations associated with pyrimethamine resistance in apicomplexan parasites.

Pyrimethamine resistance in the malaria parasite Plasmodium falciparum is characterized by specific point mutations in the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. We have previously explored the effect of these mutations by engineering homologous alleles of Toxoplasma gondii DHFR-TS, which can readily be expressed as recombinant protein for enzymatic studies, or as allelic replacements in transgenic parasites. In order to directly assess the costs of pyrimethamine-resistance in vivo, we have carried out competition studies between mixtures of T. gondii tachyzoites harbouring wild-type or mutant DHFR-TS alleles, both in tissue culture and in mice. Arg59+Asn108 mutants (using the P. falciparum numbering system) exhibit no significant fitness defects in vitro, but a fitness defect of 1.8% per generation in mice. Arg59+Ser223 mutants exhibit fitness defects of >2.8% per generation both in vitro and in vivo, which may explain why this highly pyrimethamine-resistant allele has not been observed in the field. It is important to note that long-term propagation of parasites in vitro or in vivo can produce adaptations affecting fitness by >3.7% per generation, necessitating careful attention to background in head-to-head competition studies. A sensitive PCR-based assay permits different growth rates to be assessed even in the absence of a drug resistance marker that can be scored by plaque assay.

Sequence elements in both the intergenic space and the 3' untranslated region of the Crithidia fasciculata KAP3 gene are required for cell cycle regulation of KAP3 mRNA.

mRNA levels of several Crithidia fasciculata genes involved in DNA metabolism have previously been found to cycle as cells progress through the cell cycle. Octamer consensus sequences in the 5' untranslated regions (5' UTRs) of these transcripts were shown to be required for cycling of these mRNAs. The KAP3 gene encodes a kinetoplast histone H1-like DNA binding protein, and its mRNA levels cycle in parallel with those of the kinetoplast DNA topoisomerase (TOP2), dihydrofolate reductase-thymidylate synthase (DHFR-TS), and the large subunit of the nuclear single-stranded DNA binding protein (RPA1). KAP3 mRNA contains two octamer consensus sequences in its 3' UTR but none in its 5' UTR. Mutation of these octamer sequences was not sufficient to prevent cycling of a sequence-tagged KAP3 mRNA expressed from a plasmid. Mutation of an octamer sequence contained on the precursor transcript but not on the mRNA, in addition to mutation of the two octamer sequences in the 3' UTR, was necessary to abolish cycling of the mRNA. The requirement for a sequence not present on the mature mRNA indicates that regulation of the mRNA levels by the octamer sequences occurs at or prior to splicing of the transcript. Incompletely processed RNAs containing octamer sequences were also found to accumulate during the cell cycle when the mRNA levels were lowest. These RNA species hybridize to both the KAP3 coding sequence and that of the downstream drug resistance gene, indicating a lack of processing within the intergenic region separating these genes. We propose a cell cycle-dependent interference in transcript processing mediated by octamer consensus sequences as a mechanism contributing to the cycling of such transcripts.

Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

In this study we used site-directed mutagenesis to test the hypothesis that substrate channeling in the bifunctional thymidylate synthase-dihydrofolate reductase enzyme from Leishmania major occurs via electrostatic interactions between the negatively charged dihydrofolate produced at thymidylate synthase and a series of lysine and arginine residues on the surface of the protein. Accordingly, 12 charge reversal or charge neutralization mutants were made, with up to 6 putative channel residues changed at once. The mutants were assessed for impaired channeling using two criteria: a lag in product formation at dihydrofolate reductase and an increase in dihydrofolate accumulation. Surprisingly, none of the mutations produced changes consistent with impaired channeling, so our findings do not support the electrostatic channeling hypothesis. Burst experiments confirmed that the mutants also did not interfere with intermediate formation at thymidylate synthase. One mutant, K282E/R283E, was found to be thymidylate synthase-dead because of an impaired ability to form the covalent enzyme-methylene tetrahydrofolate-deoxyuridate complex prerequisite for chemical catalysis.

The crystal structure of dihydrofolate reductase-thymidylate synthase from Cryptosporidium hominis reveals a novel architecture for the bifunctional enzyme.

Cryptosporidium hominis is an emerging pathogen that primarily affects immune-compromised patients, including those with AIDS. There is no effective cure at this point for the degenerative wasting disease that can follow infection. Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a bifunctional enzyme in C. hominis and is crucial in the folate cycle for the production of dTMP, one of the four DNA bases. DHFR-TS is an excellent drug target since its inhibition leads to the death of the pathogen. In apicomplexan protozoa, including C. hominis, the gene for DHFR-TS can be partitioned into a DHFR domain, a linker domain and a TS domain. In human cells, DHFR and TS are separate, monofunctional enzymes. We have determined the X-ray crystal structure of DHFR-TS from C. hominis. The structure reveals that the linker polypeptide between the DHFR and TS domains has important structural interactions with the opposite monomer of the homodimeric enzyme. A comparison of the structure of DHFR-TS from C. hominis and structures of DHFR-TS from Plasmodium falciparum [11] and Leishmania major [5] shows that there are significant structural differences between the apicomplexan and kinetoplastid forms of the enzyme. Additionally, the DHFR and TS domains of the C. hominis enzyme have important sequence and structural differences from the human forms of the enzymes. The species-specific differences are important for future inhibitor design targeting C. hominis.

Insights into antifolate resistance from malarial DHFR-TS structures.

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. The efficacy of this class of DHFR-inhibitor drugs is now compromised because of mutations that prevent drug binding yet retain enzyme activity. The crystal structures of PfDHFR-TS from the wild type (TM4/8.2) and the quadruple drug-resistant mutant (V1/S) strains, in complex with a potent inhibitor WR99210, as well as the resistant double mutant (K1 CB1) with the antimalarial pyrimethamine, reveal features for overcoming resistance. In contrast to pyrimethamine, the flexible side chain of WR99210 can adopt a conformation that fits well in the active site, thereby contributing to binding. The single-chain bifunctional PfDHFR-TS has a helical insert between the DHFR and TS domains that is involved in dimerization and domain organization. Moreover, positively charged grooves on the surface of the dimer suggest a function in channeling of substrate from TS to DHFR active sites. These features provide possible approaches for the design of new drugs to overcome antifolate resistance.

A Molecular Docking Strategy Identifies Eosin B as a Non-active Site Inhibitor of Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase

Protozoal parasites are unusual in that their thymidylate synthase (TS) and dihydrofolate reductase (DHFR) enzymes exist on a single polypeptide. In an effort to probe the possibility of substrate channeling between the TS and DHFR active sites and to identify inhibitors specific for bifunctional TS-DHFR, we used molecular docking to screen for inhibitors targeting the shallow groove connecting the two active sites. Eosin B is a 100 microm non-active site inhibitor of Leishmania major TS-DHFR identified by molecular docking. Eosin B slows both the TS and DHFR reaction rates. When Arg-283, a key residue to which eosin B is predicted to bind, is mutated to glutamate, however, eosin B only minimally inhibits the TS-DHFR reaction. Additionally, eosin B was found to be a 180 microm inhibitor of Toxoplasma gondii in both biochemical and cell culture assays.

Retroviral Transduction of a Mutant Dihydrofolate Reductase-Thymidylate Synthase Fusion Gene into Murine Marrow Cells Confers Resistance to Both Methotrexate and 5-Fluorouracil

Gene transfer-based myeloprotection strategies against chemotherapy require the development of effective drug resistance genes or gene combinations. Our laboratory has previously generated drug-resistant mutants of dihydrofolate reductase (DHFR F/S) and thymidylate synthase (TS G52S) for myeloprotection against methotrexate (MTX) and 5-fluorouracil (5-FU), respectively. For the purpose of conferring dual myeloprotection against both MTX and 5-FU, we have generated two retroviral constructs encoding both DHFR F/S and TS G52S as a fusion protein (DHFR F/S-TS G52S) or as individual proteins from a bicistronic gene. The DHFR F/S-TS G52S fusion protein is functional and exhibits kinetic properties similar to that of the individual mutant enzymes. NIH 3T3 cells and mouse bone marrow progenitors retrovirally transduced with the fusion DHFR F/S-TS G52S cDNA provided similar levels of resistance to MTX and 5-FU as cells expressing the individual mutant enzymes and higher levels of resistance to MTX than cells expressing DHFR F/S from the 3' end of a bicistronic gene. As MTX and 5-FU are used in combination therapy for diseases such as breast and colon cancer, this fusion gene may be useful in the clinic to reduce myelosuppressive toxicity associated with this drug combination.

Pyrimethamine analogs as strong inhibitors of double and quadruple mutants of dihydrofolate reductase in human malaria parasites.

Pyrimethamine acts against malarial parasites by selectively inhibiting their dihydrofolate reductase-thymidylate synthase. Resistance to pyrimethamine in Plasmodium falciparum is due to point mutations in the DHFR domain, initially at residue 108 (S108N), with additional mutations imparting much greater resistance. Our previous work, the development of a simple rational drug design strategy to overcome such resistance, used suitable meta-substituents in the pyrimethamine framework to avoid the unfavorable steric clash with mutant side chains at position 108. Interestingly, the meta-chloro analog of pyrimethamine not only overcame the resistance due to S108N, but also that contributed by the more remote mutation, C59R. The present work improves on this by means of other meta-substituents. Against wild type DHFR, double mutant types A16V + S108T and C59R + S108T, and the highly pyrimethamine/cycloguanil-resistant quadruple-mutant form N51I + C59R + S108N + I164L, pyrimethamine itself gave Ki values of 1.5, 2.4, 72.3 and 859 nM, respectively. The meta-substituted analogs, especially the meta-bromo analog, were much more powerful inhibitors of these DHFRs, including the quadruple-mutant form (meta-bromo analog, Ki 5.1 nM). For comparison, the dihydropyrazine antifolate, WR99210, gave Ki values of 0.9, 3.2, 0.8 and 0.9 nM, respectively. Ki values were also measured against recombinant human DHFR, as were their activities against the growth of Plasmodium falciparum cultures bearing the double mutations (FCB and K1 strains) and quadruple mutation (V1/S) and the wild type (3D7). The meta-analogs were highly active against all of these, with the meta-bromo again being the strongest, having an IC50 of 37 nM against V1/S, compared to > 5000 nM for pyrimethamine itself and 1.1 nM for WR99210.

Candidate defense genes from rice, barley, and maize and their association with qualitative and quantitative resistance in rice.

Candidate genes involved in both recognition (resistance gene analogs [RGAs]) and general plant defense (putative defense response [DR]) were used as molecular markers to test for association with resistance in rice to blast, bacterial blight (BB), sheath blight, and brown plant-hopper (BPH). The 118 marker loci were either polymerase chain reaction-based RGA markers or restriction fragment length polymorphism (RFLP) markers that included RGAs or putative DR genes from rice, barley, and maize. The markers were placed on an existing RFLP map generated from a mapping population of 116 doubled haploid (DH) lines derived from a cross between an improved indica rice cultivar, IR64, and a traditional japonica cultivar, Azucena. Most of the RGAs and DR genes detected a single locus with variable copy number and mapped on different chromosomes. Clusters of RGAs were observed, most notably on chromosome 11 where many known blast and BB resistance genes and quantitative trait loci (QTL) for blast, BB, sheath blight, and BPH were located. Major resistance genes and QTL for blast and BB resistance located on different chromosomes were associated with several candidate genes. Six putative QTL for BB were located on chromosomes 2, 3, 5, 7, and 8 and nine QTL for BPH resistance were located to chromosomes 3, 4, 6, 11, and 12. The alleles of QTL for BPH resistance were mostly from IR64 and each explained between 11.3 and 20.6% of the phenotypic variance. The alleles for BB resistance were only from the Azucena parent and each explained at least 8.4% of the variation. Several candidate RGA and DR gene markers were associated with QTL from the pathogens and pest. Several RGAs were mapped to BB QTL. Dihydrofolate reductase thymidylate synthase co-localized with two BPH QTL associated with plant response to feeding and also to blast QTL. Blast QTL also were associated with aldose reductase, oxalate oxidase, JAMyb (a jasmonic acid-induced Myb transcription factor), and peroxidase markers. The frame map provides reference points to select candidate genes for cosegregation analysis using other mapping populations, isogenic lines, and mutants.

Mechanistic Characterization of Toxoplasma gondiiThymidylate Synthase (TS-DHFR)-Dihydrofolate Reductase: EVIDENCE FOR A TS INTERMEDIATE AND TS HALF-SITES REACTIVITY

This study describes the use of rapid transient kinetic methods to characterize the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) enzyme from Toxoplasma gondii. In addition to elucidating the detailed kinetic scheme for this enzyme, this work provides the first direct kinetic evidence for the formation of a TS intermediate and for half-sites TS reactivity in human and Escherichia coli monofunctional TS and in T. gondii and Leishmania major bifunctional TS-DHFR. Comparison of the T. gondii TS-DHFR catalytic mechanism to that of the L. major enzyme reveals the mechanistic differences to be predominantly in DHFR activity. Specifically, TS ligand induced domain-domain communication involving DHFR activation is observed only in the L. major enzyme and, whereas both DHFR activities involve a rate-limiting conformational change, the change occurs at different positions along the kinetic pathway.

Transcriptional analysis of genes encoding enzymes of the folate pathway in the human malaria parasite Plasmodium falciparum.

Folate metabolism in Plasmodium falciparum is essential for cell growth and replication, and the target of important antimalarial agents. The pathway comprises a series of enzymes that convert GTP to derivatives of tetrahydrofolate, which are cofactors in one-carbon transfer reactions. We investigated the expression of five of the genes encoding these enzymes by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using a threshold detection technique. We followed changes in mRNA levels as parasites progress through the erythrocytic cell cycle and examined this process in two cloned lines of diverse origins, as well as under stress conditions, induced by either removal of important metabolites or challenge by folate enzyme inhibitors. Although conventionally regarded as performing housekeeping functions, these genes show disparate levels of and changes in expression through the cell cycle, but respond quite uniformly to folate pathway-specific stress factors, with no evidence of feedback at the transcriptional level. Overall, the two genes involved in the thymidylate cycle (encoding dihy-drofolate reductase-thymidylate synthase, dhfr-ts, and serine hydroxymethyltransferase, shmt) gave the most abundant transcripts. However, only the latter showed major variation across the cell cycle, with a peak around the time of onset of DNA replication, possibly indicative of a regulatory function.

Sequence variation in the dihydrofolate reductase-thymidylate synthase ( DHFR-TS) and trypanothione reductase ( TR) genes of Trypanosoma cruzi

Dihydrofolate reductase-thymidylate synthase ( DHFR-TS) and trypanothione reductase ( TR) are important enzymes for the metabolism of protozoan parasites from the family Trypanosomatidae (e.g. Trypanosoma spp., Leishmania spp.) that are targets of current drug-design studies. Very limited information exists on the levels of genetic polymorphism of these enzymes in natural populations of any trypanosomatid parasite. We present results of a survey of nucleotide variation in the genes coding for those enzymes in a large sample of strains from Trypanosoma cruzi, the agent of Chagas’ disease. We discuss the results from an evolutionary perspective. A sample of 31 strains show 39 silent and five amino acid polymorphisms in DHFR-TS, and 35 silent and 11 amino acid polymorphisms in TR. No amino acid replacements occur in regions that are important for the enzymatic activity of these proteins, but some polymorphisms occur in sites previously assumed to be invariant. The sequences from both genes cluster in four major groups, a result that is not fully consistent with the current classification of T. cruzi in two major groups of strains. Most polymorphisms correspond to fixed differences among the four sequence groups. Two tests of neutrality show that there is no evidence of adaptive divergence or of selective events having shaped the distribution of polymorphisms and fixed differences in these genes in T. cruzi. However, one nearly significant reduction of variation in the TR sequences from one sequence group suggests a recent selective event at, or close to, that locus.

Toward rational design of species-specific inhibitors of Pneumocystis carinii thymidylate synthase and Toxoplasma gondii dihydrofolate reductase-thymidylate synthase.

Toward rational design of species-specific inhibitors of Pneumocystis carinii thymidylate synthase and Toxoplasma gondii dihydrofolate reductase-thymidylate synthase.