Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoanParamecium tetraurelia

We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan,Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation-mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a ≈500 kb macronuclear chromosome and is transcribed as an mRNA of ≈1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of ≈12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, theP. tetraurelia gene is most similar to that fromToxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences.     

Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoanParamecium tetraurelia

We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan,Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation-mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a 500 kb macronuclear chromosome and is transcribed as an mRNA of 1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of 12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, theP. tetraurelia gene is most similar to that fromToxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences.     

Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated M _r of 124000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.     

Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene

Plasmodial bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a validated antimalarial drug target. In this study, expression of the putative dhfr-ts of Plasmodium ovale rescued the DHFR chemical knockout and a TS null bacterial strain, demonstrating its DHFR and TS catalytic functions. PoDHFR-TS was expressed in Escherichia coli BL21 (DE3) and affinity purified by Methotrexate Sepharose column. Biochemical and enzyme kinetics characterizations indicated that PoDHFR-TS is similar to other plasmodial enzymes, albeit with lower catalytic activity but better tolerance of acidic pH. Importantly, the PoDHFR from Thai isolate EU266602 remains sensitive to the antimalarials pyrimethamine and cycloguanil, in contrast to P. falciparum and P. vivax isolates where resistance to these drugs is widespread.     

Molecular cloning and analysis of a cDNA coding for the bifunctional dihydrofolate reductase-thymidylate synthase of Daucus carota

Molecular cloning of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota was achieved by immunoscreening of a cDNA library obtaining a 2 kbp clone which contains an open reading frame of 1528 bp. Comparison of the deduced amino acid sequence with those from other sources revealed the presence of motifs typical of DHFR and TS thus confirming the bifunctional nature of the carrot protein. As in other organisms, a higher degree of conservation was observed in the TS domain. Analysis of the dhfr-ts gene content in carrot revealed the presence of several copies per diploid genome.     

Combined gene deletion of dihydrofolate reductase-thymidylate synthase and pteridine reductase in Leishmania infantum

Our understanding of folate metabolism in Leishmania has greatly benefited from studies of resistance to the inhibitor methotrexate (MTX). Folates are reduced in Leishmania by the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS) and by pteridine reductase (PTR1). To further our understanding of folate metabolism in Leishmania, a Cos-seq genome-wide gain of function screen was performed against MTX and against the two thymidylate synthase (TS) inhibitors 5-fluorouracil and pemetrexed. The screen revealed DHFR-TS and PTR1 but also the nucleoside transporter NT1 and one hypothetical gene derived from chromosome 31. For MTX, the concentration of folate in the culture medium affected the enrichment pattern for genes retrieved by Cos-seq. We generated a L. infantum DHFR-TS null mutant that was thymidine auxotroph, a phenotype that could be rescued by the addition of thymidine or by transfection of the flavin dependent bacterial TS gene ThyX. In these DHFR-TS null mutants it was impossible to obtain a chromosomal null mutant of PTR1 except if DHFR-TS or PTR1 were provided episomally. The transfection of ThyX however did not allow the elimination of PTR1 in a DHFR-TS null mutant. Leishmania can survive without copies of either DHFR-TS or PTR1 but not without both. Provided that our results observed with the insect stage parasites are also replicated with intracellular parasites, it would suggest that antifolate therapy in Leishmania would only work if both DHFR-TS and PTR1 would be targeted simultaneously.     

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.     

Proliferation-dependent pattern of expression of a dihydrofolate reductase-thymidylate synthase gene from Daucus carota

The pattern of expression of a carrot dhfr-ts gene was evaluated in different plant organs, in somatic embryos, and in hypocotyl explants induced to dedifferentiate in vitro by the addition of the synthetic auxin 2,4 dichorophenoxyacetic acid. The promoter of this gene was also placed upstream of a uidA (GUS) reporter gene and, using biolistic and protoplasts transient expression assays, was shown to drive a particularly high level of expression in actively growing suspension cells. The results from these expression analyses combined with the presence of putative cell cycle-related cis-acting elements in the dhfr-ts promoter, strongly point to a cell division-dependent expression of this gene.     

New developments in malaria diagnostics: Monoclonal antibodies against Plasmodium dihydrofolate reductase-thymidylate synthase,heme detoxification protein and glutamate rich protein

Currently available rapid diagnostic tests (RDTs) for malaria show large variation in sensitivity and specificity, and there are concerns about their stability under field conditions. To improve current RDTs, monoclonal antibodies (mAbs) for novel malaria antigens have been developed and screened for their possible use in new diagnostic tests. Three antigens, glutamate rich protein (GLURP), dihydrofolate reductase-thymidylate synthase (DHFR-TS) and heme detoxification protein (HDP), were selected based on literature searches. Recombinant antigens were produced and used to immunize mice. Antibody-producing cell lines were subsequently selected and the resulting antibodies were screened for specificity against Plasmodium falciparum and Plasmodium vivax. The most optimal antibody couples were selected based on antibody affinity (expressed as dissociation constants, K_D) and detection limit of crude antigen extract from P. falciparum 3D7 culture. The highest affinity antibodies have K_D values of 0.10 nM ± 0.014 (D5) and 0.068 ± 0.015 nM (D6) for DHFR-TS mAbs, 0.10 ± 0.022 nM (H16) and 0.21 ± 0.022 nM (H18) for HDP mAbs and 0.11 ± 0.028 nM (G23) and 0.33 ± 0.093 nM (G22) for GLURP mAbs. The newly developed antibodies performed at least as well as commercially available histidine rich protein antibodies (K_D of 0.16 ± 0.13 nM for PTL3 and 1.0 ± 0.049 nM for C1–13), making them promising reagents for further test development.Key words: plasmodium, Plasmodium falciparum, malaria, diagnostics, rapid diagnostic test, monoclonal antibodies, glutamate rich protein, dihydrofolate reductase-thymidylate synthase, heme detoxification protein     

Characterization of Plasmodium knowlesi dihydrofolate reductase-thymidylate synthase and sensitivity to antifolates

Malaria caused by an infection of Plasmodium knowlesi can result in high parasitemia and deaths. Therefore, effective and prompt treatment is necessary to reduce morbidity and mortality. The study aims to characterize P. knowlesi dihydrofolate reductase-thymidylate synthase enzyme (PkDHFR-TS) and its sensitivity to antifolates. The putative Pkdhfr gene was PCR amplified from field isolates collected from the Southern Thailand. Molecular analysis showed 11 polymorphisms in the dhfr domain of the bifunctional dhfr-ts gene. Of these, 1 polymorphism was a non-synonymous substitution (R34L) that had previously been reported but not associated with antifolate resistance. The recombinant PkDHFR-TS enzyme was found to be sensitive to standard antifolates—pyrimethamine and cycloguanil—as well as P218, a registered candidate drug currently first in human clinical trial. Results suggest that antifolates class of compounds should be effective against P. knowlesi infection.     

Sequence and S1 nuclease mapping of the 5'' region of the dihydrofolate reductase-thymidylate synthase gene of Leishmania major.

The 5' structure of mRNA transcribed from the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene of the protozoan parasite Leishmania major has been characterized. S1 nuclease mapping identifies a heterogenous 5' structure which is not affected by growth phase or developmental stage. The DNA sequence of the 5' region of the DHFR-TS gene does not reveal homology with other trypanosomatid genes, eukaryotic consensus genetic elements, or the mammalian DHFR promoter element. This latter finding is especially significant as we show that the 5' region of the E. coli DHFR gene exhibits homology to the mammalian DHFR promoter element, despite their greater evolutionary distance.     

Purification and immunochemical characterization of a dihydrofolate reductase-thymidylate synthase enzyme complex from wild-carrot cells

An enzyme complex with dihydrofolate reductase (DHFR, E.C.1.5.1.3.) activity was purified to apparent homogenity from wild-carrot cells. The complex has a mol. wt of 286 kd and contains five polypeptide chains of 95, 70, 50, 45 and 26 kd. The DHFR enzyme activity and methotrexate-binding site are on the 45-kd subunit. Folate analogs (methotrexate, aminopterin and formylaminopterin) as well as SH-group inhibitors [p-hydroxymercuribenzoate, 5,5' -dithiobis(2-nitrobenzoic acid), or N-ethylmaleimide] inhibit DHFR. Thymidylate synthase (TS, E.C.2.1.1.45) activity co-purified with the enzyme complex through each of seven steps and co-eluted from gel filtration columns with the DHFR activity at the mol. wt of the enzyme complex. Further identification of TS within the complex was achieved using a Leishmania DHFR-TS antisera which specifically inhibited the carrot TS, although it immunoprecipitated both TS and DHFR. Polyclonal antisera, raised against and specific for the complex as judged by Ouchterlony double diffusion tests and Western blot analysis, inhibited and immunoprecipitated both DHFR and TS. The Leishmania antisera also identified the 70-kd polypeptide within the purified complex as TS in a Western blot experiment. The functions of the other three polypeptides have not yet been established.     

Chemical and genetic validation of dihydrofolate reductase-thymidylate synthase as a drug target in African trypanosomes

The phenotypes of single- (SKO) and double-knockout (DKO) lines of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of bloodstream Trypanosoma brucei were evaluated in vitro and in vivo. Growth of SKO in vitro is identical to wild-type (WT) cells, whereas DKO has an absolute requirement for thymidine. Removal of thymidine from the medium triggers growth arrest in S phase, associated with gross morphological changes, followed by cell death after 60 h. DKO is unable to infect mice, whereas the virulence of SKO is similar to WT. Normal growth and virulence could be restored by transfection of DKO with T. brucei DHFR-TS, but not with Escherichia coli TS. As pteridine reductase (PTR1) levels are unchanged in SKO and DKO cells, PTR1 is not able to compensate for loss of DHFR activity. Drugs such as raltitrexed or methotrexate with structural similarity to folic acid are up to 300-fold more potent inhibitors of WT cultured in a novel low-folate medium, unlike hydrophobic antifols such as trimetrexate or pyrimethamine. DKO trypanosomes show reduced sensitivity to these inhibitors ranging from twofold for trimetrexate to >10 000-fold for raltitrexed. These data demonstrate that DHFR-TS is essential for parasite survival and represents a promising target for drug discovery.