Dihydrofolate Reductase-thymidylate Synthase Research Articles

Gene targeting in the rodent malaria parasite Plasmodium yoelii

It is anticipated that the sequencing of Plasmodium falciparum genome will soon be completed. Rodent models of malaria infection and stable transformation systems provide powerful means of using this information to study gene function in vivo. To date, gene targeting has only been developed for one rodent malaria species, Plasmodium berghei. Another rodent species, Plasmodium yoelii, however, is favored to study the mechanisms of protective immunity to the pre-erythrocytic stages of infection and vaccine development. In addition, it offers the opportunity to investigate unique aspects of pathogenesis of blood stage infection. Here, we report on the stable transfection and gene targeting of P. yoelii. Purified late blood stage schizonts were used as targets for electroporation with a plasmid that contains a pyrimethamine-resistant form of the P. berghei dihydrofolate reductase-thymidylate synthase (Pb dhfr-ts) fused to green fluorescent protein ( gfp) gene. After drug selection, fluorescent parasites contained intact, non-rearranged plasmids that remain stable under drug-pressure. In addition, we used another dhfr-ts/ gfp based plasmid to disrupt the P. yoelii trap (thrombospondin-related anonymous protein) locus by site-specific integration. The phenotype of P. yoelii TRAP knockout was identical to that previously reported for the P. berghei TRAP knockout. In the absence of TRAP, the erythrocytic cycle, gametocyte and oocyst development of the mutant parasites were indistinguishable from wild type (WT). Although the sporozoites appeared morphologically normal, they failed to glide and to invade the salivary glands of mosquitoes.

Complementation of Plasmodium berghei TRAP knockout parasites using human dihydrofolate reductase gene as a selectable marker

Previously we have used the Plasmodium dihydrofolate reductase thymidylate synthase ( DHFR-TS) selectable marker to generate Plasmodium berghei TRAP null mutant parasites. These TRAP null mutants do not glide and they showed a great reduction in their ability to infect mosquito salivary glands and the hepatocytes of the vertebrate host. Thus far, complementation of these knockout parasites was not possible due to the lack of additional selectable markers. Recently, a new selectable marker, based on the human dihydrofolate reductase (h DHFR) gene, has been developed which confers resistance to the antifolate drug WR99210. This drug has been found to be highly active against pyrimethamine-sensitive and -resistant strains of P. berghei. In this study, we have used the h DHFR gene as a second selectable marker for the complementation of P. berghei TRAP null mutant parasites. Restoration of the TRAP null mutant parasites to the wild-type phenotype was achieved in this study via autonomously replicating episomes bearing a wild-type copy of the TRAP gene. This is the first report of complementation of a mutant phenotype in malaria parasites.

Blasticidin resistance: a new independent marker for stable transfection of Leishmania

Protozoan parasites of the genus Leishmania are the etiologic agents of a spectrum of human diseases referred to as leishmaniasis. Leishmania alternates life stage between an intracellular amastigote form residing within the phagolysosome of vertebrate macrophage and an extracellular promastigote form living in the gut of sand flies. Molecular characterization of the genome of Leishmania has been greatly facilitated by the development of stable transfection methods, expression vectors and gene knockouts [1,2]. Stable transfection of Leishmania and trypanosomes requires the use of dominant selectable markers, and a number of independent markers have been used successfully. These include neomycin phosphotransferase (NEO) [3]; hygromycin phosphotransferase (HYG) [4], streptothricin acetyltransferase (SAT) [5], puromycin acetyltransferase (PAC) [6] and the bleomycin binding protein from Streptoalloteichus hindustanus (PHLEO) [6], which confer resistance to G418, hygromycin B, nourseothricin, puromycin and phleomycin respectively. In addition, overexpression of N-acetylglucosamine-1-phosphate transferase (NAGT) or dihydrofolate reductasethymidylate synthase (DHFR-TS) can confer resistance to tunicamycin and methotrexate respectively, allowing their use as selectable markers on episomal vectors [7,8]. Since Leishmania is diploid and lacks an experimentally manipulable sexual cycle, generation of null mutants requires the inactivation of two alleles [4]. Typically this is accomplished by two rounds of targeting with independent selectable markers, with a third marker needed to generate an ‘add-back’ construct restoring gene expression; this is an essential control for biological function [9]. While the use of loss of heterozygosity approaches can reduce the number of markers needed for null mutant construction [10], with the current repertoire of markers one can manipulate Abbre6iations: BSD, gene encoding blasticidin deaminase. * Corresponding author. Tel.: +1-314-7472630; fax: +1314-7472634. E-mail address: beverley@borcim.wustl.edu (S.M. Beverley)

The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genome

Genetic transformation of malaria parasites has been limited by the number of selectable markers available. For the rodent malaria parasite, Plasmodium berghei, only a single selection marker has been at hand, utilising the dihydrofolate reductase-thymidylate synthase gene from either P. berghei or Toxoplasma gondii to confer resistance to the anti-malarial drug pyrimethamine. Here we report the use of the human dihydrofolate reductase (h DHFR) gene as a new selectable marker, which confers resistance to the antifolate inhibitor WR99210 upon both pyrimethamine sensitive and resistant isolates of P. berghei. Transfection with circular constructs containing the h DHFR gene resulted in the generation of highly resistant parasites containing multiple copies of episomally-maintained plasmids. These parasites showed around a 1000-fold increase in resistance to WR99210 compared to the parental parasites. We were also able to generate and select transgenic parasites harbouring only a single copy of h DHFR targeted into their genome, despite the fact that these parasites showed only a fivefold increase in resistance to WR99210 compared to the parental parasites. Importantly, and for the first time with malaria parasites, the h DHFR gene could be used in conjunction with the existing pyrimethamine selectable markers. This was demonstrated by reintroducing the circumsporozoite ( CS) gene into transgenic CS-knockout mutant parasites that contained the P. berghei DHFR-TS selectable marker. The development of h DHFR as a second selectable marker will greatly expand the use of transformation technology in Plasmodium, enabling more extensive genetic manipulation and thus facilitating more comprehensive studies on the biology of the malaria parasite.

Molecular epidemiology of malaria in Yaounde, Cameroon. VI. Sequence variations in the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene and in vitro resistance to pyrimethamine and cycloguanil.

Pyrimethamine and cycloguanil, the major human metabolite of proguanil, are inhibitors of dihydrofolate reductase that play a key role in the treatment and prevention of chloroquine-resistant Plasmodium falciparum infections in sub-Saharan Africa. Resistance to these antifolate drugs has emerged in some areas of Africa. Earlier molecular studies have demonstrated that point mutations at key positions of the dihydrofolate reductase-thymidylate synthase gene are strongly associated with antifolate resistance. However, whether the same or distinct mutations are involved in the development of resistance to both pyrimethamine and cycloguanil has not been well established in naturally occurring P. falciparum isolates. In this study, the in vitro responses to both antifolate drugs were measured in 42 Cameroonian isolates and compared with the complete sequence of the dihydrofolate reductase domain of the gene (from 34 of 42 isolates) to analyze the genotype that may distinguish between pyrimethamine and cycloguanil resistance. The wild-type profile (n = 11 isolates) was associated with low 50% inhibitory concentrations (IC50s) ranging from 0.32 to 21.4 nanamole for pyrimethamine and 0.60-6.40 nM for cycloguanil. Mutant isolates had at least one amino acid substitution, Asn-108. Only three mutant codons were observed among the antifolate-resistant isolates: Ile-51, Arg-59, and Asn-108. The increasing number of point mutations was associated with an increasing level of pyrimethamine IC50 and, to a much lesser extent, cycloguanil IC50. These results support a partial cross-resistance between pyrimethamine and cycloguanil that is based on similar amino acid substitutions in dihydrofolate reductase and suggest that two or three mutations, including at least Asn-108, may be necessary for cycloguanil resistance, whereas a single Asn-108 mutation is sufficient for pyrimethamine resistance.

Essential Protein-Protein Interactions between Plasmodium falciparum Thymidylate Synthase and Dihydrofolate Reductase Domains

In Plasmodium falciparum, dihydrofolate reductase and thymidylate synthase activities are conferred by a single 70-kDa bifunctional polypeptide (DHFR-TS, dihydrofolate reductase-thymidylate synthase) which assembles into a functional 140-kDa homodimer. In mammals, the two enzymes are smaller distinct molecules encoded on different genes. A 27-kDa amino domain of malarial DHFR-TS is sufficient to provide DHFR activity, but the structural requirements for TS function have not been established. Although the 3'-end of DHFR-TS has high homology to TS sequences from other species, expression of this protein fragment failed to yield active TS enzyme, and it failed to complement TS(-) Escherichia coli. Unexpectedly, even partial 5'-deletion of full-length DHFR-TS gene abolished TS function on the 3'-end. Thus, it was hypothesized that the amino end of the bifunctional parasite protein plays an important role in TS function. When the 27-kDa amino domain (DHFR) was provided in trans, a previously inactive 40-kDa carboxyl-domain from malarial DHFR-TS regained its TS function. Physical characterization of the "split enzymes" revealed that the 27- and the 40-kDa fragments of DHFR-TS had reassembled into a 140-kDa hybrid complex. Thus, in malarial DHFR-TS, there are physical interactions between the DHFR domain and the TS domain, and these interactions are necessary to obtain a catalytically active TS. Interference with these essential protein-protein interactions could lead to new selective strategies to treat malaria resistant to traditional DHFR-TS inhibitors.

Molecular epidemiology of malaria in Yaounde, Cameroon IV. Evolution of pyrimethamine resistance between 1994 and 1998.

Pyrimethamine, in combination with sulfadoxine, is currently one of the major alternative drugs used for the treatment of chloroquine-resistant Plasmodium falciparum malaria infections in Africa. The mechanism of pyrimethamine resistance has been strongly associated with a single, key point mutation in the dihydrofolate reductase-thymidylate synthase gene, resulting in the substitution of the wild-type allele Ser-108 by either Asn-108 or Thr-108. The pyrimethamine-resistant phenotype and/or genotype were determined in 273 Cameroonian clinical isolates obtained in Yaounde by in vitro assays and polymerase chain reaction-restriction fragment length polymorphism over a 5-year period. The in vitro assays showed that 42% (18 of 43) and 63% (69 of 110) of the isolates obtained in 1994-1995 and 1997-1998, respectively, were resistant to pyrimethamine (50% inhibitory concentration [IC50] > 100 nM). The polymerase chain reaction showed that 43% (55 of 127) and 59% (50 of 85) of the isolates in 1994-1995 and 1997-1998, respectively, had the mutant Asn-108 allele. The pyrimethamine-resistant genotype (Asn-108) corresponded with the pyrimethamine-resistant phenotype (IC50 > or = 100 nM) in a large majority (> 95%) of the isolates. The results of our study suggest an increasing prevalence of pyrimethamine resistance in Yaounde. Our study further suggests that pyrimethamine resistance can be monitored by a technique that can be adopted by malaria research centers in Africa.

Role of the locus and of the resistance gene on gene amplification frequency in methotrexate resistant Leishmania tarentolae.

The protozoan parasite Leishmania resists the antifolate methotrexate (MTX) by amplifying the R locus dihydrofolate reductase-thymidylate synthase ( dhfr-ts ) gene, the H locus ptr1 pterin reductase gene, and finally by mutation in a common folate/MTX transporter. Amplification of dhfr-ts has never been observed in Leishmania tarentolae MTX resistant mutants while ptr1 amplification is common. We have selected a L.tarentolae ptr1 null mutant for MTX resistance and observed dhfr-ts amplification in this mutant demonstrating that once a preferred resistance mechanism is unavailable, a second one will take over. By introducing the ptr1 gene at the R locus and the dhfr-ts gene at the H locus by gene targeting, we investigated the role of the resistance gene and the locus on the rate of gene amplification. Transfection studies indicated that ptr1 gave higher levels of MTX resistance than dhfr-ts. Consistent with this, when ptr1 was present as part of either the H locus or the R locus it was invariably amplified, while dhfr-ts was only amplified when ptr1 was inactivated. When dhfr-ts was present in a ptr1 null background on both the H locus and the R locus, amplification from the H locus was more frequent suggesting that both the gene and the locus are determining the frequency of gene amplification in Leishmania.

Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae.

Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX.

Stable transformation of Toxoplasma gondii based on a pyrimethamine resistant trifunctional dihydrofolate reductase-cytosine deaminase-thymidylate synthase gene that confers sensitivity to 5-fluorocytosine

To improve genetic models available for the analysis of apicomplexan protozoan parasites, bacterial sequences encoding the 427 amino acid cytosine deaminase (CD) gene were fused, in-frame, to an engineered linker domain of the high level pyrimethamine resistant form of the parasite bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. Toxoplasma gondii was transformed with the plasmid containing the fused pyrimethamine resistant dihydrofolate reductase-cytosine deaminase-thymidylate synthase (DHFRm2m3-CD-TS) gene and parasites were selected in a high level of pyrimethamine. Transfected parasites that acquired resistance to pyrimethamine were cloned and evaluated for expression of the CD genetic marker. CD transgenic parasites acquired a high sensitivity to 5-fluorocytosine due to the intraparasitic conversion of this non-toxic prodrug to the cytotoxic compound 5-fluorouracil. Exogenously supplied cytosine or uracil rescued the growth of CD transgenic T. gondii parasites that were cultured in the presence of cytotoxic concentrations of 5-fluorouracil or 5-fluorocytosine. Bacterial CD fused to the pyrimethamine resistant DHFR-TS marker provides a novel genetic tool for new positive and negative genetic selection strategies in several protozoan parasites. An advantage of the CD genetic marker is that it is derived from a bacterial gene and can therefore be used in nearly any parasite genetic background for negative selection. This novel system should facilitate new approaches for the development of improved model genetic systems for the biological investigation of apicomplexan parasites.

Plasmodium vivax:Rapid Detection by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism of the Key Mutation in Dihydrofolate Reductase-Thymidylate Synthase Gene Associated with Pyrimethamine Resistance

Tahar, R., Eldin de Pecoulas, P., Mazabraud, A., and Basco, L. K. 1998.Plasmodium vivax:Rapid detection by polymerase chain reaction and restriction fragment length polymorphism of the key mutation in dihydrofolate reductase-thymidylate synthase gene associated with pyrimethamine resistance.Experimental Parasitology89,343–346.

Sequence variations in the Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene and their relationship with pyrimethamine resistance

The gene encoding dihydrofolate reductase-thymidylate synthase of the human malaria parasite, Plasmodium vivax, was isolated by polymerase chain reaction from genomic DNA and cloned. The sequences of the dihydrofolate reductase domain of 30 clinical isolates originating from various geographic areas were compared. Interstrain analysis revealed several genotypic variations, including short tandem repeat arrays which produced length polymorphism between different parasite isolates and point mutations in the putative dihydrofolate reductase active site cavity corresponding to those associated with pyrimethamine resistance in P. falciparum and rodent malaria parasites. Amino acid substitutions Ser→Asn-117 and Ser→Arg-58 were associated with decreased level of in vitro pyrimethamine sensitivity. These findings suggest that the P. vivax dihydrofolate reductase domain is characterized by polymorphism that has not been observed in P. falciparum and may explain the resistance of some P. vivax isolates to pyrimethamine. Nucleotide sequence data reported in this paper are available in the EMBL, GenBenk™ and DDJB databases under the accession numbers X98123 (isolate ARI/Pakistan), AJ003050 (isolate CNC/Thailand), AJ003051 (isolate COU/unknown geographic origin), AJ003052 (isolate DUF/French Guiana), AJ003053 (isolate GRO/Madagascar), AJ003054 (isolate HRT/Comoros Islands), AJ003071 (isolate LFT/Cambodia), AJ003072 (isolate LGF/India), AJ003073 (isolate MAN/Comoros Islands), AJ003074 (isolate MAT/Surinam), AJ003075 (isolate PHI/Djibouti), AJ003076 (isolate PIT/Madagascar), AJ003077 (isolate YTZ/Indonesia), AJ222630 (isolate Burma-1), AJ222631 (isolate Burma-151), AJ222632 (isolate Burma-5), AJ222633 (isolate Burma-6), AJ222634 (isolate Burma-98).

Analysis of the Plasmodium vivax dihydrofolate reductase–thymidylate synthase gene sequence

A basis for the intrinsic resistance of some Plasmodium vivax isolates to pyrimethamine is suggested following the isolation of the bifunctional gene encoding dihydrofolate reductase-thymidylate synthase (DHFR-TS) of this human malaria parasite. Malaria parasites are dependent on this enzyme for folate biosynthesis. Specific inhibition of the DHFR domain of the enzyme by pyrimethamine blocks pyrimidine biosynthesis, leading to an inhibition of DNA replication. The gene was isolated by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotides designed to hybridize on the highly conserved regions of the sequence. The nucleotide sequence was completed by screening P. vivax genomic bank. Sequence analysis revealed an open reading frame (ORF) of 1872 nucleotides encoding a deduced protein of 623 amino acids (aa). Alignment with other malarial DHFR-TS genes showed that a 237-residue DHFR domain and a 286-residue TS domain were separated by a 100-aa linker region. Comparison with other malarial species showed low and essentially no isology in the DHFR and junctional domains, respectively, whereas an extensive isology was observed in the TS domain. The characteristic features of the P. vivax DHFR-TS gene sequence include an insertion of a short repetitive tandem array within the DHFR domain that is absent in another human malaria parasite, P. falciparum, and a GC-biased aa composition, giving rise to highly GC-rich DHFR (50.8%), junctional (58.7%), and TS (40.5%) domains, as compared with other malaria parasites. Analysis of the 5' noncoding region revealed the presence of a putative TATA box at 116 nucleotides upstream of the ATG start codon as well as a putative GC box at -636. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed two residue changes: Ser Arg-58 and Ser Asn-117. These aa residues correspond to codons 59 and 108 in the P. falciparum DHFR active site in which similar aa substitutions (Cys Arg-59 and Ser Asn-108) are associated with pyrimethamine resistance. These findings may explain the intrinsic resistance of some P. vivax isolates to pyrimethamine.

Molecular epidemiology of malaria in Yaounde, Cameroon I. Analysis of point mutations in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum.

Resistance to antifolate antimalarial drugs (cycloguanil, a biologically active metabolite of proguanil, and pyrimethamine) is associated with a Ser- to Asn-108 point mutation in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. The frequency of this mutation was studied in 127 clinical isolates obtained in Yaounde, Cameroon using a simple and rapid molecular technique based on the polymerase chain reaction and restriction fragment length polymorphism. Of the 127 isolates, pure wild-type Ser-108 codon, pure mutant-type Asn-108 codon, and mixed codons were observed in 66, 55, and six parasites, respectively. The proportion of antifolate-resistant, pure mutant-type codon, with respect to pure wild-type or mixed alleles, was 43% (55 of 127). The results of the molecular assay were compared with those of semimicro isotopic in vitro assay in 34 isolates. All 17 pure Ser-108 isolates and two isolates with mixed alleles were sensitive to both pyrimethamine (50% inhibitory concentration [IC50] < 100 nM) and cycloguanil (IC50 < 50 nM). Fourteen of 15 isolates with the mutant-type Asn-108 codon were resistant to pyrimethamine and cycloguanil. One isolate with Asn-108 showed a slightly elevated pyrimethamine IC50 (78 nM), which was within the sensitive range. This study provides further evidence that antifolate-resistant P. falciparum isolates are already present in Yaounde, Cameroon.

Plasmodium falciparum:Effect of Chemical Structure on Efficacy and Specificity of Antisense Oligonucleotides against Malariain Vitro

Antisense oligodeoxynucleotides (AS ODNs) have shown promise both as potential anti-malarial chemotherapeutic agents and as a means for identifying genes critical for parasite survival. Because conventional ODNs containing phosphodiester (PO) groups are subject to rapid nuclease degradation, ODNs with phosphorothioate (PS) groups are commonly used. However, at high concentration, these lose target specificity, and in some animal models, they become toxic. We compared a variety of chemical modifications (PO, PS, PO-PS hybrids, 2′-O-methyl-2′-deoxy chimeras) and structural modifications (sequence alterations favoring self-stabilizing loop formation) for their ability to inhibitPlasmodium falciparummalaria culturedin vitro.All modifications were done using an AS ODN sequence targeted against dihydrofolate reductase thymidylate synthase (DHFR). Inhibition by PO-PS hybrids containing as few as three PS groups at the 3′- and 5′-ends did not differ significantly from that obtained using compounds containing all-PS groups. Similarly, inhibition by PS chimeric compounds containing 2′-O-methyl modifications did not differ significantly from that of conventional PS compounds. In contrast, while inhibition by PO-PS hybrid chimeras did not differ significantly from that of all-PS compounds at low concentrations, at 1 μMthey inhibited parasite growth 25% less (P< 0.001) than all-compounds or PS 2′-O-methyl-2′-deoxy chimeras. Extension of the nucleotide sequence to increase stem—loop formation yielded two compounds which inhibited parasite growth about 20% more than unmodified compounds, though this difference was not significant. Furthermore, most of this increase appears to correlate with the greater number of PS groups associated with the increased ODN length. We conclude that limiting the number of PS groups and inclusion of PO 2′-O-methyl groups may yield compounds with high antisense activity but low non-sequence-dependent effects. Such compounds are currently being testedin vivo.

Dihydrofolate reductase and antifolate resistance in malaria

The dihydrofolate reductase (DHFR, EC 1.5.1.3) domain of Plasmodium falciparum bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is an attractive target of two important antifolate antimalarials: pyrimethamine (Pyr) and cycloguanil (Cyc). Over recent years, knowledge of malarial DHFR and mechanism(s) of antifolate resistance have increased substantially. These observations have provided an important framework for better understanding the molecular basis of antifolate resistance in malaria. This article provides a brief review and update on molecular aspects relevant to antifolate resistance in malaria.

Stable transgene expression in Plasmodium falciparum

Plasmid vectors designed to express transgenes and a selectable marker in Plasmodium falciparum were constructed. These consist of a selectable gene cassette comprising the Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene mutated to confer pyrimethamine resistance flanked by either Plasmodium chabaudi DHFR-TS or P. falciparum calmodulin promoter sequences and the P. falciparum histidine rich protein 2 3′ region. Also, each vector includes a different expression cassette driven by various Plasmodium transcriptional control sequences. Initially, the chloramphenicol acetyl transferase (CAT) reporter gene was cloned into the expression site of two vectors, pCC6-CAT and pCC13-CAT, which were identical except for the orientation of the expression cassette with respect to the selectable gene cassette. Approximately 8-fold more CAT activity was detected when the direction of transcription of the expression cassettes was in a head to head, rather than a tail to head, orientation. Importantly, it was found that stable transfection could only be achieved when the gene cassettes were in the head to head direction suggesting that this orientation also has an effect on the level of expression of the selectable marker. All other plasmids were designed with the cassettes in a head to head orientation. With the exception of pCC6-CAT and a second vector pHC4-CAT, stable transfectants were obtained with each vector in which the CAT gene had been inserted into the expression cassette. This is the first time vectors for the stable expression in Plasmodium parasites of transgenes other than a selectable marker have been described.

Biochemical and Genetic Tests for Inhibitors ofLeishmaniaPteridine Pathways

The study of antifolate-resistant mutants of the protozoan parasiteLeishmaniahas provided useful information about genetic processes such as gene amplification and mutation and knowledge of the unique features of the pteridine metabolic pathway in this primitive eukaryote. The novel bifunctional dihydrofolate reductase–thymidylate synthase (DHFR-TS) is an essential enzyme, yet most DHFR-TS inhibitors show little promise as potential drugs.Leishmaniapossess a novel alternative pteridine reductase (PTR1) which is relatively insensitive to methotrexate. We have proposed that the ability of PTR1 to serve as a metabolic bypass and thus modulate drug inhibition of DHFR-TS activity may be responsible for the poor efficacy of many antifolates. In this work, we have sought inhibitors ofL. majorPTR1 from a collection of 74 compounds. The most potent inhibitors were also tested againstL. majorDHFR-TS and human DHFR and several compounds showing good activity for PTR1 alone, or for all three reductases, were identified. The activity of these compounds was tested against wild-type promastigotes, and those which were potent inhibitors of both PTR1 and DHFR-TS (but not those active against only PTR1) showed good potencies. Growth inhibition tests ofL. majormutants, lacking PTR1 or DHFR-TS (ptr1−anddhfr-ts−knockouts) or overexpressing PTR1, were used as a “genetic screen” to assess whether these two pteridine reductases were targetsin vivo.Remarkably, only one compound showed a methotrexate-like pattern of inhibition. Six compounds showed good inhibition ofLeishmaniagrowth regardless of PTR1 or DHFR-TS levels. These findings suggest thatLeishmaniacells contain multiple targets for a diverse set of antifolates, with one or more significant targets in addition to DHFR-TS and PTR1. This emphasizes the necessity of combined biochemical and genetic screens in efforts to rationally design chemotherapeutic strategies inLeishmania.

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil.

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR-thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.

New approaches to Leishmania chemotherapy: pteridine reductase 1 (PTR1) as a target and modulator of antifolate sensitivity

Leishmania and other trypanosomatid protozoa require reduced pteridines (pterins and folates) for growth, suggesting that inhibition of these pathways could be targeted for effective chemotherapy. This goal has not yet been realized, indicating that pteridine metabolism may be unusual in this lower eukaryote. We have investigated this possibility using both wild type and laboratory-selected antifolate-resistant strains, and with defined genetic knockouts of several pteridine metabolic genes. In Leishmania, resistance to the antifolate methotrexate is mediated through several mechanisms singly or in combination, including alterations in transport leading to reduced drug influx, overproduction (R-region amplification) or point mutation of dihydrofolate reductase-thymidylate synthase (DHFR-TS), and amplification of a novel pteridine reductase (PTR1, encoded by the H-region). All of the proteins involved are potential targets for antifolate chemotherapy. Notably, parasites in which the gene encoding dihydrofolate reductase (DHFR) has been deleted (dhfr-ts- knockouts) do not survive in animal models, validating this enzyme as a target for effective chemotherapy. However, the properties of pteridine reductase 1 (PTR1) suggest a reason why antifolate chemotherapy has so far not been successful in trypanosomatids. PTR1, by its ability to provide reduced pterins and folates, has the potential to act as a by-pass and/or modulator of DHFR inhibition under physiological conditions. Moreover, PTR1 is less sensitive to many antifolates targeted primarily against DHFR. These findings suggest that successful antifolate chemotherapy in Leishmania will have to target simultaneously both DHFR and PTR1.