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In the protozoan parasite Leishmania, drug resistance can be a complex phenomenon. Several metabolic pathways and membrane transporters are implicated in the resistance phenotype. To monitor the expression of these genes, we generated custom DNA microarrays with PCR fragments corresponding to 44 genes involved with drug resistance. Transcript profiling of arsenite and antimony resistant mutants with these arrays pinpointed a number of genes overexpressed in mutants, including the ABC transporter PGPA, the glutathione biosynthesis genes gamma-glutamylcysteine synthetase (GSH1) and the glutathione synthetase (GSH2). Competitive hybridisations with total RNA derived from sensitive and methotrexate resistant cells revealed the overexpression of genes coding for dihydrofolate reductase (DHFR-TS), pteridine reductase (PTR1) and S-adenosylmethionine synthase (MAT2) and a down regulation of one gene of the folate transporter (FT) family. By labelling the DNA of sensitive and resistant parasites we could also detect several gene amplification events using DNA microarrays including the amplification of the S-adenosyl homocysteine hydrolase gene (SAHH). Alteration in gene expression detected by microarrays was validated by northern blot analysis, while Southern blots indicated that most genes overexpressed were also amplified, although other mechanisms were also present. The microarrays were useful in the study of resistant parasites to pinpoint several genes linked to drug resistance.

Folates are polyglutamylated in most organisms, and in cancer cells the polyglutamylation of folates and of the antifolate methotrexate (MTX) is an important determinant of MTX susceptibility. The folylpolyglutamate synthetase (FPGS) responsible for polyglutamylation of folates was recently characterized in the parasite Leishmania. We show here that MTX is polyglutamylated in Leishmania tarentolae and that triglutamates are the predominant form. The glutamate chain length of MTX increases significantly in Leishmania cells transfected with the FPGS gene and decreases in cells with one FPGS allele disrupted. Modulation in the expression of the FPGS gene also has a profound effect on MTX susceptibility and this effect was found to be dependent on the folate concentration of the medium. In the folate-rich medium SDM-79, overexpression of FPGS will confer MTX resistance while in M-199 medium, which has much less folates, FPGS transfectants are more sensitive to MTX. Cells with one allele of FPGS disrupted are more resistant to MTX in low folate medium. The modulation of FPGS expression affects both the short-term and long-term accumulation of folate and MTX, showing a marked decrease in accumulation in the FPGS haploid mutant. This differential accumulation was mediated by decreased influx of the drug into the cell. Finally, the analysis of MTX-resistant Leishmania mutants indicated that the presence of shorter glutamate chains on MTX is correlated with MTX resistance.

The protozoan parasite Leishmania is a folate auxotroph and thus depends on the uptake of folate from the environment to meet its folate requirement. We show here that Leishmania contains several putative pteridine transporter genes. Some of these genes are deleted in methotrexate-resistant Leishmania cells where there is no measurable uptake of methotrexate. Transport studies suggest that Leishmania has more than one active folate transporter, and one of these, named FT5, corresponds to a very high affinity folate transporter (K(m) 84 nm). The uptake of both folate and methotrexate was impaired in an FT5 null mutant at low substrate concentrations (50 nm), although transport properties at higher concentrations (1000 nm) were not statistically different between wild-type and the FT5 null mutant. Modulation of the expression of FT5 also changes the susceptibility of Leishmania cells to methotrexate. These results have permitted the characterization of a novel class of folate transporters and suggest that the parasite Leishmania has several gene products possibly transporting folates and related molecules under varying conditions.

The folate metabolic pathway has been exploited successfully for the development of antimicrobial and antineoplasic agents. Inhibitors of this pathway, however, are not useful against Leishmania and other trypanosomatids. Work on the mechanism of methotrexate resistance in Leishmania has dramatically increased our understanding of folate and pterin metabolism in this organism. The metabolic and cellular functions of the reduced form of folates and pterins are beginning to be established and this work has led to several unexpected findings. Moreover, the currently ongoing sequencing efforts on trypanosomatid genomes are suggesting the presence of several gene products that are likely to require folates and pterins. A number of the properties of folate and pterin metabolism are unique suggesting that these pathways are valid and worthwhile targets for drug development.

The protozoan parasite Leishmania relies on the up- take of folate and pterin from the environment to meet its nutritional requirements. We show here that a novel gene (folate transporter 1 (FT1)) deleted in a Leishmania infantum methotrexate-resistant mutant corresponds to the main folate transporter (Km, 410 nM). FT1 was established as the main folate transporter by both gene transfection and by targeted gene deletion. Modu- lation of the expression of FT1 by these manipulations altered the susceptibility of Leishmania cells to methotrexate. Folate transport was stage-regulated with higher activity in the logarithmic phase and less in the stationary phase. FT1 fused to green fluorescent protein led to the observation that FT1 was located in the plasma membrane in the logarithmic phase but was re- targeted to an intracellular organelle followed by a degradation of the protein in stationary phase. Leishmania has several folate transporters with different characteristics, and the growth stage-related activity of at least one transporter is regulated post-translationally. [ABSTRACT FROM AUTHOR]