title: Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae.
abstract: 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.
authors.affiliation: Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada G1V 4G2.
authors.forename: C
authors.initials: C
authors.lastname: Kündig
authors.forename: A
authors.initials: A
authors.lastname: Haimeur
authors.forename: D
authors.initials: D
authors.lastname: Légaré
authors.forename: B
authors.initials: B
authors.lastname: Papadopoulou
authors.forename: M
authors.initials: M
authors.lastname: Ouellette
mesh_terms: D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism; D017125:Genes, Protozoan; D007891:Leishmania / Q000187:drug effects / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism / Q000494:pharmacology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D011621:Pteridines / Q000378:metabolism*; D011956:Receptors, Cell Surface
chemical_list: D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011621:Pteridines; D011956:Receptors, Cell Surface; D005492:Folic Acid; D008727:Methotrexate