Changes in folate and pterin metabolism after disruption of the Leishmania H locus short chain dehydrogenase gene.
The short chain dehydrogenase gene ltdh, which is derived from the H locus of Leishmania, confers high level resistance to the dihydrofolate reductase inhibitor methotrexate via a novel mechanism. Resistance is correlated with LTDH overproduction. High level resistance of ltdh transfectants was observed even in poor media where reduced folates are essential for the synthesis of thymidylate precursors. The ltdh transfectants were also capable of growing for several passages, at slightly reduced rates, in unsupplemented folate-deficient medium (fdDME-L), unlike the wild-type cells that could grow only in fdDME-L if supplemented with various folates or pterins. An homozygous ltdh mutant was obtained by gene targeting. This mutant became hypersensitive to methotrexate, but unlike wild-type cells, methotrexate toxicity could not be circumvented by the addition of thymidine. Although the homozygous mutant was capable of growing in fdDME-L supplemented with folate derivatives, it lost its ability to thrive in fdDME-L supplemented with pterins. Our results support the model in which LTDH is a key enzyme responsible for the conversion of a pterin derivative into an essential cofactor. This essential cofactor can also be converted into reduced folates at sufficient levels for growth when LTDH is overproduced, rendering dihydrofolate reductase dispensable. The novelty and uniqueness of LTDH opens the possibility of developing parasite-specific inhibitors.
Papadopoulou
B
B
Centre de Recherche du Centre Hospitalier, Université Laval, Québec, Canada.
Roy
G
G
Mourad
W
W
Leblanc
E
E
Ouellette
M
M
D000818:Animals; D004171:Diploidy; D005492:Folic Acid / Q000378:metabolism*; D017125:Genes, Protozoan; D007891:Leishmania / Q000201:enzymology*; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D011622:Pterins / Q000378:metabolism*; D014162:Transfection
D011622:Pterins; D005492:Folic Acid; D010088:Oxidoreductases; C085592:pteridine reductase