10 CHARACTERIZATION OF A FOLATE-METHOTREXATE TRANSPORT-DEFICIENT CLONE OF LEISHMANIA DONOVANI

Abstract

Leishmania donovani is the causative agent of visceral leishmaniasis or kala azar. A clonal mutant strain of Leishmania donovani was derived in a single step from a wild type population by virtue of its resistance to 1 millimolar methotrexate. This cell line, MTXA5, was cross-resistant to aminopterin but equally sensitive to two other inhibitors of certain dihydrofolate reductases, pyrimethamine and trimethoprim. In contrast to the wild type parental cells, MTXA5 cells were incapable of taking up or transporting radiolabeled methotrexate and folate from the cell culture medium. Surprisingly, however, both wild type and mutant cells grew equally well in increasing concentrations of folate, although only the wild type parental cells were capable of growing in folate-deficient growth medium supplemented with either biopterin or neopterin. In order to attempt to analyze the transport system of Leishmania biochemically, an affinity labelling technique was developed using radiolabelled methotrexate and folate that had been ‘activated’ with 1-ethyl-3(3-dimethyl aminopropyl)carbodiimide. Using this protocol, a protein with MW = 46 kd was labelled in wild type cells. This 46 kd protein was associated with plasma membrane fractions. No band was observed in wild type cytosolic fractions. These data have biochemically and genetically identified a common folate/methotrexate carrier in this genus of parasites.

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