Plasmodium falciparum: Induction, selection, and characterization of pyrimethamine-resistant mutants

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Abstract

We have selected eight pyrimethamine resistant mutants of a cloned, drug sensitive, Plasmodium falciparum malaria parasite, strain FCR3. The mutants exhibited resistance to between 10 and 200 times higher concentrations of drug than the wild type parasite. The mutants were selected from cultured parasites that were either unmutagenized or N-methyl N′-mtro-N-nitrosoguanidine mutagenized. One mutant was shown to contain a mutant dihydrofolate reductase enzyme in parasite extracts that exhibited (1) a five- to ninefold reduction in its binding of methotrexate, (2) an undetectable enzyme activity based on the spectrophotometric conversion of dihydrofolate to tetrahydrofolate, and (3) essentially normal amounts of the parasite's bifunctional thymidylate synthetase-dihydrofolate reductase enzyme. Other mutants exhibited both normal dihydrofolate reductase specific activity and normal enzyme sensitivity to the inhibitory activity of the drug.

Keywords

Plasmodium falciparum
Protozoa, parasitic
Malaria, human
Pyrimethamine, antimalarial
Drug resistance
mutagenesis
Mutants
Drug effect
Bifunctional enzyme protein

Abbreviations

N-methyl-N′-nitro-N-nitro-soguanidine
(MMNG)
Dihydrofolate reductase
DHFR
Thymidylate synthetase
TS
5-fluoro-2-deoxy[6-3H]Uridine-5′-monophosphate
([6-3H]FdUMP)
l-5,10-Methylene-tetrahydrofolate
(CH2FAH4)
[5-3H]deoxyuridine-5′-phosphate
([5-3H]dUMP)
[3H]methotrexate
(3HMTX)
Erythrocytes, red blood cells
RBC
Ethylenediaminetetraacetic acid
EDTA
(N-Tris[hydroxymethyl] methyl-2-aminomethane sulfonic acid)
TES
N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid
Hepes
Phosphate buffered saline
PBS

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Present address: Department of Biosciences, H.P. University, Shimla, H.P., India.