Novel antifolate resistant mutations of Plasmodium falciparum dihydrofolate reductase selected in Escherichia coli.

A simple and effective system has been developed from which a number of Plasmodium falciparum dihydrofolate reductase (pfDHFR) mutants conferring resistance to antifolates were randomly generated and characterized. The system exploited error-prone PCR to generate random mutations in the pfDHFR. Using the synthetic gene encoding for wild-type and quadruple mutant (N51I+C59R+S108N+I164L) pfDHFRs as templates, mutants resistant to pyrimethamine (Pyr), m-Cl analogue of Pyr (SO3) and WR99210 were selected by bacterial complementation system in which the endogenous DHFR activity of bacterial host cells, but not of Plasmodium, is selectively inhibited by trimethoprim (Tmp).