ALLELIC EXCHANGE AT THE ENDOGENOUS GENOMIC LOCUS IN PLASMODIUM-FALCIPARUM PROVES THE ROLE OF DIHYDROPTEROATE SYNTHASE IN SULFADOXINE-RESISTANT MALARIA

We have exploited the recently developed ability to transfect the mala ria parasite Plasmodium falciparum to investigate the role of polymorp hisms in the enzyme dihydropteroate synthase (DHPS), identified in sul fadoxine-resistant field isolates. By using a truncated form of the dh ps gene, specific mutations were introduced into the endogenous gene b y allelic replacement such that they were under the control of the end ogenous promoter. Using this approach a series of mutant dhps alleles that mirror P. falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis sh ows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5-fold increase in sulfadoxine resistance and addition of further mut ations increases the level of resistance to 24-fold above that seen fo r the transfectant expressing the wild-type dhps allele. This indicate s that resistance to high levels of sulfadoxine in P. falciparum has a risen by an accumulation of mutations and that Gly437 is a key residue , consistent with its occurrence in most dhps alleles from resistant i solates. These studies provide proof that the mechanism of resistance to sulfadoxine in P. falciparum involves mutations in the dhps gene an d determines the relative contribution of these mutations to this phen otype.