SEQUENCE VARIATION OF THE HYDROXYMETHYLDIHYDROPTERIN PYROPHOSPHOKINASE - DIHYDROPTEROATE SYNTHASE GENE IN-LINE SO THE HUMAN MALARIA PARASITE, PLASMODIUM-FALCIPARUM, WITH DIFFERING RESISTANCE TO SULFADOXINE

Dihydropteroate synthase (H(2)Pte synthase) is the target of the sulfu r-based antimalarial drugs, which are frequently used in synergistic c ombination with inhibitors of dihydrofolate reductase (H(2)folate redu ctase) to combat chloroquine-resistant malaria. We have isolated the H (2)Pte synthase coding sequence of the most pathogenic human parasite Plasmodium falciparum. It forms part of a longer coding sequence, loca ted on chromosome 8, that also specifies 6-hydroxymethyl-7,8-dihydropt erin pyrophosphokinase (CH2OH-H(2)pterinPP kinase) at its 5' proximal end. This domain is unusually large, with two long insertions relative to other CH2OH-H(2)pterinPP kinase molecules. To investigate a possib le genetic basis for clinical resistance to sulfa drugs, we sequenced the complete H(2)Pte synthase domains from eleven isolates of P. falci parum with diverse geographical origins and levels of sulfadoxine resi stance. Overall, point mutations in five positions were observed, affe cting: four codons. Parasite lines exhibiting high-level resistance we re found to carry either a double mutation, altering both Ser436 and A la613, or a single mutation affecting Ala581. The mutations at positio ns 436 and 581 have the same location relative to each of two degenera te repeated amino acid motifs that are conserved across all other know n H(2)Pte synthase molecules. The amino acid alteration at residue 613 is identically positioned relative to a different conserved motif. Th e fourth amino acid residue (437) affected by mutation, though adjacen t to the apparently crucial residue 436, shows no obvious correlation with resistance. Although these mutations have no exact counterparts i n any other organism, that at position 581 falls within a region of th ree amino acids where H(2)Pte synthase is modified in various ways in a number of sulfonamide-resistant pathogenic bacteria. Copy-number ana lysis indicated that there was no amplification of the H(2)Pte synthas e domain in resistant parasite lines of P. falciparum, compared to sen sitive lines.