Transfection studies to explore essential folate metabolism and antifolate drug synergy in the human malaria parasite Plasmodium falciparum

Summary Folate metabolism in Plasmodium falciparum is the target of important antimalarial agents. The biosyn- thetic pathway converts GTP to polyglutamated deriv- atives of tetrahydrofolate (THF), essential cofactors for DNA synthesis. Tetrahydrofolate can also be acquired by salvage mechanisms. Using a transfec- tion system adapted to studying this pathway, we investigated modulation of dihydropteroate syn- thase (DHPS) activity on parasite phenotypes. Dihy- dropteroate synthase incorporates p- aminobenzoate (pABA) into dihydropteroate, the precursor of dihy- drofolate. We were unable to obtain viable parasites where the dhps gene had been truncated. However, parasites where the protein was full-length but mutated at two key residues and having < 10% of normal activity were viable in folate-supplemented medium. Metabolic labelling showed that these para- sites could still convert pABA to polyglutamated folates, albeit at a very low level, but they could not survive on pABA supplementation alone. This degree of disablement in DHPS also abolished the synergy of the antifolate combination pyrimethamine/sulfa- doxine. These data indicate that DHPS activity above a low but critical level is essential regardless of the availability of salvageable folate and formally prove the role of this enzyme in antifolate drug synergy and folate biosynthesis in vivo . However, we found no evidence of a significant role for DHPS in folate sal- vage. Moreover, when biosynthesis was compro- mised by the absence of a fully functional DHPS, the parasite was able to compensate by increasing flux through the salvage pathway.