ISOLATION AND MOLECULAR CHARACTERIZATION OF THE BIFUNCTIONAL HYDROXYMETHYLDIHYDROPTERIN PYROPHOSPHOKINASE-DIHYDROPTEROATE SYNTHASE GENE FROM TOXOPLASMA-GONDII

Toxoplasma gondii is an important cause of AIDS-related opportunistic infection, manifest as toxoplasmic encephalitis. The clinical treatmen t of choice is the synergistic combination of antifolate agents, pyrim ethamine and sulphadiazine, of which the latter targets the parasite's dihydropteroate synthase (DHPS) activity. Here, we describe the isola tion of the gene encoding this activity in T. gondii. The nucleotide s equence contains an open reading frame interrupted by five introns, wh ich encodes a protein of 664 amino acids with an M-r of 72991. Sequenc e analysis revealed that, in addition to DHPS, the predicted protein c ontains a second enzyme function, hydroxymethyldihydropterin pyrophosp hokinase (PPPK). This enzyme immediately precedes DHPS in the folate b iosynthetic pathway. The bifunctional arrangement of the T. gondii ppp k-dhps gene is the same as that observed in the related protozoan para site, Plasmodium falciparum, and confirms previous biochemical data th at these activities were inseparable. Recently, specific mutations wit hin conserved motifs of the DHPS gene of P. falciparum have been ident ified which give rise to sulphonamide drug resistance. Analysis of sev en clinical isolates of T. gondii did not reveal anp similar mutations in this limited sample of organisms that had been subjected to drug p ressure. (C) 1997 Elsevier Science B.V.