Purine salvage pathways in the apicomplexan parasite Toxoplasma gondii.

We have exploited a variety of molecular genetic, biochemical, and genomic techniques to investigate the roles of purine salvage enzymes in the protozoan parasite Toxoplasma gondii. The ability to generate defined genetic knockouts and target transgenes to specific loci demonstrates that T. gondii uses two (and only two) pathways for purine salvage, defined by the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK). Both HXGPRT and AK are single-copy genes, and either one can be deleted, indicating that either one of these pathways is sufficient to meet parasite purine requirements. Fitness defects suggest both pathways are important for the parasite, however, and that the salvage of adenosine is more important than salvage of hypoxanthine and other purine nucleobases. HXGPRT and AK cannot be deleted simultaneously unless one of these enzymes is provided in trans, indicating that alternative routes of functionally significant purine salvage are lacking. Despite previous reports to the contrary, we found no evidence of adenine phosphoribosyltransferase (APRT) activity when parasites were propagated in APRT-deficient host cells, and no APRT ortholog is evident in the T. gondii genome. Expression of Leishmania donovani APRT in transgenic T. gondii parasites yielded low levels of activity but did not permit genetic deletion of both HXGPRT and AK. A detailed comparative genomic study of the purine salvage pathway in various apicomplexan species highlights important differences among these parasites.

Fitness effects of DHFR-TS mutations associated with pyrimethamine resistance in apicomplexan parasites.

Pyrimethamine resistance in the malaria parasite Plasmodium falciparum is characterized by specific point mutations in the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. We have previously explored the effect of these mutations by engineering homologous alleles of Toxoplasma gondii DHFR-TS, which can readily be expressed as recombinant protein for enzymatic studies, or as allelic replacements in transgenic parasites. In order to directly assess the costs of pyrimethamine-resistance in vivo, we have carried out competition studies between mixtures of T. gondii tachyzoites harbouring wild-type or mutant DHFR-TS alleles, both in tissue culture and in mice. Arg59+Asn108 mutants (using the P. falciparum numbering system) exhibit no significant fitness defects in vitro, but a fitness defect of 1.8% per generation in mice. Arg59+Ser223 mutants exhibit fitness defects of >2.8% per generation both in vitro and in vivo, which may explain why this highly pyrimethamine-resistant allele has not been observed in the field. It is important to note that long-term propagation of parasites in vitro or in vivo can produce adaptations affecting fitness by >3.7% per generation, necessitating careful attention to background in head-to-head competition studies. A sensitive PCR-based assay permits different growth rates to be assessed even in the absence of a drug resistance marker that can be scored by plaque assay.