Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii

Abstract

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.Like all parasitic protozoa, the obligate intracellular parasite Toxoplasma gondii lacks the ability to synthesize the purine ring de novo, and thus relies entirely on the salvage of purines from the host cell to meet its nutritional needs (1-3). This requirement, coupled with the shortcomings of conventional therapies for treating congenital toxoplasmosis and opportunistic infections associated with AIDS and other immunosuppressive conditions (4 -8), makes purine salvage an attractive target for chemotherapy.The purine metabolism of T. gondii has previously been examined biochemically, resulting in the identification of various activities capable of assimilating nucleosides and nucleobases from the host cell into the purine nucleotide pools of the parasite (2, 3). (See "Discussion" for a model of the purine salvage pathway in Toxoplasma and other apicomplexan parasites.) Reported salvage activities include the phosphoribosylation of adenine, guanine, hypoxanthine, and xanthine, and the phosphorylation of adenosine. The latter seems to contribute most significantly to parasite purine economy, as adenosine is incorporated into nucleotide pools at a considerably higher rate than any purine nucleobase (2, 3).Most of the reported salvage activities can be accounted for by two enzymes: hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) 1 and adenosine kinase (AK). The genes for both have been cloned and expressed in bacterial systems, and the purified proteins have be...

Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

“…No additional candidate orthologues were found. These results have been independently confirmed by other groups (13,15). In addition, no potential homologues were found for the Bacillus subtilis proteins expressed by the mtnK, mtnY, mtnW, mtnX, mtnZ, and mtnV genes that are proposed to constitute the methionine salvage pathway (16).…”

“…We were also unable to find orthologues of other potential purine salvage enzymes that have been reported in other organisms but not in the Plasmodium species. Other groups have independently come to similar conclusions (13,15).…”

“…The expression pattern suggests that PfADA, PfPNP, and hypoxanthineguanine-xanthine phosphoribosyltransferase form the major path for purine salvage in P. falciparum. Other groups have independently drawn similar conclusions (13,15). Purine salvage in malaria is unlike that in most other protozoa, including other Apicomplexa such as T. gondii (2, 3) and C. parvum (13) that are rich in AK and rely on adenosine salvage to AMP for a major purine source.…”

Host cell manipulation by the human pathogen Toxoplasma gondii

“…T. gondii is an auxotroph for tryptophan [19][20][21], arginine [22,23], polyamines [24], purines [25,26], cholesterol [27,28], iron [23,29], and other essential nutrients [30]. Although parasites surrounded by the PVM are protected against the acidification from the fusion of endocytic vesicle, this non-fusion state deprives parasites of an abundant source of nutrients from the host's endocytic and exocytic system.…”

Synthesis, biological evaluation and molecular modeling studies of N6-benzyladenosine analogues as potential anti-toxoplasma agents

“…First, T. gondii is a purine auxotroph and depends on the salvage pathways for its purine requirements, and the salvage of adenosine is the major source of purines in T. gondii [14,15]. Specifically, the activity of adenosine kinase (EC.2.7.1.20), the enzyme responsible for the salvage of adenosine in this parasite, is 10-fold higher than those of other enzymes in the purine salvage pathways, and hence contributes more significantly to the salvage of purines in T. gondii than any other enzyme of the purine salvage pathways [12][13][14][15][16]. Secondly, structure-activity relationships [17,18], comparative enzymatic [18][19][20], metabolic [19][20][21], molecular [22], and X-ray structural studies [23][24][25] have demonstrated that there are sufficient differences between the active sites and substrate specificities of human and T. gondii adenosine kinases that can be exploited to design specific "subversive substrates" for the T. gondii enzyme [12,13].…”