Treatment of human fibroblasts with human recombinant y interferon blocked the growth of Toxoplasma gondii, an obligate intracellular protozoan parasite. Growth Interferons are currently assigned to three classes on the basis of the cells that produce them (reviewed in ref. 1); a and X interferons (IFN-a and IFN-f) are produced by leukocytes and fibroblasts, respectively, in response to viral infection, whereas y interferon (IFN-y) is produced by T-lymphocytes when stimulated by mitogens or by antigens to which they had previously been sensitized. Each of these interferons was originally identified through its inhibition of viral growth. In addition to their well-documented antiviral activities, crude interferons also occasionally have been observed to suppress the growth of other nonviral intracellular infectious agents (reviewed in ref.2). In studying the antiviral-like action of interferon against Toxoplasma gondii, an obligate intracellular protozoan parasite, it is important that macrophages not be used as the host cells. Activated macrophages are known to kill intracellular T. gondii (3), and macrophageactivating factor might be present in crude interferon preparations. Indeed, macrophage-activating factor and IFN-y may well be the same substance. Several laboratories have reported that interferon blocked the growth of T. gondii in cultured fibroblast or epithelial cells (4-6). Shirahata and Shimizu (6) have presented evidence that IFN-y is the active substance in their studies of the inhibition of T. gondii. Experiments done in this laboratory showed that human recombinant IFN-a and IFN-,B had no effect on T. gondii. However, treatment of human fibroblasts with 8 to 16 units of human recombinant IFN-y per ml blocked the growth of T. gondii. This IFN-y inhibited vesicular stomatitis virus at 4 units/ml with the same line of human fibroblast as host cells (unpublished data).The biochemical basis of the antiviral state induced by the various interferons is now reasonably well understood (reviewed in ref. 7). In reviewing the action of interferon on nonviral agents, Vildek and Jahiel (2) suggested 13 yr ago that inhibitory mechanisms different from those that block viral growth were likely to be involved. This report presents evidence for an antitoxoplasma mechanism induced by IFNy that is totally different from the well-studied antiviral state induced by interferons. MATERIALS AND METHODSHost Cells and Parasites. Human fibroblasts were grown in Eagle's (8) minimal essential medium supplemented with antibiotics and 10% fetal bovine serum. The serum concentration was reduced to 3% for infection or for treatment with interferon. Medium without tryptophan was prepared from a kit supplied by GIBCO and supplemented with dialyzed fetal bovine serum. Cloned T. gondii of the RH strain were maintained by serial passage in human fibroblast cultures and assayed by plaque formation, with the results reported as plaque-forming units as described (9).Interferons and Their Titration. Human recombinant IFNa, IFN-p,...

Toxoplasma gondii is a highly successful protozoan parasite in the phylum Apicomplexa, which contains numerous animal and human pathogens. T.gondii is amenable to cellular, biochemical, molecular and genetic studies, making it a model for the biology of this important group of parasites. To facilitate forward genetic analysis, we have developed a high-resolution genetic linkage map for T.gondii. The genetic map was used to assemble the scaffolds from a 10X shotgun whole genome sequence, thus defining 14 chromosomes with markers spaced at ∼300 kb intervals across the genome. Fourteen chromosomes were identified comprising a total genetic size of ∼592 cM and an average map unit of ∼104 kb/cM. Analysis of the genetic parameters in T.gondii revealed a high frequency of closely adjacent, apparent double crossover events that may represent gene conversions. In addition, we detected large regions of genetic homogeneity among the archetypal clonal lineages, reflecting the relatively few genetic outbreeding events that have occurred since their recent origin. Despite these unusual features, linkage analysis proved to be effective in mapping the loci determining several drug resistances. The resulting genome map provides a framework for analysis of complex traits such as virulence and transmission, and for comparative population genetic studies.

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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...