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. 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 (1Perotto J. Keister D.B. Gelderman A.H. J. Protozool. 1971; 18: 470-473Google Scholar, 2Schwartzman J.D. Pfefferkorn E.R. Exp. Parasitol. 1982; 53: 77-86Google Scholar, 3Krug E.C. Marr J.J. Berens R.L. J. Biol. Chem. 1989; 264: 10601-10607Google Scholar). This requirement, coupled with the shortcomings of conventional therapies for treating congenital toxoplasmosis and opportunistic infections associated with AIDS and other immunosuppressive conditions (4Haverkos H.W. Am. J. Med. 1987; 82: 907-914Google Scholar, 5Leport C. Raffi F. Matheron S. Katlama C. Regnier B. Saimot A.G. Marche C. Vedrenne C. Vilde J.L. Am. J. Med. 1988; 84: 94-100Google Scholar, 6Laughon B.E. Allaudeen H.S. Becker J.M. Current W.L. Feinberg J. Frenkel J.K. Hafner R. Hughes W.T. Laughlin C.A. Meyers J.D. J. Infect. Dis. 1991; 164: 244-251Google Scholar, 7Lane H.C. Laughon B.E. Falloon J. Kovacs J.A. Davey Jr., R.T. Polis M.A. Masur H. Ann. Intern. Med. 1994; 120: 945-955Google Scholar, 8Brooks R.G. Remington J.S. Luft B.J. Antimicrob. Agents Annu. 1987; 2: 297-306Google Scholar), 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 (2Schwartzman J.D. Pfefferkorn E.R. Exp. Parasitol. 1982; 53: 77-86Google Scholar, 3Krug E.C. Marr J.J. Berens R.L. J. Biol. Chem. 1989; 264: 10601-10607Google Scholar). (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 (2Schwartzman J.D. Pfefferkorn E.R. Exp. Parasitol. 1982; 53: 77-86Google Scholar, 3Krug E.C. Marr J.J. Berens R.L. J. Biol. Chem. 1989; 264: 10601-10607Google Scholar). Most of the reported salvage activities can be accounted for by two enzymes: hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) 1The abbreviations used are: HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyltransferase; AK, adenosine kinase; AMP, adenosine 5′-monophosphates; APRT, adenine phosphoribosyltransferase; 6TX, 6-thioxanthine; 8-azaA, 8-azaadenine; 2-FA, 2-fluoroadenine. and adenosine kinase (AK). The genes for both have been cloned and expressed in bacterial systems, and the purified proteins have been examined biochemically and structurally (9Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Google Scholar, 10Sullivan Jr., W.J. Chiang C.W. Wilson C.M. Naguib F.N. el Kouni M.H. Donald R.G. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 1-14Google Scholar, 11Darling J.A. Sullivan Jr., W.J. Carter D. Ullman B. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 15-23Google Scholar, 12Schumacher M.A. Carter D. Ross D.S. Ullman B. Brennan R.G. Nat. Struct. Biol. 1996; 3: 881-887Google Scholar, 13Schumacher M.A. Scott D.M. Mathews I.I. Ealick S.E. Roos D.S. Ullman B. Brennan R.G. J. Mol. Biol. 2000; 296: 549-567Google Scholar). Genetic studies indicate that neither enzyme is essential for parasite viability, suggesting that the purine salvage pathways of the parasite are functionally redundant. The incorporation of labeled inosine and hypoxanthine into adenine nucleotides, and of labeled adenosine into IMP, indicates that AMP and IMP are interconvertible. The conversion of AMP to IMP (via AMP deaminase), and of IMP to AMP (via adenylosuccinate synthetase/lyase) probably predominate, although adenine and adenosine deaminase activities (3Krug E.C. Marr J.J. Berens R.L. J. Biol. Chem. 1989; 264: 10601-10607Google Scholar) may also play a role. Despite the previously measured incorporation of adenine by Toxoplasma, no adenine phosphoribosyltransferase gene has been identified in this or any other apicomplexan parasite. In the present study, we have integrated genetic, biochemical, and genomic approaches to explore the contributions of HXGPRT and AK in maintaining a robust purine salvage pathway. The ability of T. gondii to grow in virtually any nucleated mammalian cell (14Pfefferkorn E.R. Schwartzman J.D. Kaspar L.H. Cytopathology of Parasitic Disease. 99. Putnam Books, London1983: 74-91Google Scholar), combined with the availability of various mammalian somatic cell mutants and the ability to genetically manipulate the parasite (15Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Google Scholar), has allowed for a comprehensive analysis of the role played by individual parasite enzymes in overall purine nutrition. These studies provide formal proof that all purine salvage in T. gondii proceeds via HXGPRT or AK (there is no functional APRT activity), and fitness assays support the suggestion that AK is metabolically more important than HXGPRT. We have also exploited the large-scale genomic datasets now available for multiple apicomplexan species to create detailed maps of purine salvage pathways for several of these parasites, an important first step toward identifying potential drug targets for broad-spectrum anti-parasitic chemotherapy. Parasites, Cells, Chemicals, and Reagents—T. gondii strain RH (available through Ogden Bioservices Corporation, Rockville, MD) or mutants derived from this strain were used for all in vitro tissue culture experiments and were cultivated in primary human foreskin fibroblasts (HFF cells) or other fibroblast cells, as described previously (15Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Google Scholar, 16Wigler M. Pellicer A. Silverstein S. Axel R. Urlaub G. Chasin L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1373-1376Google Scholar). 14CAdenine (56 Ci mol–1), 14Cxanthine (57 Ci mol–1), 3Hadenosine (48 Ci mmol–1), and 3Huracil (20 Ci mmol–1) were purchased from Moravek Biochemicals (Brea, CA). All unlabeled subversive substrates of purine salvage enzymes were purchased from Sigma. DNA modifying enzymes were acquired from New England Biolabs (Beverly, MA). DE81 anion-exchange filters were obtained from Whatman (Hillsboro, OR). Molecular Genetic Manipulations—All molecular manipulations were performed according to standard protocols (15Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Google Scholar). Isolation of parasite genomic DNA by with in the of by and genomic DNA to and as The AK of the of the AK from the J.A. Sullivan Jr., W.J. Carter D. Ullman B. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 15-23Google Scholar) by with and The HXGPRT from a (9Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Google Scholar) by with were purified from by and labeled of for defined knockouts at the HXGPRT or AK loci and have been described as have the to parasites (9Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Google Scholar, 10Sullivan Jr., W.J. Chiang C.W. Wilson C.M. Naguib F.N. el Kouni M.H. Donald R.G. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 1-14Google Scholar). The by an AK by the T. gondii into the The Jr., W.J. Chiang C.W. Wilson C.M. Naguib F.N. el Kouni M.H. Donald R.G. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 1-14Google Scholar) with and to the AK by and and the resulting and the The with and by of a by with the AK The with and the resulting by into the of the AK of this AK to the HXGPRT as described (9Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Google Scholar) by in of the host cell parasites were from host cell by through were by at for at and in in of and by on or the of were by the and for all All enzyme assays were according to the described previously Biochem. Scholar, R.L. D. Biochem. Parasitol. Scholar). AK were performed in a at in with activity), of 3Hadenosine (48 Ci mmol–1), and of parasite were by of the anion-exchange were in and in at by were a phosphoribosyltransferase and adenine phosphoribosyltransferase (APRT) were performed with of 14Cxanthine (57 Ci or of (56 Ci mol–1), and of of T. gondii to of intracellular T. gondii measured in of either human foreskin fibroblasts or APRT-deficient fibroblasts by the incorporation of 3Huracil into were in the of either to or to parasites were to a of of 3Huracil (20 Ci mmol–1), and were as described (15Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Google Scholar). In of were at a of into of APRT-deficient mammalian host of (56 Ci to and adenine incorporation allowed to for were as described for of incorporation (15Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Google Scholar). measured by Expression of Leishmania donovani APRT in T. of the L. donovani APRT T. Wilson S. A. Ullman B. Biochem. Parasitol. Scholar) a and a in The the of from to for in Toxoplasma M. M. Roos D.S. Biochem. Parasitol. 120: Scholar). The with and and into and D. S. in the with that of the This also a the of the D. Scholar). The resulting into and parasites as described (15Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Google Scholar). parasites were allowed to fibroblast in and of were for of were in in and with parasites were also to parasites of in drug were cloned by in drug (15Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Google Scholar) and for APRT Fitness assays the and and parasites were performed as described Roos D.S. Mol. Scholar). of or parasites at various assays were performed in in the of (15Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Google Scholar). or used as for the for parasites, the latter for parasites. adenine used to for mutants in parasites. of all available genomic and expressed by of used for this This or genomic for and T. gondii and and genomic and expressed from T. and are available on the and are from T. gondii is from and are from is from is from is from T. and are from and and obtained from of purine salvage and enzymes were identified by with a of for the purine salvage genes were used as A of and is provided as Genetic of genetic of either the AK or HXGPRT loci have been described previously and to be in the purine salvage activities (9Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Google Scholar, 10Sullivan Jr., W.J. Chiang C.W. Wilson C.M. Naguib F.N. el Kouni M.H. Donald R.G. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 1-14Google Scholar). The of mutants to both loci in a parasite. A of the from experiments is provided in of either individual or and In in we were to the AK in the on adenine of parasites with the or to the HXGPRT in the on of parasites with the to both adenine and were in the latter but DNA analysis no at the HXGPRT and levels of HXGPRT These parasites are to at the HXGPRT or at other loci that of to AK and HXGPRT of in a The to both HXGPRT and AK that the may be for T. gondii. this we to genetically both AK activity in a that is The to the of T. gondii AK by the T. gondii parasites were obtained of parasites, and the resulting were by analysis to and enzyme assays to purine salvage as in with AK demonstrates genomic of and in and parasites and deletion of the in the Jr., W.J. Chiang C.W. Wilson C.M. Naguib F.N. el Kouni M.H. Donald R.G. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 1-14Google Scholar). This in the of the In a also of the from of the at the HXGPRT The with an HXGPRT also that the HXGPRT in parasites and in the The of the HXGPRT in this to the is to molecular manipulations associated with of the and assays no AK activity in the but activity with that of parasites (and when AK to the HXGPRT in the assays activity in the but in parasites, to the mutants previously (9Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Google Scholar). these that is to both the AK and HXGPRT genetic loci but only one of these activities is provided in this by of AK at the HXGPRT APRT and to of to generate parasites is the previous identification of APRT activity in T. gondii (3Krug E.C. Marr J.J. Berens R.L. J. Biol. Chem. 1989; 264: 10601-10607Google Scholar), provide for an alternative of purine for the parasite. with T. gondii cultivated in human foreskin fibroblasts were to both and as in These adenine are to as subversive substrates of APRT, and both have been to in other Jr., J.A. J.A. Biochem. 1987; Scholar, Scholar, M.H. S. S. F. Exp. Cell Scholar). parasites were in APRT-deficient M. Pellicer A. Silverstein S. Axel R. Urlaub G. Chasin L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1373-1376Google Scholar), however, for both to a in of and for and 8-azaA, These suggest that of and is than parasite, APRT although levels of APRT activity were in of parasites cultivated in human foreskin fibroblasts not we were to phosphoribosylation of adenine when parasites were in APRT-deficient fibroblasts the suggestion that parasites an APRT In T. gondii in APRT-deficient host only low levels of activities for purine salvage enzymes in and in a Expression of L. donovani APRT in T. purine salvage pathways in T. we to L. donovani APRT in and T. gondii. and expressed in the parasite and the APRT activity both in and in vitro and parasites into when in APRT-deficient host cells, in to transgenic parasites L. donovani APRT APRT activity virtually in of parasites in APRT-deficient host but significant levels of activity were in and and of other purine salvage activities in parasites levels of indicates that HXGPRT and AK activities are by the of the transgenic APRT activity the of these salvage activities suggest that adenosine in T. gondii is significantly more than for other the activity of transgenic APRT is but considerably than either AK or HXGPRT. explore parasites be to genetic deletion of both the AK and HXGPRT and parasites were with or and to for deletion of either in no have been identified to Fitness for and studies on and mutants no differences in as with the parasites from were derived (9Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Google Scholar, 10Sullivan Jr., W.J. Chiang C.W. Wilson C.M. Naguib F.N. el Kouni M.H. Donald R.G. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 1-14Google Scholar). however, we have found that assays the of fitness defects in other mutants Roos D.S. Mol. Scholar). in both the and significant fitness with parasites a fitness of and a of These indicate that both AK and HXGPRT play an important role in parasite metabolism and although the of neither gene is essential for viability, as The in the suggestion that this enzyme the more important role in purine of in of the large-scale genomic datasets that have available for various apicomplexan species A. B. R.L. J. D. R.L. L. Roos D.S. J. Jr., Scholar, B. L. Roos D.S. Scholar, D. S. Scholar), we have a detailed model for purine salvage pathway in these parasites, as in The T. also in this as and are to be in the T. Scholar). genes de purine enzymes are evident in any of these protozoa, suggesting that all are of purine This is for the T. previous studies on the incorporation of and R.L. of and Ross Scholar). of these genes are and pathways have not been that purine pathways were in this salvage the ability to nucleosides and have previously been reported for both T. gondii C.W. Carter Sullivan Jr., W.J. Donald R.G. Roos D.S. Naguib F.N. el Kouni M.H. Ullman B. Wilson C.M. J. Biol. Chem. 1999; Scholar) and N.S. C. Ullman B. J. Biol. Chem. 2000; Scholar). and nucleosides C.W. Carter Sullivan Jr., W.J. Donald R.G. Roos D.S. Naguib F.N. el Kouni M.H. Ullman B. Wilson C.M. J. Biol. Chem. 1999; Scholar), and is for and nucleobase M. G. Mol. Biochem. Parasitol. Scholar, J.M. J. Parasitol. Scholar). The parasite evidence for both purine and and the also evidence for at one A is evident in the genome. found in the or Toxoplasma a and robust both AK and HXGPRT, but not The pathway in seems to be In and all a of this pathway. relies on HXGPRT, and on AK. All of these parasites APRT, but the of an APRT gene as but not for AMP and IMP are present in all of these species is to IMP from AMP These also to in ability to nucleosides and nucleobases. Toxoplasma and purine (and probably adenosine uses a purine and a and all of these This an integrated genetic, biochemical, and genomic to the of salvage pathways to the purine of T. gondii and T. gondii two functionally salvage HXGPRT and AK, and previous have that this parasite can of either activity neither HXGPRT AK is essential for T. gondii (9Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Google Scholar, 10Sullivan Jr., W.J. Chiang C.W. Wilson C.M. Naguib F.N. el Kouni M.H. Donald R.G. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 1-14Google Scholar). to both HXGPRT and AK in a parasite were evidence that functional of at one of these two enzymes is essential support for this is provided by the and parasites of AK activity in of the genomic loci for both AK and HXGPRT. These experiments that AK and HXGPRT provide the only two routes for purine in T. gondii. parasites a fitness than mutants that through AK is probably than HXGPRT. studies have APRT activity in T. gondii (3Krug E.C. Marr J.J. Berens R.L. J. Biol. Chem. 1989; 264: 10601-10607Google Scholar), with parasite higher specific activity of adenine phosphoribosylation than that of than hypoxanthine or In these however, we that adenine incorporated into nucleotide pools with a virtually to that for hypoxanthine (3Krug E.C. Marr J.J. Berens R.L. J. Biol. Chem. 1989; 264: 10601-10607Google Scholar), suggesting that adenine may have been to hypoxanthine and incorporated via HXGPRT, than APRT the levels of APRT present in mammalian host W.J. J. Biol. Chem. Scholar) may have in of parasite In parasites in host were to adenine but in APRT-deficient host M. Pellicer A. Silverstein S. Axel R. Urlaub G. Chasin L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1373-1376Google Scholar) were not we found no APRT activity in parasites or parasite when APRT-deficient host were used for parasite Expression of a APRT allowed transgenic parasites to low levels of adenine although this activity to permit of both HXGPRT and AK APRT activity has also been reported in 1989; Scholar, R. S. J. Exp. Parasitol. Scholar), Proc. Natl. Acad. Sci. U. S. A. Scholar), and J. Exp. Parasitol. Scholar). purine and the of purines via is to J. Biol. Chem. Scholar), a significant role for APRT genes were evident in the of any apicomplexan parasite and seems that any genes were as all APRT genes are in primary (and no alternative enzymes are to We from the available genetic, biochemical, and genomic that APRT is from all of these apicomplexan parasites. the T. a APRT, suggesting that the of the may have purine salvage APRT, AK, or HXGPRT. analysis no evidence for of any of these genes, in to B. C. Jr., J.M. C. Proc. Natl. Acad. Sci. U. S. A. Scholar). studies indicate that this also other differences from most of the a and the of and activity J. these are by the analysis also a gene to the that can on or as a purine R.L. of and Ross Scholar). A also in but this enzyme seems to have been in other In HXGPRT AK to provide the routes for purine in but species may the or both of these Toxoplasma and parasites genes to both as as enzymes by in deaminase not in these parasite but this gene is not and the conversion of adenosine into inosine (3Krug E.C. Marr J.J. Berens R.L. J. Biol. Chem. 1989; 264: 10601-10607Google Scholar, 10Sullivan Jr., W.J. Chiang C.W. Wilson C.M. Naguib F.N. el Kouni M.H. Donald R.G. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 1-14Google Scholar, Mol. Biochem. Parasitol. Scholar) that is probably studies also indicate an adenine deaminase in Mol. Biochem. Parasitol. Scholar), although this not evident in the genome. and species AK, but to have HXGPRT. HXGPRT activity in J. Exp. Parasitol. Scholar), but this is not by the parasite or more and studies B. J. C. L. Proc. Natl. Acad. Sci. U. S. A. Scholar). We were to AMP deaminase in the T. or T. but that this activity be present as the for nucleotide a and HXGPRT activity G. Jr., R.L. Sullivan M.A. Scholar) but lacks AK. subversive purine and have protozoan B. Carter D. Infect. Agents Dis. Scholar, Marr J.J. Am. J. Med. Scholar, S. Marr J.J. J. Med. Scholar), but functional in purine salvage pathways a potential to drug as therapies may have to multiple activities to and the of The of an APRT activity in T. gondii indicates that only HXGPRT and AK have to be and both of these enzymes have previously been biochemically and structurally (9Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Google Scholar, 11Darling J.A. Sullivan Jr., W.J. Carter D. Ullman B. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 15-23Google Scholar, 13Schumacher M.A. Scott D.M. Mathews I.I. Ealick S.E. Roos D.S. Ullman B. Brennan R.G. J. Mol. Biol. 2000; 296: 549-567Google Scholar, M.A. Carter D. Scott D.M. Roos D.S. Ullman B. Brennan R.G. J. Scholar). The significant fitness of genetically either that this may be a drug in T. gondii. The more purine salvage pathways in other parasites makes these targets more any of the activities from host cell adenosine to in the in adenosine AMP IMP be to the parasite. The in of and for both the parasite and mammalian enzymes is available (9Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Google Scholar, 11Darling J.A. Sullivan Jr., W.J. Carter D. Ullman B. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 15-23Google Scholar, 12Schumacher M.A. Carter D. Ross D.S. Ullman B. Brennan R.G. Nat. Struct. Biol. 1996; 3: 881-887Google Scholar, 13Schumacher M.A. Scott D.M. Mathews I.I. Ealick S.E. Roos D.S. Ullman B. Brennan R.G. J. Mol. Biol. 2000; 296: 549-567Google Scholar, G. Jr., R.L. Sullivan M.A. Scholar, Jr., J. Biol. Chem. Scholar, R.L. J.L. J.J. J. Biol. Chem. 1979; Scholar, I.I. Ealick S.E. Scholar, G. C. 1994; Scholar, C.M. C. 1999; Scholar) and the of with We for with genomic and and for of the with