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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 approximately 300 kb intervals across the genome. Fourteen chromosomes were identified comprising a total genetic size of approximately 592 cM and an average map unit of approximately 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.

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.

In contrast to the cytocidal effect of 6-thiopurines on mammalian cells, the action of 6-thioxanthine on Toxoplasma gondii was only parasitostatic. 6-Thioxanthine was a substrate of the parasite's hypoxanthine-guanine phosphoribosyltransferase. That enzyme converted 6-thioxanthine to 6-thioxanthosine 5'-phosphate which accumulated to near millimolar concentrations within parasites incubated intracellularly in medium containing the drug. 6-Thioxanthosine 5'-phosphate was the only detectable metabolite of 6-thioxanthine. The absence of 6-thioguanine nucleotides explains the lack of a parasitocidal effect because the incorporation of 6-thiodeoxyguanosine triphosphate into DNA is the mechanism of the lethal effect of 6-thiopurines on mammalian cells. Extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate incorporated more labeled hypoxanthine or xanthine into their nucleotide pools than did control parasites. The basis for this increased nucleobase salvage remains unexplained. It was not due to up-regulation of hypoxanthine-guanine phosphoribosyltransferase and could not be explained by reduced use of labeled nucleotides for nucleic acid synthesis. Extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate used labeled hypoxanthine almost entirely to make adenine nucleotides while control parasites made both adenine and guanine nucleotides. Both extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate and control parasites efficiently used labeled xanthine to make guanine nucleotides. These observations suggested that inosine 5'-phosphate-dehydrogenase was inhibited while guanosine 5'-phosphate synthase was not. Assay of inosine 5'-phosphate dehydrogenase in soluble extracts of T. gondii confirmed that 6-thioxanthosine 5'-phosphate was an inhibitor. We conclude that 6-thioxanthine blocks the growth of T. gondii by a depletion a guanine nucleotides., (Copyright 2001 Elsevier Science (USA).)

2,4-Diaminopteridines (21 compounds) and 2,4-diamino-5-methyl-5-deazapteridines (34 compounds) along with three 2,4-diamino-5-unsubstituted-5-deazapteridines and four 2,4-diaminoquinazolines, each with an aryl groups attached to the 6-position of the heterocyclic moiety through a two-atom bridge (either CH2NH, CH2N(CH3),CH2S, or CH2CH2), were synthesized and evaluated as inhibitors of the growth of Toxoplasma gondii in culture and as inhibitors of dihydrofolate reductase enzymes from T. gondii, Pneumocystis carinii, and rat liver. Exceptionally high levels of combined potency and selectivity as growth inhibitors of T. gondii and as inhibitors of the microbial enzymes relative to the mammalian enzyme were found among the 5-methyl-5-deazapteridines but not for the other heterocyclic types. Thirty of the 34 5-methyl-5-deaza compounds gave growth inhibition IC50 values lower than that of pyrimethamine (0.4 microM) with 14 compounds below 0.1 microM, values that compare favorably with those for piritrexim and trimetrexate (both near 0.02 microM). As inhibitors of T gondii DHFR, all but three of the 34 5-methyl-5-deaza compounds gave IC50 values in the order of magnitude with those of piritrexim (0.017 microM) and trimetrexate (0.010 microM), and 17 compounds of this group gave IC50 values versus P. carinii DHFR similarly comparable with those of piritrexim (0.031 microM) and trimetrexate (0.042 microM). Thirteen of these congeners gave both T. gondii growth inhibition and DHFR inhibition IC50 values of 0.10 microM or less, thus indicating facile penetration of the cell membrane. Eleven of these inhibitors of both T. gondii growth and DHFR have selectivity ratios (IC50 rat liver divided by IC50 T. gondii) of 5 or greater for the parasite DHFR. The highest selectivity ratio of nearly 100 belongs to the 5-methyl-5-deaza compound whose 6-substituent is CH2CH2C6H3(OCH3)2-2,5. This compound is over 10(3)-fold more selective for T. gondii DHFR than bridge homologue piritrexim (selectivity ratio 0.088), a compound now in clinical trials. The candidate with CH2NHC6H3(CH3)2-2,5 in the 6-position gave the highest P. carinii DHFR selectivity ratio of 4.0, which is about 60-fold more selective than trimetrexate (0.071) and 80-fold more selective than piritrexim (0.048) toward this enzyme. The 10 best compounds with respect to potency and selectivity includes six compounds bearing 2,5-disubstituted phenyl groups in the side chain (with little, if any, difference in effects of methyl, methoxy, or ethoxy), two side chains bearing 1-naphthyl groups, and two with 5,6,7,8-tetrahydro-1-naphthyl groups. Bridge groups represented in the 10 choice compounds are CH2NH, CH2N(CH3), CH2CH2, and CH2S. The high levels of both potency and selectivity among these agents suggest that in vivo studies now underway may lead to agents that could replace trimetrexate and piritrexim in treatment of toxoplasmosis and P. carinii pneumonia.

6-Thioxanthine caused 50% inhibition of the growth of Toxoplasma gondii in human fibroblasts at a concentration of 5 micrograms/ml. A mutant induced by treatment with ethylnitrosourea (ThxR-1) was 20-fold more resistant than the wildtype. Wild-type parasites grown in Lesch-Nyhan fibroblasts efficiently incorporated hypoxanthine, guanine, and xanthine, but ThxR-1 incorporated each of these precursors less than 2% as well as the wildtype did. Soluble extracts of wild-type parasites had potent phosphoribosyltransferase activities for hypoxanthine, guanine, and xanthine, while extracts of ThxR-1 had barely detectable activity with any of these substrates. The basis for the resistance of ThxR-1 to 6-thioxanthine is, therefore, the lack of the enzyme hypoxanthine-guanine phosphoribosyltransferase. Thus, salvage pathways that employ this enzyme are not essential for the acquisition of purines, which the parasite must obtain from the host cell. Incubation in a medium containing mycophenolic acid and xanthine allowed the efficient recovery of wild-type T. gondii in the presence of many ThxR-1 parasites. Together with the use of 6-thioxanthine to detect resistant mutants in the presence of many wild-type parasites, this procedure provides a simple selection and back-selection for mutations that affect the hypoxanthine-guanine phosphoribosyltransferase gene of T. gondii.

Studies were carried out to evaluate the proposed role of indoleamine 2,3-dioxygenase (INDO) induction in the antimicrobial and antiproliferative effects of gamma interferon (IFN-gamma) in human fibroblasts. The INDO cDNA coding region was cloned in the pMEP4 expression vector, containing the metallothionein (MTII) promoter in the sense (+ve) or the antisense (-ve) orientation. Human fibroblasts (GM637) stably transfected with the sense construct expressed INDO activity after treatment with CdCl2 or ZnSO4, but cells transfected with the antisense construct did not. The growth of Chlamydia psittaci was strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+ or Zn2+. The inhibition correlated with the level of INDO activity induced and could be reversed by the addition of excess tryptophan to the medium. The growth of Toxoplasma gondii was also strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+. Expression of Cd(2+)-induced INDO activity also inhibited thymidine incorporation and led to cytotoxicity in INDO +ve cells but not in INDO -ve cells. Thus, the induction of INDO activity by IFN-gamma may be an important factor in the antimicrobial and antiproliferative effects of IFN-gamma in human fibroblasts.

Azithromycin and spiramycin markedly inhibited the growth of Toxoplasma gondii in cultured human fibroblasts. However, 3 days of treatment were required to reveal their full antitoxoplasma activity. This delayed onset of inhibition was similar to that previously reported for clindamycin. Mutants of T. gondii resistant to azithromycin (AziR-1) and spiramycin (SprR-1) were isolated and compared with a previously described mutant resistant to clindamycin (ClnR-2). Mutant ClnR-2 was cross-resistant to all three antibiotics, while AziR-1 was cross-resistant only to spiramycin and SprR-1 was cross-resistant only to azithromycin. In short-term studies of protein synthesis by freshly prepared extracellular parasites, clindamycin and azithromycin were effective only at concentrations much greater than their 50% inhibitory concentrations in infected cultures and the resistant mutants did not differ from the wild type in antibiotic sensitivity. Thus, protein synthesis on cytoplasmic ribosomes of the parasite did not seem to be the target of these antibiotics. To determine whether mitochondrial protein synthesis in T. gondii was inhibited by clindamycin or azithromycin, wild-type parasites were grown in cultured cells in the presence of antibiotic concentrations well above the 50% inhibitory concentrations. Mitochondrial function, measured by oxygen uptake per purified extracellular parasite, did not decrease substantially, after the parasites had multiplied 11-fold in the presence of antibiotic. Thus, mitochondrial protein synthesis did not seem to be the target of clindamycin or azithromycin. An alternative target is protein synthesis in the putative apicomplexan organelle that has a 35-kb genome.

Anticoccidial drugs were evaluated for activity and for the development of resistance in a model of Toxoplasma gondii growing in human fibroblast cultures. Of 13 anticoccidial drugs tested, 9 had selective antitoxoplasma activity (50% inhibitory concentration, in micrograms per milliliter): decoquinate (0.005), arprinocid-N-oxide (0.015), robenidine (0.03), the aryl triazine CP-25,415 (0.2), toltrazuril (0.4), clopidol (1), dinitolmide (Zoalene; Dow) (10), and the carboxylic acid ionophores monensin (0.001) and salinomycin (0.04). Glycarbylamide, amprolium, nicarbazin, and the 6-(p-bromophenoxy)-7-chloro analog of halofuginone (Stenorol; Roussel-UCLAF) (CP-63,567) were toxic for the fibroblasts. Since Eimeria tenella has a similar drug susceptibility profile, anticoccidial drugs can be viewed as a potential source of new antitoxoplasma therapies. The development of resistance has limited the usefulness of most of these drugs as anticoccidial agents; in coccidia, resistance to all except the ionophores occurs readily in vivo. We explored the development of resistance in T. gondii by attempting to select mutants in vitro from parasites mutagenized with ethylnitrosourea. Mutants that had 20- to 50-fold-reduced susceptibility to decoquinate, arprinocid-N-oxide, and CP-25,415 were obtained. Ionophore-resistant T. gondii mutants were also selected in vitro; however, there was only a twofold difference in susceptibility between these mutants and the wild type. For three drugs (clopidol, robenidine, and toltrazuril), we were unable to select resistant mutants. For experimental anticoccidial drugs, there is currently no in vitro method for assessing the risk of development of resistance in Eimeria species. Our results suggest that T. gondii may offer a useful surrogate for this assessment.

The protozoan parasite Toxoplasma gondii has recently emerged as an important opportunistic pathogen in humans. Toxoplasma also shares a number of biological features with Plasmodium and Eimeria, which are important pathogens of humans and animals. Because o f the ease o f experimental use, David Sibley, Elmer Pfefferkom and John Boothroyd have undertaken the development of genetics in Toxoplasma as a model intracellular parasite. Toxoplasma is presently the only parasitic protozoan where both classical and molecular genetics are feasible. The recent advances in this system are highlighted here, along with potential applications of genetics for understanding intracellular parasitism.

Mutants of Toxoplasma gondii resistant to drugs that appear to affect the mitochondrial bc1 complex were isolated with the aid of mutagenesis with ethylnitrosourea. Mutant DeqR-1 was > 1,000-fold more resistant to decoquinate than was the wild type but more sensitive to atovaquone (formerly called 566C80). Mutant AtoR-1 was 20-fold more resistant to atovaquone than was the wild type and was also partially cross-resistant to decoquinate. Both drugs rapidly inhibited oxygen uptake by freshly prepared extracellular parasites, and the mutants were resistant to this inhibition. Neither the addition of uracil to the medium nor the use of a mutant of T. gondii with a defect in pyrimidine salvage had a substantial effect on the in vitro anti-parasitic activity of these drugs, suggesting that de novo pyrimidine synthesis was not the major biochemical target of either drug.

We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen.

Clindamycin, which has been reported to have no significant in vitro activity against Toxoplasma gondii, actually markedly inhibits the growth of this parasite in infected human fibroblasts. When measured 3 days after treatment, the concentration required to reduce parasite growth by 50% is about 1 ng/ml. Some observers failed to note this inhibition because of its markedly delayed onset. At 6 ng/ml, clindamycin is parasiticidal, and the rate and extent of parasite killing increase with higher drug concentrations. With the aid of chemical mutagenesis, we isolated a parasite mutant that is approximately 100-fold more resistant to clindamycin than is the wild type. Lincomycin inhibits T. gondii at a higher 50% inhibitory concentration, about 100 ng/ml. The clindamycin-resistant mutant is partially cross-resistant to lincomycin.

Sulfadiazine was a potent inhibitor of the in vitro growth of Toxoplasma gondii, although it had little effect during the first 24 hr of treatment. A mutant parasite (R-SulR-5) with a 300-fold increase in sulfadiazine resistance was selected by a combination of chemical mutagenesis and growth in gradually increased sulfadiazine concentrations. This mutant was completely cross-resistant to several other sulfonamides and to dapsone. The same concentration of p-aminobenzoic acid reversed the sulfadiazine inhibition of both mutant and wild-type parasites even though much higher concentrations of sulfadiazine were used to inhibit the mutant. Dihydropteroate synthase, a sulfonamide-sensitive enzyme in the pathway leading to dihydrofolic acid, had similar activities in wild-type and R-SulR-5 parasites. However, the mutant enzyme was 40-fold more resistant to sulfadiazine and had higher apparent Kms for both substrates, p-aminobenzoic acid and dihydropteridine pyrophosphate. The mutant was slightly less active than the wild type in the uptake of sulfadiazine.

The three types of IgG FcR (Fc gamma RI, Fc gamma RII, Fc gamma RIII) on human leukocytes play an important role in elimination of antibody-coated infectious agents. To further understand the role of the different Fc gamma R in mediating this killing, we examined the ability of human myeloid and lymphoid cells to kill the protozoan Toxoplasma gondii in the presence of antitoxoplasma IgG or bispecific antibodies. Although human myeloid cells (monocytes, macrophages, neutrophils, and eosinophils) all lysed unsensitized T. gondii, killing by these cells was significantly enhanced by opsonization with antitoxoplasma rabbit IgG. Human lymphocytes, however, did not lyse T. gondii unless the parasites were coated with antibody. The role of antibody and Fc gamma R in mediating ADCC of T. gondii was then examined using bispecific antibodies made by chemically cross-linking Fab fragments of antitoxoplasma antibodies to Fab fragments of antibodies specific for human leukocyte surface Ag, including Fc gamma R. Thus, simultaneous binding of these bispecifics to parasites and effector cells allowed an evaluation of killing when T. gondii were targeted to each Ag independently. Bispecifics which targeted T. gondii to Fc gamma RI, II or III enhanced lysis by monocytes. However, similar results were obtained with bispecifics targeting T. gondii to non-Fc gamma R Ag (CD11b or beta 2-microglobulin) on monocytes. Likewise, polymorphonuclear leukocytes mediated significantly more lysis in the presence of bispecifics linking T. gondii to Fc gamma RII, Fc gamma RIII, or the two non-Fc gamma R Ag CD11b and beta 2-microglobulin. Thus, although human myeloid cells did not require antibody-Fc gamma R triggering to kill T. gondii, antibody appeared to enhance lysis by capturing and directing the parasites to the effector cell surface. Human lymphocytes, in contrast, mediated significant lysis of T. gondii only in the presence of bispecifics targeting T. gondii to Fc gamma RIII, indicating a requirement for specific triggering of Fc gamma RIII for killing by large granular lymphocytes. Consequently, using bispecifics to compare targeting to specific Ag, both non-Fc gamma R and Fc gamma R, allowed determination of the role of antibody-Fc gamma R interactions in T. gondii killing. In addition, these studies demonstrate the potential of bispecifics in determining the role of specific Ag in killing of or infection by pathogens.

Kittens are the principal disseminators of Toxoplasma gondii. They can shed greater than 10(8) oocysts in the feces after initial infection with bradyzoites in tissue cysts. Thereafter, most kittens develop protective immunity and do not shed oocysts again if they are reinfected. Bradyzoites of a T gondii mutant, designated T-263, were used to vaccinate kittens. Their use did not result in oocyst shedding, but successfully prevented 84% (31/37) of the kittens from shedding oocysts when challenge exposed with a normal isolate of T gondii. Vaccination of outdoor-roaming cats and kittens would be a useful public health measure to prevent transmission of toxoplasmosis near homes, on farms, and in zoos. It is anticipated that several years will be required for a lyophilized bradyzoite vaccine to be ready for licensing and possible commercial availability.

L-651,582 (5-amino-[4-(4-chlorobenzoyl)-3,5-dichlorobenzyl]-1,2,3-triazole-4- carboxamide), which is active in vivo against coccidiosis caused by the intracellular parasitic protozoan Eimeria tenella, is also effective against this organism in tissue culture in Maden Darby bovine kidney (MDBK) host cells. L-651,582 inhibited 45Ca++ uptake, as well as [3H]hypoxanthine incorporation into soluble nucleotide pools, in MDBK cells at concentrations similar to those required for antiparasitic activity (IC50 = 0.3 microgram/ml). However, the drug did not inhibit the [3H] hypoxanthine incorporation into the nucleotide pools of the intracellular E. tenella under the same conditions. The antiparasitic activity of several L-651,582 analogs paralleled their ability to inhibit [3H]hypoxanthine incorporation in MDBK cells. L-651,582 similarly inhibited the growth of the intracellular parasitic protozoan Toxoplasma gondii in vitro with either HeLa cells or Normal Human Fibroblasts as host cells. The IC50 for inhibition of T. gondii growth in normal human fibroblasts was similar to that for [3H]hypoxanthine incorporation inhibition. In HeLa cells, however, 40-fold higher levels were required for the inhibition of [3H]hypoxanthine incorporation than were required to inhibit parasite growth, showing that antiparasitic activity was not a consequence of the alteration in host nucleotide metabolism. This was also consistent with the observation that the effect of L-651,582 against E. tenella in MDBK cells was not reversed by the addition of nutrients involved in nucleotide biosynthesis. This study suggests that L-651,582 has antiparasitic activity which correlates with host cell effects and which may be a result of blocking Ca++ entry in the host cells, but is not the result of an alteration of host nucleotide biosynthesis.

Although treatment of human macrophages or fibroblasts with human gamma interferon results in the inhibition of intracellular Toxoplasma gondii, murine gamma interferon stimulated only murine macrophages, not murine fibroblasts, to inhibit T. gondii. This species difference may be important in understanding the control of acute and chronic toxoplasmosis.

Mutants resistant to sinefungin (SF) and hydroxyurea (HU) were isolated from an oocyst-producing strain of Toxoplasma gondii with the aid of mutagenesis with ethylnitrosourea. These mutants were used with previously described mutants resistant to adenine arabinoside (araA) and fluorodeoxyuridine (FUDR) in genetic crosses in cats. In order to interpret the data from crosses in which all four mutants were used to infect the same cat, it was necessary to devise a mathematical expression to predict the recombination frequency for unlinked markers. This frequency was shown in theory to be half of the product of the two parental phenotype frequencies. A series of crosses in which the parental frequencies were systematically varied yielded frequencies of recombination that were in accord with this calculation. The four-way crosses in the same cat showed unlinked recombination between all markers except HU and FUDR. This pair of markers yielded no doubly resistant recombinants, suggesting complete linkage. However, linkage was excluded when a binary cross between the HU- and FUDR-resistant mutants resulted in the normal number of doubly sensitive recombinants. The lack of doubly resistant recombinants was shown to be a consequence of phenotypic suppression of HU resistance by FUDR resistance. This suppression was first demonstrated by showing that an FUDR-resistant mutant selected from an HU-resistant parasite lost the HU resistance. The phenotypically suppressed HU-resistant gene was revealed by genetic crosses with wild type T. gondii. Although both parental stains were sensitive to HU, some of the progeny parasites were resistant.

Treatment of human fibroblasts with human recombinant gamma interferon blocked the growth of Toxoplasma gondii, an obligate intracellular protozoan parasite. Growth of the parasite was measured by a plaque assay 7 days after infection or by the incorporation of [3H]uracil 1 or 2 days after infection. The antitoxoplasma activity induced in the host cells by gamma interferon was strongly dependent upon the tryptophan concentration of the medium. Progressively higher minimal inhibitory concentrations of gamma interferon were observed as the tryptophan concentration in the culture medium was increased. Treatment with gamma interferon did not make the cells impermeable to tryptophan. The kinetics of [3H]tryptophan uptake into the acid-soluble pools of control and gamma interferon-treated cultures were identical during the first 48 sec. Thereafter uptake of [3H]tryptophan into the acid-soluble pool of control fibroblasts reached the expected plateau after 96 sec. In contrast, uptake of [3H]tryptophan continued for at least 12 min in the gamma interferon-treated cultures. At that time, the acid-soluble pool of the gamma interferon-treated cultures contained 8 times the radioactivity of the control cultures. This continued accumulation was the result of rapid intracellular degradation of [3H]tryptophan into kynurenine and N-formylkynurenine that leaked slowly from the cells. These two metabolites were also recovered from the medium of cultures treated for 1 or 2 days with gamma interferon. Human recombinant alpha and beta interferons, which have no antitoxoplasma activity, did not induce any detectable degradation of tryptophan. Several hypotheses are presented to explain how the intracellular degradation of tryptophan induced by gamma interferon could restrict the growth of an obligate intracellular parasite.

Certain temperature-sensitive mutants of the RH strain of Toxoplasma gondii and a "back mutant," all maintained in human fibroblasts, were studied in mice. Most mutants gave rise to acute, fatal infections, and after sulfonamide prophylaxis rarely persisted as chronic infections in mice. However, the ts-4 strain was nonfatal in doses up to 10(3) to 10(5) tachyzoites, elicited high titers of antibody, and did not persist beyond 2 mo. No Toxoplasma cysts were found. There was no evidence that a febrile reaction of the mice was restrictive, because the highest temperatures, 37.9 to 38.4 C, occurred 3 days after infection, whereas the organisms were recoverable for 16 to 32 days. Because doses of Toxoplasma, survived by 11 of 12 normal BALB/c mice, and by one of five thymic transplanted athymic mice, killed six of six athymic mice, it appears that the limited persistence of ts-4 is related to the immunologic response.

The principal iodinatable surface protein (P30) of our cloned RH strain of Toxoplasma gondii has an apparent molecular weight of 30,000, as measured by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Monoclonal antibody B specifically immunoprecipitated protein P30 from a detergent extract of surface radioiodinated T. gondii. Monoclonal antibody B in the presence of complement was also parasiticidal for T. gondii, and this parasiticidal effect could be blocked by protein P30. Monoclonal antibody B was purified from mouse ascitic fluid and linked to cyanogen bromide-activated Sepharose. The resulting immunoabsorbent was used to purify 1.7 mg of protein P30 from a large number of parasites. The efficiency of recovery of protein P30 was measured by assays of radioactivity and of parasiticidal blocking activity. Protein P30 represented 3 to 5% of the total protein. It is also present in a recently isolated strain of T. gondii. A convalescent human antitoxoplasma serum immunoprecipitated radiolabeled protein P30. Three convalescent antisera when quantitated by an ELISA test had a high anti-protein P30 titer. Charge shift electrophoresis showed that protein P30 has an extensive hydrophobic region and thus is probably an integral membrane protein. Electrophoresis under nonreducing conditions showed no evidence that protein P30 exists as a disulfide linked homo- or heterodimer, although it probably has intramolecular disulfide bonds.

Hydroxyurea inhibited the growth and DNA synthesis of Toxoplasma gondii growing in human fibroblast cells. A concentration of 18 micrograms/ml totally suppressed plaque formation. The synthesis of T. gondii RNA was not acutely inhibited. The parasite was equally sensitive to hydroxyurea when grown in wild type or hydroxyurea resistant host cells. With the aid of chemical mutagenesis, we isolated a stable hydroxyurea resistant mutant of T. gondii. This mutant showed no increased ability to incorporate [3H]uracil into its pyrimidine deoxynucleotide pool. Hydroxyurea depressed the [3H]uracil labeling of the pyrimidine deoxynucleotide pool in the wild type parasite but not in the mutant, suggesting that the mutant ribonucleotide reductase was resistant to the inhibitory effect of the drug.

We have previously observed that gamma-interferon (IFN-gamma) inhibited the growth of the intracellular protozoan parasite Toxoplasma gondii in cultured human fibroblasts and that this inhibition was related to the disappearance of tryptophan from the medium with the concomitant appearance of kynurenine and N-formylkynurenine. In this report, we show that IFN-gamma induced an indoleamine 2,3-dioxygenase in human fibroblasts that converts tryptophan to N-formylkynurenine. The induction of this enzyme was a function of IFN-gamma concentration over the range of 1 to at least 32 NIH reference units/ml. The induction was also a function of time, with the greatest increase in indoleamine 2,3-dioxygenase seen 8-24 h after treatment of cultures with IFN-gamma. The induction of indoleamine 2,3-dioxygenase by IFN-gamma was inhibited by treatment of the cultures with either actinomycin D or cycloheximide, and thus was dependent on both RNA and protein synthesis. The indoleamine 2,3-dioxygenase induced by IFN-gamma appeared to differ from other mammalian enzymes that degrade tryptophan. It had a Km for tryptophan that was 100-fold lower than that for rat liver tryptophan 2,3-dioxygenase and its substrate specificity was narrower than that of rabbit intestine indoleamine 2,3-dioxygenase. N-Formylkynurenine formamidase, the enzyme that produces kynurenine, was a constitutive enzyme and its activity was not further increased by treatment of human fibroblasts with IFN-gamma. The indoleamine 2,3-dioxygenase induced by IFN-gamma did not appear to play a major role in the antiviral activity of IFN-gamma in human fibroblasts.

Aphidicolin, a mycotoxin that inhibits eucaryotic DNA polymerase alpha, blocked the growth of Toxoplasma gondii in confluent cultured human fibroblasts. Aphidicolin immediately inhibited DNA synthesis by T. gondii while it had a delayed and less dramatic effect on RNA synthesis. A mutant of T. gondii resistant to aphidicolin was isolated with the aid of mutagenesis by ethylnitrosourea. Parasite growth measured three days after drug treatment and parasite DNA synthesis measured immediately after drug treatment were, respectively, five- and four-fold more resistant to aphidicolin in the mutant as compared with the wild type parasite. The mutant had a three-fold greater capacity than the wild type to incorporate uracil into its deoxycytidine triphosphate pool. This increased deoxycytidine triphosphate pool is the probable explanation for the mutant's resistance because this deoxynucleotide is known, in mammalian cells, to reverse the inhibition of DNA synthesis by aphidicolin in a competitive manner.

Purified P30, the principal iodinatable membrane protein of Toxoplasma gondii, induced proliferation of peripheral blood mononuclear cells from seropositive individuals but not from seronegative individuals. Culture supernatants from stimulated cells of seropositive individuals blocked the growth of T. gondii in human fibroblasts, whereas those from antibody-negative individuals failed to do so. The anti-toxoplasmic effect of culture supernatants correlated with the induction of indoleamine 2,3-dioxygenase and the destruction of tryptophan, as previously described for fibroblasts treated with recombinant gamma interferon (IFN-gamma). The anti-toxoplasmic effect was blocked by monoclonal antibody to IFN-gamma. The protective effect correlated with the amount of IFN-gamma in the culture supernatant, as measured by inhibition of viral CPE. Thus, the level of IFN-gamma appears to be an important immune factor in protection against toxoplasmosis in humans.

Several monoclonal antibodies against Toxoplasma gondii were parasiticidal in the presence of normal human serum as measured by reduction in plaque titer or in assays that detected lysis. One monoclonal antibody, G, was used to select a resistant mutant from a large population of chemically mutagenized wild-type parasites. This mutant retained the wild-type sensitivity to other monoclonal antibodies and to polyclonal antisera. A comparison of surface radioiodinated wild-type and mutant parasites by using one and two-dimensional electrophoresis showed that the mutant lacked a protein (or proteins) of approximate m.w. 22,000. An immunoprecipitation procedure using monoclonal antibody G yielded a protein(s) of this m.w. from surface radioiodinated wild-type T. gondii but not from surface radioiodinated mutant parasites.

The replication of both alpha- and picornaviruses results in the production of plus-stranded viral progeny. Each of these two classes of virus devotes about the same amount of viral genetic information to structural proteins but the alphaviruses use more genetic information for non-virion proteins. This additional information may be required because the alphaviruses have a much broader host range. Alternatively, the additional information may be required for interrupted transcription that yields a subgenomic mRNA that encodes all of the alphaviral structural proteins. This subgenomic mRNA allows preferential synthesis of a precursor af all virion proteins. Cleavage of this precursor has been observed during in vitro protein synthesis and can be prevented by an appropriate temperature-sensitive mutation.

The effect of human recombinant interferon-gamma (IFN-gamma) on Toxoplasma gondii in cultured human fibroblasts is predominantly parasitostatic. This effect is dependent upon the induction in the host cell of a potent indoleamine 2,3-dioxygenase that converts tryptophan to N-formylkynurenine. This product is, in turn, degraded to kynurenine by a formamidase. Within 24 h of treatment with IFN-gamma most of the tryptophan originally present in the medium is converted to these products together with some minor metabolites. When added to the medium of infected cultures at concentrations equimolar to the tryptophan content, neither N-formylkynurenine nor kynurenine suppresses the growth of T. gondii, although at higher concentrations they are effective. The medium of uninfected cultures treated with IFN-gamma for 24 h has no effect on the growth of T. gondii, when transferred to fresh cultures provided that the residual IFN-gamma is first removed by ultrafiltration or neutralized with a specific monoclonal antibody. Thus minor metabolites produced from tryptophan in response to IFN-gamma and excreted into the medium are not parasitostatic. When cultures treated with IFN-gamma for 24 h are incubated with medium that contains [3H]tryptophan, the radioactive amino acid is converted to N-formylkynurenine and kynurenine as rapidly as it enters the cell. This degradation not only results in a very low intracellular concentration of tryptophan but also produces intracellular concentrations of tryptophan metabolites that are significantly higher than the tryptophan concentration in control cells. However, it is unlikely that either metabolite reaches intracellular concentrations that are sufficient to suppress the growth of the parasite. The parasitostatic effect of IFN-gamma is most likely to result from the starvation of T. gondii for tryptophan.

Adenine arabinoside, which inhibits the synthesis of DNA by intracellular Toxoplasma gondii, is more inhibitory to the parasite than it is to cultured human fibroblast host cells. A single-step mutant of T. gondii that is 50-fold more resistant to adenine arabinoside was isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Cytosine arabinoside is notably more inhibitory to cultured human cells than it is to T. gondii. Thus in infected cells treated with cytosine arabinoside and labeled with 3H-deoxyuridine nearly all of the label is specifically incorporated into the intracellular T. gondii.

Pyrimidine biosynthesis was studied in actively dividing, intracellular Toxoplasma gondii, in mutant Chinese hamster ovary cells blocked in pyrimidine biosynthesis to eliminate any contribution by the host cell. The parasite grew normally in these cells even though pyrimidines were not supplied in the medium. Uninfected, mutant cultures showed negligible pyrimidine synthesis. However, mutant cultures infected wit T. gondii efficiently incorporated 14C from glucose or aspartic acid into the pyrimidine bases of nucleic acids. Thus T. gondii is capable of de novo pyrimidine biosynthesis. The parasite may also be able to use pyrimidines of the host cell, because of pyrazofurin, an antimetabolite that blocks pyrimidine biosynthesis, markedly inhibited only a mutant parasite defective in the salvage of pyrimidines.

Indirect immunofluorescence using anti-myosin rabbit sera showed myosin localized in a characteristic pattern at the anterior pole of Toxoplasma gondii. This polar fluorescent staining was abolished by pre-absorption of the anti-sera with myosin extracted from avian muscle. Both intracellular and extracellular T. gondii showed similar patterns when formaldehyde-fixed, but neither showed polar fluorescence when acetone was used as the sole fixative. Immunofluorescent staining of live T. gondii revealed no polar fluorescence, suggesting that myosin is not present on the outer parasite membrane. Anti-myosin serum did not prevent host cell invasion and plaque formation in the presence of human complement. Inhibition of contractile proteins with cytochalasin D inhibited T. gondii motility and infectivity in a plaque formation assay. The pattern of polar fluorescence described here resembles the IgM-associated polar staining frequently detected in human sera, but we believe it is a different phenomenon because human sera that showed such staining retained their activity after pre-absorption with avian myosin. The unusual localization of myosin at the anterior pole of T. gondii tachyzoites may play a role in the function of anterior organelles, which are thought to facilitate the invasion of host cells.

Stage-specific antigens of Toxoplasma gondii have not been reported. We developed a panel of monoclonal antibodies that identified unique surface membrane antigens on the sporozoite and oocyst wall of T. gondii. These monoclonal antibodies failed to react with T. gondii tachyzoite surface membrane antigens in several immunologic assays. A comparison of surface membrane radioiodinated tachyzoites and sporozoites by one- and two-dimensional electrophoresis showed that T. gondii sporozoites have two major membrane proteins of approximate m.w. 67,000 and 25,000, not present in the tachyzoite stage. Two of the stage-specific monoclonal antibodies (2E4 and 5A4) were directed against the sporozoite 67,000 m.w. protein. In addition, T. gondii sporozoites are deficient in the major radioiodinated tachyzoite protein P30, which is identified by both human and mouse convalescent antitoxoplasma antisera. A convalescent antitoxoplasma serum failed to recognize sporozoite antigens by both immunoprecipitation and reaction with nitrocellulose transfer blots.

We have previously reported the isolation and preliminary characterization of a mutant of Toxoplasma gondii that was resistant to adenine arabinoside. Fiftyfold higher concentrations of adenine arabinoside were required to inhibit the growth of the resistant parasite in human fibroblast cultures. To determine the enzymic basis for resistance, we measured the kinases and deaminases that act on adenosine or deoxyadenosine. All of these enzymic activities were found in uninfected human fibroblast cells. The mutant and wild type parasite proved to have similar activities of adenosine deaminase, deoxyadenosine deaminase, and deoxyadenosine kinase. However, the adenine arabinoside resistant mutant had less than 0.1% of the adenosine kinase activity observed in the wild type T. gondii. The mutant parasite is presumably resistant because without adenosine kinase to phosphorylate adenine arabinoside it cannot carry out the first step in the conversion of the analogue to adenine arabinoside triphosphate, the active form. A mutant of 3T6 (mouse) cells previously selected for a loss of adenosine kinase also proved to be resistant to adenine arabinoside.

The growth of Toxoplasma gondii in cultured human fibroblasts was inhibited by recombinant human gamma interferon at concentrations of 8 to 16 U/ml. The interferon was titrated by observing a total inhibition of parasite plaque formation 7 days after infection. Inhibition of the growth of T. gondii in the early days after infection was measured by marked reductions in the incorporation of radioactive uracil, a precursor that can only be used by the parasites. This assay showed that when cells were pretreated with gamma interferon for 1 day and then infected, inhibition of T. gondii growth could be readily detected 1 or 2 days after infection. When the pretreatment was omitted and parasites and gamma interferon were added at the same time, no inhibition of parasite growth could be detected 1 day later, although it was apparent after 2 days. Cultures from which the gamma interferon had been removed by washing after a 1-day treatment showed inhibition of T. gondii growth. Gamma interferon had no effect on the viability of extracellular parasites, but it did inhibit the synthesis of host cell RNA and protein by ca. 50% 3 days after treatment. This degree of inhibition is unlikely, of itself, to compromise the growth of T. gondii. Recombinant alpha and beta interferons had no effect on the growth of T. gondii.

The anticoccidial drug arprinocid and arprinocid-N-oxide, a metabolite produced in vivo, blocked the growth of Toxoplasma gondii in human fibroblasts. The more potent arprinocid-N-oxide inhibited growth by 50% at 20 ng/ml while arprinocid inhibited at 2 micrograms/ml. For both drugs, the host cell was less sensitive than was the parasite. Hypoxanthine did not reverse the antitoxoplasma activity of either drug. We isolated a parasite mutant, R-AnoR-1 that was 16- to 20-fold more resistant to arprinocid-N-oxide than was the wild type RH T. gondii. This mutant was not resistant to arprinocid in vitro. Arprinocid in a daily oral dose of 136 micrograms regularly protected mice against an otherwise fatal infection with RH T. gondii and 55 micrograms had some protective effect. However, all mice infected with R-AnoR-1 and treated with 360 micrograms arprinocid per day died. Since this mutant is fully sensitive to arprinocid, the form of the drug that is therapeutically active in vivo cannot be arprinocid and is likely to be arprinocid-N-oxide.

Radioactive uracil was not significantly incorporated into the nucleic acids of human fibroblast cells. Infection of these cells with Toxoplasma gondii resulted in an exponential increase in the rate of uracil incorporation that paralleled the exponential growth of the parasite. One day after infection the rate of uracil incorporation was increased 100-fold. It was established by autoradiography that all of the [3H] uracil was incorporated into the intracellular parasites. A possible explanation for this difference in ability to use uracil is our observation that the specific activity of uridine phosphorylase was 100-fold greater in partially purified parasites than in the host cell.

Toxoplasma gondii, an obligate intracellular parasite, is readily grown in nearly all cultured cells. The host-parasite relationship in these cultures can often be explored by using mutant host cells or mutant parasites. Host cells incapable of incorporating uracil or hypoxanthine, which were excellent precursors for T. gondii, allowed the demonstration that the host cell had no access to the purine or pyrimidine nucleotide pools of the parasite. Conversely, a T. gondii mutant that was defective in the principal pyrimidine salvage pathway allowed the demonstration that the parasite had no access to pyrimidine nucleotide or deoxynucleotide pools of the host cell. One metabolite that must pass from the host cytoplasm to T. gondii is a purine. An absolute defect in purine biosynthesis by the parasite was disclosed by growing T. gondii in a mutant host cell that was, itself, incapable of purine synthesis. T. gondii grew normally at 40 degrees C in a mutant host cell that was incapable of protein synthesis at that temperature. Thus, the parasite did not depend on concomitant protein synthesis in the host cell. An antigenic mutant of T. gondii was isolated with the aid of parasiticidal monoclonal antibody. This mutant lacked a major parasite surface protein, of relative molecular mass (Mr) 22 000. The antibody used to select this mutant immunoprecipitated a protein of this Mr from the wild-type parasite.

Emimycin was a potent and selective inhibitor of the growth and nucleic acid synthesis of Toxoplasma gondii in human fibroblasts. An emimycin-resistant mutant of T. gondii lost the pyrimidine salvage enzyme uracil phosphoribosyltransferase, the same enzyme absent in parasites resistant to fluorodeoxyuridine. The mutant resistant to emimycin was completely cross-resistant to fluorodeoxyuridine. Emimycin was as good a substrate as uracil for the uracil phosphoribosyltransferase of T. gondii. [3H]Emimycin supplied in the medium of cultures with actively growing intracellular parasites was converted to emimycin riboside-5'-phosphate in the soluble pool of T. gondii. All other emimycin analogs of uracil-containing nucleotides were also formed but little emimycin riboside diphosphate-N-acetylhexosamine was found. [3H]Emimycin was not converted to analogs of the cytidine nucleotides. When intracellular T. gondii were treated with a concentration of [3H]emimycin that partially inhibited parasite RNA synthesis, much less [3H]emimycin was incorporated into RNA than would be predicted by the amount of intracellular [3H]emimycin riboside triphosphate.

The induction of mutants resistant to 5-fluorodeoxyuridine (FUDR) was used to measure the efficiency of various physical and chemical mutagens on extracellular and intracellular Toxoplasma gondii. The frequency of resistant mutant was measured by plaque assay in human fibroblast cultures in the presence and absence of FUDR. When considered as a function of lethality, the most efficient mutagenesis was obtained with nitrosoguanidine treatment of extracellular parasites and with ethylmethane sulfonate treatment of actively growing intracellular parasites. Each of these treatments increased the frequency of FUDR-resistant mutants from less than one to more than 200 per million parasites. Ultraviolet irradiation, X-rays, and the alkylating mustard ICR-191 also induced FUDR-resistant mutants in a dose-dependent fashion.