Compositions and methods for treatment of Toxoplasma gondii and other apicomplexans

Pyrimidine auxotroph mutants of apicomplexans are provided which are mutated in one of six enzymes of the de novo pyrimidine biosynthesis pathway. Also provided are methods of protecting an animal against infection by apicomplexans by administering a pyrimidine auxotroph mutant and methods for screening for inhibitors of pyrimidine salvage enzymes in apicomplexans.

EXAMPLES 
 Example 1 
 Parasite Material Most of the work described herein used the RH strain of Toxoplasma gondii which is the most commonly used laboratory strain amongst Toxoplasma researchers (Pfefferkorn et al. Exp. Parasitol. 1976 39:365-376). Due to its long history of continuous passage in the laboratory, this strain is highly virulent in animals and grows rapidly in culture making it ideal for obtaining large amounts of material. However, it has lost the ability to go through the complete sexual cycle in cats. Parasites were grown in vitro in monolayers of cultured human foreskin fibroblasts (HFF) in accordance with well known procedures, for example, Fox et al. Mol. Biochem. Parasitol. 1999 98:93-103. Typically, using the RH strain, infected cultures were maintained by seeding uninfected monolayers; at about a 1:50 dilution every 48-72 hours. This yields about 10 9 parasites from three T175 flasks of infected cultures. Parasites were harvested just as host lysis occurred filter purifying parasites through 3 micron nucleopore filters. Detailed methods for growth, harvesting, passage, purification of tachyzoite parasites, storage, and replication assays of T. gondii are routine and well known, for example, Roos et al. Meth. Microb. Path. 1994 45:23-65, and Fox et al. Mol. Biochem. Parasitol. 1999 98:93-103. All media reagents were purchased from Gibco-BRL (Rockville, Md.), Bio Whittaker (Walkersville, Md.) and Sigma (St. Louis, Mo.). 
 Example 2 
 Chemicals and Enzyme Assays Most chemical or biochemical reagents were purchased from Sigma (St. Louis, Mo.). Ganciclovir was obtained from Roche Labs, Nutley, N.J. The linked assay system for cpsII activity was performed in accordance with known methods, for example, Asai et al. Mol. Biochem. Parasitol. 1983 7:89-100 and Hill et al. Mol. Biochem. Parasitol. 1981 2:123-134. For cpsII assays parasites were lysed in M-PER extraction buffer (Pierce Inc., Rockford, Ill.) or by osmotic shock in 4 volumes (w/v) of 10 mM potassium phosphate (pH 7), 0.05 mM dithiothreitol and protease inhibitors antipain, leupeptin, chymostatin, and pepstatin A, each at 0.1 mM. After 1 to 2 minutes of lysis, glycerol 7.5% (w/v) was added back to the extracts. The lysed parasite extracts were centrifuged at 20,000× g for 15 minutes and the supernatants used in cpsII enzyme assay. CpsII reaction assays contained 50 mM HEPES (pH 7.2), 10% (w/v) glycerol, 20 mM MgCl 2 , 20 mM ATP, 3 mM L-glutamine, 0.5 mM L-ornithine, 10 mM KCl, 0.05 mM dithiothreitol, 1 unit ornithine carbamyl transferase, 10 mM bi&lsqb; 14 C&rsqb;carbonate(1 &mgr;Ci/&mgr;M) and extract in a final volume of 0.1 ml. Sodium bi&lsqb; 14 C&rsqb;carbonate was 30-60 mCi/mmol and obtained from ICN. Reactions were run for 30 minutes at 37° C. and then terminated by addition of 10 &mgr;l of 5 M formic acid. A small piece of solid CO 2 was dropped into the stopped reaction and excess CO 2 was removed by standing the open solution in a fume hood. Reaction volumes were removed, dried and redissolved in 0.1 ml water prior to addition of liquiscint scintillant and counting of &lsqb; 14 C&rsqb; in a Beckman scintillation counter. Thymidine kinase assays were performed in accordance with known methods, for example, Maga et al. (Biochemical Journal 1994 302:279-282). Briefly, parasite extracts were lysed by sonication (1 minute, kontes microtip) or in M-PER extraction buffer plus a cocktail of protease inhibitors (antipain, leupeptin, chymostatin, and pepstatin A; 10 &mgr;g each) and 1 mM phenylmethylsulfonyl fluoride. TK assays were run in a 50 &mgr;l volume at 37° C. for 30 minutes in a mixture containing 30 mM potassium HEPES (pH 7.5), 6 mM ATP, 6 mM MgCl 2 , 0.5 mM dithiothreitol and 3.3 &mgr;M &lsqb; 3 H&rsqb;Thymidine (20-40 Ci/mmol from ICN). The reaction was terminated by transferring 25 &mgr;l of the incubation mixture to DE81 ion exchange paper (Whatman, Clifton, N.J.). The spotted paper was washed in 1 mM ammonium formate (pH 3.6) to remove unconverted nucleoside, distilled water, and then a final ethanol wash prior to drying of the paper and scintillation counting in liquiscint. Protein determination for parasite protein extracts was determined using Bio-Rad protein assay reagents and bovine serum albumin in accordance with standard procedures (Bio-Rad, Hercules, Calif.). 
 Example 3 
 Molecular Methods Molecular methods including DNA isolation, restriction, Southern blotting, hybridization, and PCR reactions used herein are all well known, for example, Bzik et al. Proc. Natl. Acad. Sci. USA 1987 84:8360-8364 and Fox et al. Mol. Biochem. Parasitol. 1999 98:93-103. Transfection of T. gondii was also performed in accordance with routine procedures (Roos et al. Meth. Microb. Path. 1994 45:23-65 and Fox et al. Mol. Biochem. Parasitol. 1999 98:93-103). The gene libraries were developed from HindII or PstI digested genomic DNA cloned into bluescript KSII digested with the same enzyme and treated with alkaline phosphatase prior to ligation with T. gondii DNA fragments. Libraries were manipulated in accordance with known methods (Bzik et al. Proc. Natl. Acad. Sci. USA 1987 84:8360-8364). Total mRNA was isolated from T. gondii using TRIZOL-LS reagent (Gibco-BRL, Rockville, Md.) and mRNA was converted to cDNA using a cDNA kit from Pharmacia (Piscataway, N.J.) with polydT or random hexamers primers. DNA sequencing was done using classical dideoxy chain termination or automated sequencing using fluorescent dyes (ABI sequencer, Foster City, Calif.). DNA sequences were analyzed using the MacVector suite of programs (Oxford Molecular, Beaverton, Oreg.) and resources at NCBI, such as blast search. The DHFRm2m3-TS allele was obtained from the NIH AIDS Reference and Reagent Center (Rockville, Md.). The TK75 allele which was described by Black et al. Proc. Natl Acad. Sci. USA 1996 93:3525-3529 was obtained from Darwin (Seattle, Wash.). Bluescript plasmid was from Stratagene (La Jolla, Calif.). Restriction enzymes, nucleic acid modifying enzymes and transfer membranes were from Boehringer Mannheim, Indianapolis, Ind. 
 Example 4 
 Experimental Infection and Animal Studies Balb/c inbred mice and balb/c mice bearing a homozygous knock-out of interferon gamma (gko) were obtained from Jackson Labs (Bar Harbor, Me.). Tachyzoite parasites were aseptically handled and purified from freshly lysed monolayers of infected HFF cells through a sterilized 3 micron polycarbonate membrane (Nucleopore, Cambridge, Mass.). Parasite concentration was scored microscopically in a hemocytometer. Purified parasites were pelleted at 1500 g for 10 minutes and washed in sterile EMEM media with no supplements and without disturbing the parasite pellet. The centrifuge tube was centrifuged once more for 2 minutes and the supernatant removed and replaced with EMEM media containing no supplements in a volume of EMEM to give a 10 times higher concentration (per/ml) of parasites than the highest dose. This was done so inoculation of 0.1 ml of this solution would equal the highest parasite dose. Parasites were gently resuspended in sterile EMEM (no additions). Mice, in groups of four, were inoculated with appropriate doses of tachyzoite parasites in EMEM. Following inoculation of mice the residual volume of unused tachyzoite parasites was returned to the sterile hood and dilutions were made to represent 200 and 400 parasite plaques on 25 cm 2 HFF flasks assuming 100% recovery of parasites after centrifugation/resuspension and 100% percent viability. Then, following a 7 day plaque assay, actual plaques were counted, post-inoculation of mice, and the percent viable PFU ratio to parasite counts in the hemocytometer were determined microscopically in every experimental infection. Uniformly, all of the mutants described herein as well as RH parasites always fell in the range of 0.4 to 0.6 viable PFU per parasite counted using these conditions. Following inoculation of mice, mice were observed daily for signs of infection (or distress) or death.