Toxoplasmic encephalitis is a common complication of AIDS. Although the initial response to conventional chemotherapy is generally good, prolonged treatment is often difficult because of hypersensitivity to the drugs, particularly to the sulfonamides. Thus, toxoplasmic encephalitis is a significant factor in the morbidity and mortality of AIDS patients. The long term goal is to develop new drugs to treat this opportunistic infection. Toxoplasma gondii is absolutely dependent upon an exogenous source of preformed purines and usually obtains them from its host cell. Identification of the critical bottleneck steps in purine acquisition by the parasite should define new targets for chemotherapy. Preliminary studies suggest that a parasite ecto-AMPase (or 5'-nucleotidase) is a bottleneck through which all purine nucleotides from the host cell must pass and is thus an inviting target for chemotherapy.
The specific aims with respect to T.gondii AMPase are to (1) study this gene by molecular approaches, (2) select enzyme-negative parasite mutants and determine if they become absolutely dependent on the addition of purines to the medium, thus confirming the essential role of ecto-AMPase, (3) purify and characterize the AMPase, and (4) study the antitoxoplasma activity of AMPase inhibitors. Preliminary studies suggest that the purine nucelobases and nucleosides derived from the host cell pool are reconverted to nucleotides within the T.gondii cytoplasm by only two enzymes, adenosine kinase (AK) or hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Thus, these two enzymes represent a double bottleneck in purine acquisition by T.gondii.
The specific aims with respect to these two enzymes are to (1) isolate temperature sensitive mutants that will allow both enzymes to be shut down at the restrictive temperature and thus test the double bottleneck hypotheses, (2) determine the antitoxoplasma activity of inhibitors of each enzyme using parasite mutants that lack the other enzyme, and (3) test the anti-wild type-toxoplasma activity of dual drug treatment that blocks both enzymes. Preliminary studies show that 6-thioxanthine is a specific inhibitor of T.gondii because only the parasite can convert this analogue to nucleotide form.
The specific aims with respect to 6- thioxanthine are to (1) assay all of the nucleotide interconversion enzymes of T.gondii, several of which have a potential role in the antitoxoplasma activity of 6-thioxanthine, and (2) determine if 6- thioxanthine acts primarily by inhibiting GMP synthesis or by being converted to the toxic GMP analogue, 6-thioguanosine-5-'phosphate. The investigators view the primary goal of this proposal to be the definitive characterization of those steps in toxoplasma purine metabolism that are targets for the chemotherapy of toxoplasmosis.
| Belperron, A A; Fox, B A; Horii, T et al. (2001) Toxoplasma gondii: genetic selection of tethered dihydrofolate reductase-thymidylate synthase fusion proteins. Exp Parasitol 98:167-70 |
| Pfefferkorn, E R; Bzik, D J; Honsinger, C P (2001) Toxoplasma gondii: mechanism of the parasitostatic action of 6-thioxanthine. Exp Parasitol 99:235-43 |
| Fox, B A; Belperron, A A; Bzik, D J (2001) Negative selection of herpes simplex virus thymidine kinase in Toxoplasma gondii. Mol Biochem Parasitol 116:85-8 |
