Abstract
Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of k(cat) = 0.28 s(-1) and K(m)(,ATP) = 140 microm are similar to those of human TK1, the value of K(m)(thymidine) = 48 microm is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.
Highlights
Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals
Wheat germ lysate CpTK has a Km of 48 M for dT and kcat ϭ 0.28 sϪ1 when the production of dTMP was monitored at saturating concentrations of ATP
These observations suggest that Cryptosporidium must possess another deoxynucleoside kinase
Summary
These compounds are converted to the mono-, di-, and triphosphates and incorporated into DNA and RNA. TFT, AraT, IUdR, IDC, and AraC, but not AZT and acyclovir, have been reported to have anticryptosporidial activity in a cell culture model of infection [15]. All of these nucleosides are potential substrates for CpTK, suggesting that CpTK may have very broad specificity like a viral TK. The antiparasitic effects are observed at concentrations that do not affect the host cells, which is surprising because the host cells contain these same deoxynucleoside salvage enzymes Perhaps this selective antiparasitic activity derives from the catalytic properties of CpTK. TFT inhibits C. parvum growth in a mouse model of C. parvum infection, suggesting that the pyridimidine deoxynucleoside salvage pathways can be subverted for chemotherapy
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