Prodrug activation by Cryptosporidium thymidine kinase

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.

FIGURE 1.

C. parvum pyrimidine nucleotide salvage pathways. Enzymes that appear to have been obtained via horizontal gene transfer are shown in red (bacteria) and green (plant). T, nucleoside transporter.

FIGURE 2.

Determination of the CpTK oligomeric state using gel filtration chromatography. Untagged CpTK elutes at a molecular mass of ∼70 kDa, consistent with being a trimer under the three conditions tested: enzyme alone (diamonds), CpTK + 100 μm dCTP (triangles), and CpTK + 100 μm Thd + 1 mm ATP (squares). The elution volumes of the standards are as follow: blue dextran, 96 ml; bovine γ-globulin, 158 kDa, 107 ml; chicken ovalbumin, 44 kDa, 142 ml; equine myoglobin, 17 kDa, 180 ml; and vitamin B₁₂, 1.35 kDa, 303 ml.

FIGURE 3.

Effect of TFT and FUdR on parasite and host cell growth in a cell culture model of C. parvum infection. A and B, C. parvum growth was assayed by high-content imaging. A, TFT; B, FUdR. C and D, host cell cytotoxicity was assessed using the LIVE/DEAD® assay (Invitrogen). Data are representative of two independent experiments. C, TFT; D, FUdR.

FIGURE 4.

Treatment with TFT in a IL-12 mouse model of C. parvum infection. Number of parasite oocysts in mouse feces 4 and 7 days postinfection with treatment with phosphate-buffered saline (Mock), 200 mg/kg of TFT, and 2000 mg/kg of paromomycin (Pm) is shown. ***, p < 0.0006; *, p < 0.02. The bars denote the averages and S.E.