IMP dehydrogenase from the protozoan parasite Toxoplasma gondii

Abstract

The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied in T. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme in T. gondii. Northern blot analysis shows that two IMPDH transcripts are present in T. gondii tachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD+, with a intercept inhibition constant (Kii) of 0.03+/-0.004 microM. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable beta-methylene derivatives of TAD or SAD were inhibitory, with Kii values 13- to 60-fold higher than that of mycophenolic acid.

FIG. 1.

T. gondii IMPDH gene. A. Schematic diagram of TgIMPDH locus. Gene-specific primers used for 5′- and 3′-RACE are designated by arrows. Black boxes denote exons, white boxes denote introns, and gray boxes denote untranslated regions. B, BamHI; Bg, BglII; H, HindIII. B. Southern blot analysis. Genomic DNA from T. gondii was digested with the indicated restriction enzymes and probed with labeled cDNA representing TgIMPDH. Numbers at left are sizes in kilobases. C. Northern analysis. Full-length cDNA representing the entire ∼1.6-kb coding region of TgIMPDH was used to probe a Northern blot containing T. gondii mRNA (Tg) and host cell (HFF) total RNA.

FIG. 2.

IMPDH amino acid sequence alignments. Protein sequence alignments from various IMPDH homologues were compiled with CLUSTALW with manual adjustment. Tg, Toxoplasma gondii; Pf, Plasmodium falciparum; Tb, Trypanosoma brucei; Ld, Leishmania donovani; Pc, Pneumocystis carinii; Sc, Saccharomyces cerevisiae; H1 and H2, Homo sapiens isoforms 1 and 2, respectively. Indicated are the CBS domains as well as the active-site loop and flap. The atypical serine-rich insertion in TgIMPDH is shown in italics. Asterisks denote residues that are identical across these species. Colons represent conserved amino acid substitutions, while periods represent less conserved residue substitutions.

FIG. 3.

Localization of recombinant TgIMPDH. Recombinant TgIMPDH containing a C-terminal FLAG-tag fusion localizes to the parasite cytoplasm. Parasites were costained with DAPI, which stains the nuclei of parasites (pN) and host cells (hN).

FIG. 4.

Expression and purification of recombinant TgIMPDH. Transgenic parasite clones stably expressing recombinant TgIMPDH_FLAG were selected in mycophenolic acid and xanthine following the electroporation of plasmid into RHΔHX parasites. TgIMPDH was purified by FLAG-based affinity chromatography and eluted from the resin with 3× FLAG peptide. Lysate (L), the unbound fraction (U), and the eluate (E) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were visualized by SimplyBlue stain. Molecular mass markers are shown in kilodaltons.

FIG. 5.

Mycophenolic acid inhibition of recombinant TgIMPDH. The dependence of the enzymatic reaction on NAD⁺ was assessed for purified recombinant TgIMPDH_FLAG with and without a fixed concentration of mycophenolic acid (MPA). Results from four independent experiments were combined; means with error bars representing the standard errors of the means are shown. Data were fitted to the equation describing substrate inhibition, since high concentrations of NAD⁺ were inhibitory (see Materials and Methods). Nonlinear regression (Prism 3.0) was used to calculate V_max and K_m values for both curves. The V_max values (means ± standard errors of the means) are 0.570 ± 0.16 and 0.168 ± 0.04 μM/min for the top and bottom curves, respectively; the K_m values are 158.1 ± 86.7 and 46.7 ± 25.7 μM for the top and bottom curves, respectively.

FIG. 6.

Analogues of tiazofurin. All compounds were obtained from the Drug Development and Clinical Sciences Branch of the National Institute of Allergy and Infectious Diseases (Mohamed Nasr). A commercial preparation of tiazofurin was also tested. Tiazofurin is 2-β-d-ribofuranosylthiazole-4-carboxamide; Se-tiazofurin is 2-β-d-ribofuranosylselenazole-4-carboxamide; TAD is thiazole-4-carboxamide adenine dinucleotide; β-methylene TAD is 4-carboxamido-2-β-d-ribofuranosylthiazolyl adenosine methylenediphosphonic acid; β-methylene SAD is 4-carboxamido-2-β-d-ribofuranosylselenazolyl (5′ + 5′) adenosine methylenediphosphonic acid.