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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.

SUBMITTER: Sullivan WJ

PROVIDER: S-EPMC1140536 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

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IMP dehydrogenase from the protozoan parasite Toxoplasma gondii.

Sullivan William J WJ Dixon Stacy E SE Li Catherine C Striepen Boris B Queener Sherry F SF

Antimicrobial agents and chemotherapy 20050601 6

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 b ...[more]

PMID: 15917510

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Project description:BackgroundLysine lactylation (Kla) is a novelposttranslational modification (PTM) identified in histone and nonhistone proteins of several eukaryotic cells that directly activates gene expression and DNA replication. However, very little is known about the scope and cellular distribution of Kla in apicomplexan parasites despite its significance in public and animal health care.MethodsToxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular apicomplexan parasite that can infect different nucleated cell types of animals and humans. We used this parasite as a model organism and extracted the total protein of tachyzoites to produce the first global lysine lactylome profile of T. gondii through liquid chromatography-tandem mass spectrometry. We also investigated the level and localization of the Kla protein in T. gondii using western blotting and the indirect fluorescent antibody test (IFA), respectively.ResultsA total of 983 Kla sites occurring on 523 lactylated proteins were identified in the total protein extracted from Toxoplasma tachyzoites, the acute toxoplasmosis-causing stage. Bioinformatics analysis revealed that the lactylated proteins were evolutionarily conserved and involved in a wide variety of cellular functions, such as energy metabolism, gene regulation and protein biosynthesis. Subcellular localization analysis and IFA results further revealed that most of the lactylated T. gondii proteins were localized in the nucleus, indicating the potential impact of Kla on gene regulation in the T. gondii model. Notably, an extensive batch of parasite-specific proteins unique to phylum Apicomplexa is lactylated in T. gondii.ConclusionsThis study revealed that Kla is widespread in early dividing eukaryotic cells. Lactylated proteins, including a batch of unique parasite proteins, are involved in a remarkably diverse array of cellular functions. These valuable data will improve our understanding of the evolution of Kla and potentially provide the basis for developing novel therapeutic avenues.

Project description:Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. The ability of T. gondii and other apicomplexan parasites to invade into, egress from, and move between cells of the hosts they infect is critical to parasite virulence and disease progression. An unusual and highly conserved parasite myosin motor (TgMyoA) plays a central role in T. gondii motility. The goal of this work was to determine whether the parasite's motility and lytic cycle can be disrupted through pharmacological inhibition of TgMyoA, as an approach to altering disease progression in vivo. To this end, we first sought to identify inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motor's actin-activated ATPase activity. The top hit to emerge from the screen, KNX-002, inhibited TgMyoA with little to no effect on any of the vertebrate myosins tested. KNX-002 was also active against parasites, inhibiting parasite motility and growth in culture in a dose-dependent manner. We used chemical mutagenesis, selection in KNX-002, and targeted sequencing to identify a mutation in TgMyoA (T130A) that renders the recombinant motor less sensitive to compound. Compared to wild-type parasites, parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002. Finally, we present evidence that KNX-002 can slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. Taken together, these data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target in infections with T. gondii. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, pharmacological inhibition of MyoA offers a promising new approach to treating the devastating diseases caused by T. gondii and other apicomplexan parasites.

Dataset Information

IMP dehydrogenase from the protozoan parasite Toxoplasma gondii.

Publications

IMP dehydrogenase from the protozoan parasite Toxoplasma gondii.

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