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Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii.

ABSTRACT: New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage, suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of uridine 5'-monophosphate (UMP) and thymidine 5'-monophosphate (dTMP) pools, thereby inhibiting parasite RNA and DNA synthesis. Disruption of Toxoplasma gondii carbamoyl phosphate synthetase II (CPSII) induces a severe uracil auxotrophy with no detectable parasite replication in vitro and complete attenuation of virulence in mice. Here we show that a CPSII cDNA minigene efficiently complements the uracil auxotrophy of CPSII-deficient mutants, restoring parasite growth and virulence. Our complementation assays reveal that engineered mutations within, or proximal to, the catalytic triad of the N-terminal glutamine amidotransferase (GATase) domain inactivate the complementation activity of T. gondii CPSII and demonstrate a critical dependence on the apicomplexan CPSII GATase domain in vivo. Surprisingly, indels present within the T. gondii CPSII GATase domain as well as the C-terminal allosteric regulatory domain are found to be essential. In addition, several mutations directed at residues implicated in allosteric regulation in Escherichia coli CPS either abolish or markedly suppress complementation and further define the functional importance of the allosteric regulatory region. Collectively, these findings identify novel features of T. gondii CPSII as potential parasite-selective targets for drug development.

SUBMITTER: Fox BA

PROVIDER: S-EPMC3682482 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii.

Fox Barbara A BA Ristuccia Jessica G JG Bzik David J DJ

International journal for parasitology 20081021 5

New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage, suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of uridine 5'-monophosphate (UMP) and thymidine 5'-monophosphate (dTMP) pools, thereby inhibiting parasite RNA and DNA synthesis. Disruption of ...[more]

PMID: 18992249

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Project description:Toxoplasma gondii is an obligate intracellular parasite that acquires all necessary nutrients from the hosts, but the exact nutrient acquisition mechanisms are poorly understood. Here, we identified three putative phosphate transporters in T. gondii. TgPiT and TgPT2 are mainly on the plasma membrane, whereas TgmPT is localized to the mitochondrion. TgPiT and TgmPT are widely present and conserved in apicomplexan parasites that include Plasmodium and Eimeria species. Nonetheless, they are dispensable for the growth and virulence of Toxoplasma. TgPT2, on the other hand, is restricted to coccidia parasites and is essential for Toxoplasma survival. TgPT2 depletion led to reduced motility and invasion, as well as growth arrest of the parasites both in vitro and in vivo. Both TgPiT and TgPT2 have phosphate transport activities and contribute to parasites' inorganic phosphate (Pi) absorption. Interestingly, the Pi importing activity of Toxoplasma parasites could be competitively inhibited by ATP and AMP. Furthermore, direct uptake of 32P-ATP was also observed, indicating the parasites' ability to scavenge host ATP. Nonetheless, ATP/AMP import is not mediated by TgPiT or TgPT2, suggesting additional mechanisms. Together, these results show the complex pathways of phosphate transport in Toxoplasma, and TgPT2 is a potential target for antitoxoplasmic intervention design due to its essential role in parasite growth. IMPORTANCE To grow and survive within host cells, Toxoplasma must scavenge necessary nutrients from hosts to support its parasitism. Transporters located in the plasma membrane of the parasites play critical roles in nutrient acquisition. Toxoplasma encodes a large number of transporters, but so far, only a few have been characterized. In this study, we identified two phosphate transporters, TgPiT and TgPT2, to localize to the plasma membrane of Toxoplasma. Although both TgPiT and TgPT2 possess phosphate transport activities, only the novel transporter TgPT2 was essential for parasite growth, both in vitro and in vivo. In addition, TgPT2 and its orthologs are only present in coccidia parasites. As such, TgPT2 represents a potential target for drug design against toxoplasmosis. In addition, our data indicated that Toxoplasma can take up ATP and AMP from the environment, providing new insights into the energy metabolism of Toxoplasma.

Dataset Information

Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii.

Publications

Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii.

Similar Datasets

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