The adenosine transporter of Toxoplasma gondii. Identification by insertional mutagenesis, cloning, and recombinant expression

Chi Wu Chiang, Nicola Carter, William Sullivan, Robert G K Donald, David S. Roos, Fardos N M Naguib, Mahmoud H. El Kouni, Buddy Ullman, Craig M. Wilson

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

Purine transport into the protozoan parasite Toxoplasma gondii plays an indispensable nutritional function for this pathogen. To facilitate genetic and biochemical characterization of the adenosine transporter of the parasite, T. gondii tachyzoites were transfected with an insertional mutagenesis vector, and clonal mutants were selected for resistance to the cytotoxic adenosine analog adenine arabinoside (Ara-A). Whereas some Ara-A- resistant clones exhibited disruption of the adenosine kinase (AK) locus, others displayed normal AK activity, suggesting that a second locus had been tagged by the insertional mutagenesis plasmid. These Ara-A(r) AK+ mutants displayed reduced adenosine uptake capability, implying a defect in adenosine transport. Sequences flanking the transgene integration point in one mutant were rescued from a genomic library of Ara-A(r) AK+ DNA, and Southern blot analysis revealed that all Ara-A(r) AK+ mutants were disrupted at the same locus. Probes derived from this locus, designated TgAT, were employed to isolate genomic and cDNA clones from wild-type libraries. Conceptual translation of the TgAT cDNA open reading frame predicts a 462 amino acid protein containing 11 transmembrane domains, a primary structure and membrane topology similar to members of the mammalian equilibrative nucleoside transporter family. Expression of TgAT cRNA in Xenopus laevis oocytes increased adenosine uptake capacity in a saturable manner, with an apparent K(m) value of 114 μM. Uptake was inhibited by various nucleosides, nucleoside analogs, hypoxanthine, guanine, and dipyridamole. The combination of genetic and biochemical studies demonstrates that TgAT is the sole functional adenosine transporter in T. gondii and a rational target for therapeutic intervention.

Original languageEnglish (US)
Pages (from-to)35255-35261
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number49
DOIs
StatePublished - Dec 3 1999
Externally publishedYes

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Vidarabine
Mutagenesis
Adenosine Kinase
Insertional Mutagenesis
Cloning
Toxoplasma
Organism Cloning
Adenosine
Nucleosides
Molecular Biology
Parasites
Complementary DNA
Clone Cells
Nucleoside Transport Proteins
Complementary RNA
Hypoxanthine
Dipyridamole
Genomic Library
Xenopus laevis
Guanine

ASJC Scopus subject areas

  • Biochemistry

Cite this

The adenosine transporter of Toxoplasma gondii. Identification by insertional mutagenesis, cloning, and recombinant expression. / Chiang, Chi Wu; Carter, Nicola; Sullivan, William; Donald, Robert G K; Roos, David S.; Naguib, Fardos N M; El Kouni, Mahmoud H.; Ullman, Buddy; Wilson, Craig M.

In: Journal of Biological Chemistry, Vol. 274, No. 49, 03.12.1999, p. 35255-35261.

Research output: Contribution to journalArticle

Chiang, CW, Carter, N, Sullivan, W, Donald, RGK, Roos, DS, Naguib, FNM, El Kouni, MH, Ullman, B & Wilson, CM 1999, 'The adenosine transporter of Toxoplasma gondii. Identification by insertional mutagenesis, cloning, and recombinant expression', Journal of Biological Chemistry, vol. 274, no. 49, pp. 35255-35261. https://doi.org/10.1074/jbc.274.49.35255
Chiang, Chi Wu ; Carter, Nicola ; Sullivan, William ; Donald, Robert G K ; Roos, David S. ; Naguib, Fardos N M ; El Kouni, Mahmoud H. ; Ullman, Buddy ; Wilson, Craig M. / The adenosine transporter of Toxoplasma gondii. Identification by insertional mutagenesis, cloning, and recombinant expression. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 49. pp. 35255-35261.
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