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

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