THE ADENOSINE TRANSPORTER OF TOXOPLASMA GONDII: IDENTIFICATION BY INSERTIONAL MUTAGENESIS, CLONING, AND RECOMBINANT EXPRESSION

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