Authors

Discussion

Toxoplasma gondii are unable to synthesize purines de novo, and instead salvages them from its environment, for which they need high affinity carriers because the concentration of free unphosphorylated nucleosides and nucleobases is very low inside the host cell. Apart from the essential role in purine salvage, nucleoside analogues have shown promise as potential therapeutics against T. gondii. In order to optimise the targeting of such analogues, the characterization of the purine transporters is required.

Here, we report the expression of one of the four T. gondii Equilibrative Nucleoside Transporter genes, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters had been deleted. Tg244440 transported nucleobases hypoxanthine and guanine with similar affinity (K_m ~1 µM), while inosine and guanosine displayed K_i values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter.

A large number of purine analogues were used to probe the substrate-transporter binding interactions, mostly through competitive inhibition of [³H]-guanine transport, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases. The unprecedented flexibility of the binding pocket, apparently allowing oxopurine bases and nucleosides to bind with very similar affinity but different orientations, shows the potential of T. gondii purine transporters for the uptake of cytotoxic purine analogues.