Identification and characterisation of high affinity nucleoside and nucleobase transporters in Toxoplasma gondii

Abstract

The protozoan parasite Toxoplasma gondii depends upon salvaging the purines that it requires. We have re-analysed purine transport in T. gondii and identified novel nucleoside and nucleobase transporters. The latter transports hypoxanthine (TgNBT1; K m =0.91±0.19 μM ) and is inhibited by guanine and xanthine: it is the first high affinity nucleobase transporter to be identified in an apicomplexan parasite. The previously reported nucleoside transporter, TgAT1, is low affinity with K m values of 105 and 134 μM for adenosine and inosine, respectively. We have now identified a second nucleoside transporter, TgAT2, which is high affinity and inhibited by adenosine, inosine, guanosine, uridine and thymidine ( K m values 0.28–1.5 μM) as well as cytidine ( K i =32 μM ). TgAT2 also recognises several nucleoside analogues with therapeutic potential. We have investigated the basis for the broad specificity of TgAT2 and found that hydrogen bonds are formed with the 3′ and 5′ hydroxyl groups and that the base groups are bound through H-bonds with either N3 of the purine ring or N(3)H of the pyrimidine ring, and most probably π–π-stacking as well. The identification of these high affinity purine nucleobase and nucleoside transporters reconciles for the first time the low abundance of free nucleosides and nucleobases in the intracellular environment with the efficient purine salvage carried out by T. gondii.