Authors

Discussion

Toxoplasma gondii is one of the most widely distributed parasites in the world, causing toxoplasmosis in warm-blooded animals. T. gondii is a very rapidly proliferating intracellular parasite that uses the cat as a definitive host and many mammalian species including humans as intermediate host. All protozoan parasites including Toxoplasma gondii are unable to synthesise purines like adenosine and guanosine and must thus take them up from the host cell. This process requires highly efficient purine transport, which makes them vulnerable to nucleoside analogue drugs. Four ENT sequences were found in the genome sequence of T. gondii; in the VEG strain they are named as TGVEG359630, TGVEG288540, TGVEG244440 and TGVEG233130. TGVEG244440 was amplified from T. gondii cDNA of the RH strain and was successfully expressed in T. brucei procyclic forms TbNBT-KO (nucleobase transport null mutant). The other 3 ENTs toxoplasma genes could not be amplified from RH strain and were instead synthesised and expressed in nucleoside transport null background, i.e. ΔNT1.1, ΔNT1.2, ΔNT2, as well as in an L. mexicana NT3-KO and TbNB-KO strains for nucleobase null expression. This work is ongoing.

Chiang et al., 1999 reported that TgVEG_244440 (which they called TgAT1) is an adenosine transporter of low affinity. However, our results demonstrated that TGVEG244440 is a high-affinity oxopurine transporter (guanine/hypoxanthine transporters) with K_m values of 1.25 ± 0.16 µM for guanine and 0.79 ± 0.07 µM for hypoxanthine, as well as quite high affinity for guanosine and inosine with Ki values (3.30 ± 0.30 µM; 4.05 ± 1.37 µM) respectively. However, it has not been yet established conclusively whether these nucleosides are substrates or inhibitors of this transporter. We did confirm the low affinity for adenosine but the earlier study seems to have missed the more important nucleobase transport function. We have now extensively characterised TGVEG244440 using different purine analogues of nucleobases and nucleosides and elucidated the binding modes of the various substrates, finding that TgVEG_244440 binds purine nucleobases and nucleosides in different orientations.