(T1330-10-67) Assay Development and Characterization of T. gondii Nucleoside Transporters in MDCK-II Cells

Purpose: Toxoplasma gondii lack the ability for de novo synthesis of purines thus rely on salvage of host purines. Blockade of purines uptake through T. gondii purine transporters is poised to a viable therapy for toxoplasmosis. The purpose of this study is to develop an in vitro cellular model for T. gondii nucleoside transporters in modal MDCK-II cells suitable for assessing the T. gondii purine transporter inhibitors with therapeutical potentials.

Methods: Three putative nucleoside transporters of ME49 strain of T. gondii, Tg_233130, Tg_244440 and Tg_288540 were selected from Toxo database (ToxoDB.org). Synthesized DNA was inserted into gWiz plasmids and transfected into MDCK-II cells. Human ENT1 transporter was used for comparison. Cellular uptake of the purine nucleosides adenosine and hypoxanthine were assessed and inhibition by putative inhibitors were evaluated.

Results: Among the three T. gondii purine transporters, strikingly, only Tg_244440, the smallest protein with 462 amino acid, demonstrated the uptake of purine nucleoside adenosine. Tg_244440 also transported the pyrimidine nucleotide uridine, and to a less extent the purine nucleobase hypoxanthine. The rest of the T. gondii transporters failed to show uptake of any of these prototypical substrates of human ENT1. The kinetics study showed a higher affinity of Tg_244440 toward adenosine with K_m of 3.60 µM, than urdine with a K_m of 214 µM. Adenosine transport was inhibited by dipyridamole with an IC₅₀ value of 1.44 µM but was insensitive to NBMPR. In contrast, human ENT1 transport of adenosine was readily blunted by both dipyridamole (IC₅₀=0.098 µM) and NBMPR (IC₅₀=0.042 µM).

Conclusion: Tg_244440 is the functional T. gondii nucleoside transporter expressed in MDCK-II cells, with substrate preference for adenosine than uridine, and is sensitive to dipyridamole inhibition. MDCK-II is a valid test system to evaluate T. gondii purine transporter inhibitors.

Transport of nucleoside/nucleobase by Tg_244440

Km and Vmax of adenosine and uridine transport mediated by Tg_244440

IC50 of dipyridamole and NBMPR against adenosine transport mediated by Tg_244440