Identification of TgENT1 as the TgUUT1 Uracil/Uridine Transporter of Toxoplasma gondii

The protozoan pathogen <i>Toxoplasma gondii</i> is responsible for toxoplasmosis, a disease that can be deadly in immunocompromised patients and the developing fetus during pregnancy. Current treatments are widely considered to be suboptimal. We have recently reported that 5-fluoropyrimidines have highly promising anti-toxoplasmosis effects and are internalized by the parasite by a high-affinity uracil/uridine transporter, TgUUT1. Here, we attempt to identify the gene encoding this transport protein. The only nucleoside or nucleobase family identified in the <i>T. gondii</i> genome was the Equilibrative Nucleoside Transporter (ENT) family, with four members. Of these, TgAT1 is known to be purine-specific, and deletion of the <i>TgENT2</i> and <i>TgENT3</i> genes, either separately or jointly, did not affect uridine transport or sensitivity to 5-fluoropyrimidines. In contrast, depletion of <i>TgENT1</i>, an essential gene, resulted in a significant reduction in the uptake of both uracil and uridine but not of the amino acid tryptophan. Moreover, expression of <i>TgENT1</i> in a <i>Leishmania mexicana</i> cell line with low endogenous uracil uptake rates significantly increased uracil uptake for these cells. We conclude that it is highly probable that <i>TgENT1</i> encodes the <i>T. gondii</i> uracil/uridine transporter. On the basis of our previous results, we infer that TgENT1 likely also mediates the uptake of 5-fluoropyrimidines.