The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from Leishmania mexicana (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells’ ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of L. mexicana NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of T. cruzi NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the Aspergillus nidulans uracil transporter FurD.

Trichomoniasis is a common and widespread sexually‐transmitted infection, caused by the protozoan parasite Trichomonas vaginalis. T. vaginalis lacks the biosynthetic pathways for purines and pyrimidines, making nucleoside metabolism a drug target. Here we report the first comprehensive investigation into purine and pyrimidine uptake by T. vaginalis. Multiple carriers were identified and characterized with regard to substrate selectivity and affinity. For nucleobases, a high‐affinity adenine transporter, a possible guanine transporter and a low affinity uracil transporter were found. Nucleoside transporters included two high affinity adenosine/guanosine/uridine/cytidine transporters distinguished by different affinities to inosine, a lower affinity adenosine transporter, and a thymidine transporter. Nine Equilibrative Nucleoside Transporter (ENT) genes were identified in the T. vaginalis genome. All were expressed equally in metronidazole‐resistant and ‐sensitive strains. Only TvagENT2 was significantly upregulated in the presence of extracellular purines; expression was not affected by co‐culture with human cervical epithelial cells. All TvagENTs were cloned and separately expressed in Trypanosoma brucei. We identified the main broad specificity nucleoside carrier, with high affinity for uridine and cytidine as well as purine nucleosides including inosine, as TvagENT3. The in‐depth characterization of purine and pyrimidine transporters provides a critical foundation for the development of new anti‐trichomonal nucleoside analogues.