Authors

Discussion

Trichomonas vaginalis is a highly prevalent human urogenital protozoan parasite and the causative agent of trichomoniasis, the most common non-viral sexually transmitted infection (STI) globally. Chronic infection of the reproductive tract leads to an increased risk of secondary infections, such as human immunodeficiency virus and other STIs. As with all protozoan parasites, T. vaginalis lacks de novo synthesis of purines. It is also unable to synthesise pyrimidine nucleotides, instead relying on salvage pathways as the main source of nutrients from the host. The salvage of nucleosides and nucleobases is believed to be dependent on the equilibrative nucleoside transporter (ENT) family, marking these proteins out as potential drug targets to disrupt the uptake of nutrients by the parasite. An ideal strategy to explore this further would be to express T. vaginalis ENT genes (TvENTs) by cloning them into a compatible heterologous system, where they can be studied individually to characterise the transport of nucleosides. All nine TvENT genes were successfully sub-cloned into the vector pHD1336 for expression in Trypanosoma brucei brucei, from which the adenosine transporter 1 had been deleted (TbAT1-KO). Transport assays established that TVENT3 transported cytidine the most efficiently, and TvENT6 transported uridine. However, high background levels of uridine and of purine nucleoside transport by P1-type trypanosome transporters confounded the results, prompting the search for a more suitable expression system.  As such, a Leishmania mexicana strain from which all the nucleoside transporter genes (NT1.1, NT1.2, NT2) were deleted was constructed using CRISPR/cas9. So far, the nucleoside transport null line of L. mexicana has been confirmed for uptake of adenosine, uridine, thymidine, guanosine and inosine, and three TvENTs have been introduced in this cell line for characterisation. As we have recently identified adenosine analogues with powerful antitrichomonal activity the identification and characterisation of the adenosine transporters is expected to contribute to this emerging drug strategy.