A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

Abstract

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

Nucleoside Transport and Nucleobase Uptake Null Mutants in Leishmania mexicana for the Routine Expression and Characterization of Purine and Pyrimidine Transporters

“…For instance, we have previously expressed the T. congolense transporter TcoAT1 in the T. brucei cell line lacking the TbAT1/P2 transporter [ 25 ], showing it did not have the proposed role in diminazene resistance [ 49 ], a conclusion recently confirmed by meticulous work on diamidine uptake in T. congolense [ 50 ]. We have also recently published the first characterization of Trichomonas vaginalis nucleoside transporters by expression in T. brucei [ 26 ] and reported a unique substrate binding mode for Toxoplasma gondii oxopurine transporter Tg244440 [ 28 ] expressed in a T. brucei cell line from which the main nucleobase transporter locus has been knocked out [ 27 ]. The characterization of individually expressed transporters is important because it unambiguously links the transport activity to a specific gene product, and disentangles the (potentially) multiple gene products contributing to the uptake of a certain substrate.…”

“…However, well-characterized null-backgrounds for the expression of nucleobase and nucleoside transporters in an easy-to-culture protozoan cell type would be much preferable. We have previously expressed protozoan transporters in T. brucei lacking either the TbAT1 or the NT8.1/NT8.2/NT8.3 nucleobase transporter locus [ 25 , 26 , 27 , 28 , 29 ].…”

Cloning and Characterization of Trypanosoma congolense and T. vivax Nucleoside Transporters Reveal the Potential of P1-Type Carriers for the Discovery of Broad-Spectrum Nucleoside-Based Therapeutics against Animal African Trypanosomiasis

“…Therefore, these parasites have developed highly specialized transporters in their membrane for the internalization of the hydrophilic nucleosides from the host environment [ 26 ]. To date, all protozoan purine and pyrimidine transporter genes identified have been of the Equilibrative Nucleoside Transporter (ENT) family [ 25 , 26 , 27 , 28 , 29 , 30 ], although there is a quite strong likelihood that some protozoan purine and/or pyrimidine carriers are expressed by other gene families [ 31 , 32 ]. Where this has been investigated, it was found that the trypanosomatid nucleoside transporters are concentrative proton symporters that exclude uric acid; acidification of the cytosol is prevented by H + -ATPases in the plasma membrane [ 33 , 34 , 35 ].…”

Purine Transporters as Efficient Carriers for Anti-kinetoplastid Molecules: 3′-Deoxytubercidin versus Trypanosomes

“…Finally, it is noteworthy that the knowledge obtained on the T. brucei purine transporters combined with its genetic amenability allowed the use of this parasite as a surrogate system for the expression and functional characterization of purine transporters from different protozoan species, namely, T. congolense, Leishmania spp., T. cruzi, Toxoplasma gondii, and Trichomonas vaginalis. 5,25,26 Additionally, the current wide use of CRISPR/ Cas9 technology for genome editing in Leishmania parasites in combination with its well-defined network of ENT transporters allowed the creation of lineages deficient in the transport of purine nucleosides or nucleobases, which can also be used for the expression and functional characterization of ENT genes from other protozoa. 27 This, combined with the well stablished strategies for drug screening in T. brucei and Leishmania spp., should accelerate the discovery of suitable parasiticidal permeants of the heterologously expressed transporters.…”

“…congolense, Leishmania spp., T. cruzi, Toxoplasma gondii, and Trichomonas vaginalis. ,, Additionally, the current wide use of CRISPR/Cas9 technology for genome editing in Leishmania parasites in combination with its well-defined network of ENT transporters allowed the creation of lineages deficient in the transport of purine nucleosides or nucleobases, which can also be used for the expression and functional characterization of ENT genes from other protozoa . This, combined with the well stablished strategies for drug screening in T.…”