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

Abstract

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.

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

“…However, both surrogates exhibited background nucleoside and nucleotide uptake, thus limiting the extent to which the exogenous transporter can be characterized. Here we used a L. mexicana promastigote line with null nucleoside (NT1.1, NT1.2 and NT2-KO; ‘SUPKO’) or null nucleobase uptake (NT3-KO) as surrogates [ 29 ]. TcoAT1 and each of the four T. vivax ENT family putative nucleoside transporters (TvxNT1–4) and TcoAT1 were PCR-amplified from genomic DNA, cloned into the pNUS-HcN vector, and transfected into SUPKO as described [ 72 ].…”

“…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 ].…”

“…We recently reported a sequential knock-out of the LmexNT1.1 , NT1.2 and NT2 genes using CRISPR/Cas9 technology [ 64 ] in promastigotes, resulting in a mutant model with null background purine and pyrimidine nucleoside uptake named SUPKO, which was validated as a surrogate system that allows the functional transgenic expression and characterization of protozoan nucleoside transporters [ 29 ]. Here, we use this system to express TcoAT1 and the putative T. vivax ENTs in order to establish their substrate preferences.…”

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

“…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. However, this does not diminish the need to follow up the initial screening with further testing on the targeted parasite, particularly in organisms phylogenetically distant from the expression system, as it is likely that the intracellular targets differ between species.…”

“…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.…”