Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
Department of Biology, National and Kapodistrian University of Athens, Panepistimioupolis, 15784 Athens, Greece.
Int J Mol Sci. 2022 Jul 23;23(15):8139. doi: 10.3390/ijms23158139.
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 (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 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 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 uracil transporter FurD.
转运蛋白的研究极具挑战性，因为它们不能被分离或在悬浮液中进行研究，需要细胞或囊泡系统，并且当由多种载体介导时，难以解释。核苷类似物是重要的药物候选物，所有原生动物病原体都表达多种平衡核苷转运蛋白（ENT）基因。因此，我们开发了一种常规表达核苷转运蛋白的系统，使用 CRISPR/cas9 从 （ΔNT1.1/1.2/2 (SUPKO)）中删除所有三种核苷转运蛋白的两个拷贝。SUPKO 的生长速度与亲本菌株相同，并且由于细胞能够合成嘧啶和 LmexNT3 嘌呤核苷碱基转运蛋白的表达，没有表现出明显的缺陷。SUPKO 中的核苷转运几乎无法测量，但重新引入 NT1.1、NT1.2 和 NT2 恢复了摄取。因此，SUPKO 为单独表达和表征单个 ENT 转运蛋白基因提供了理想的空背景。同样，LmexNT3-KO 菌株为嘌呤碱基的转运提供了空背景，并用于功能表征 NB2，确定其为腺嘌呤特异性。氟尿嘧啶抗性菌株（Lmex5FURes）对尿嘧啶和 5FU 的转运呈空状态，并用于表达尿嘧啶转运蛋白 FurD。
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