A nucleoside transporter on the mitochondria of T. gondii is essential for maintaining normal growth of the parasite

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BACKGROUND: Toxoplasma gondii invades almost all nucleated cells of warm-blooded animals, but T. gondii lacks the ability to synthesize purines; therefore, it must scavenge purines from host cells to fuel its proliferation and propagation. Through exogenous expression, the adenosine transporter TgAT1 has been validated for its ability to transport both oxypurine nucleosides and nucleobases across the parasite plasma membrane, although its affinity for the substrate is low. Further studies have shown that T. gondii also has a high-affinity purine and pyrimidine nucleoside transport system (TgAT2), but this protein has not been identified thus far. METHODS: Here, we identified three novel nucleoside transporters in T. gondii by homology alignment. Using immunofluorescence staining, we found that TgNT1, TgNT2, and TgNT3 are localized to the mitochondria, plasma membrane, and endoplasmic reticulum (ER), respectively. We also performed conditional knockout of TgNT1 and direct knockout of TgNT2/TgNT3 using CRISPR/Cas9 technology. RESULTS: TgNT1 is crucial for the in vitro growth and proliferation of T. gondii, whereas the other two genes are dispensable. Conditional depletion of TgNT1 impairs multiple metabolic pathways in both the mitochondria and cells of the parasite, with the most significant changes manifesting in the levels of various nucleosides. Carbon metabolism is also affected, as evidenced by alterations in metabolite levels within both the electron transport chain and the tricarboxylic acid cycle. The impairment of adhesion and invasion functions appears to be strongly associated with a reduction in the content of the initial lipid involved in glycosylphosphatidylinositol (GPI)-anchored lipid modification, which may underlie the inhibition of T. gondii's invasive capacity. Notably, TgNT1 is the first nucleoside transporter protein that is localized in the mitochondria of T. gondii. Given that TgNT1 has no homologous proteins in mammals, it holds promise as a potential drug target. CONCLUSIONS: TgNT1, a nucleoside transporter located in the mitochondria of T. gondii, is essential for maintaining the normal growth of the parasite.

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