Impact of equilibrative nucleoside transporters on Toxoplasma gondii infection and differentiation.

Toxoplasma gondii cannot synthesize purines de novo and must import them; however, the functional interplay among its four equilibrative nucleoside transporters (ENTs) homologs remains unclear. We systematically deconstructed this network by combining CRISPR-Cas9 knockouts with an auxin-inducible degron. Across all phenotypic assays, tachyzoite replication, nucleoside-analog sensitivity, alkaline-stress-induced differentiation, and murine cyst formation, the ΔTgENT2 strain was indistinguishable from the parental line, indicating that TgENT2 is dispensable under the conditions tested. In contrast, the double mutant ΔTgAT1ΔTgENT3 exhibited delayed bradyzoite differentiation in vitro and produced smaller brain cysts in vivo. This double deletion triggered approximately threefold transcriptional upregulation of TgENT1, whose product we partially localized to the plant-like vacuolar compartment (PLVAC). Conditional depletion of TgENT1 caused complete intracellular growth arrest, PLVAC swelling, and a purine-starvation-like transcriptomic program enriched for nucleoside phosphatases and cyclic-nucleotide phosphodiesterases. These findings reveal a compensatory salvage pathway in which the parasite reroutes purine acquisition through a vacuolar route when plasma-membrane import is compromised. Although this response sustains tachyzoite proliferation, it fails during the energetically demanding transition to bradyzoites, creating a metabolic bottleneck that impairs chronic infection. Our work reveals an adaptable yet ultimately limited purine-import network and identifies TgENT1, along with the vacuolar salvage axis it mediates. IMPORTANCE: In this manuscript, we demonstrate that Toxoplasma gondii employs a flexible transporter network that redirects to a vacuolar salvage route when primary transporters are compromised. Disrupting this backup pathway disrupts parasite growth, exposing an Achilles' heel in purine homeostasis. Because nucleoside transporters are druggable, these findings suggest that the purine import machinery and TgENT1 may be potential targets for therapies against T. gondii infections.