A two-membrane gateway for monocarboxylates couples host glycolysis to Toxoplasma mitochondrial fitness and virulence.

Intracellular pathogens must coordinate their metabolism with nutrient supplies from the host cell, yet the specific metabolites and transport pathways that sustain parasite bioenergetics remain incompletely defined. In the apicomplexan parasite Toxoplasma gondii, infection increases host glycolytic flux and elevates cytosolic lactate and pyruvate, suggesting that these intermediates are co-opted as carbon and energy sources. Here, we show that T. gondii imports host-derived lactate and pyruvate across both the parasitophorous vacuole membrane and the parasite plasma membrane to maintain mitochondrial function, extracellular survival, and acute virulence. Using a hexokinase knockout (Δhk) to abolish endogenous pyruvate production, we find that parasites preserve basal oxygen consumption but become strictly dependent on exogenous monocarboxylates to stimulate mitochondrial respiration. By disrupting the parasite formate-nitrite transporters TgFNT1-3, we identify TgFNT1 and TgFNT2 as the principal monocarboxylate transporters required for lactate- and pyruvate-driven respiratory responses. Furthermore, genetic ablation of TgGRA17, a parasitophorous vacuole pore protein, compromises the growth advantage conferred by elevated exogenous lactate, implicating this pore as the entry route for host-derived monocarboxylates into the vacuole. Conversely, host cells lacking the monocarboxylate exporter MCT1 accumulate cytosolic lactate/pyruvate and enhance parasite growth, linking host monocarboxylate export to parasite fitness. When both endogenous pyruvate production and exogenous uptake are disrupted, parasites display severely reduced mitochondrial basal respiratory capacity, membrane potential, ATP levels, extracellular survival, and virulence in mice. Collectively, these findings define a dual-step pyruvate acquisition pathway in T. gondii and reveal host monocarboxylates as critical fuels that buffer parasite bioenergetic stress during infection.