Figure 6.

Fractional abundance of the select isotopomers in the Δtggt1/iΔtgpepck_mt tachyzoites labeled with [U-¹³C]glutamine. Isotope inclusion was evaluated by metabolic labeling of intracellular tachyzoites with [U-¹³C]glutamine (4 h, 37 °C, 5% CO₂). Polar metabolites were isolated from isotope-labeled parasites and subjected to GC-MS analysis. M, unlabeled fraction; Sum, collective abundance of all ¹³C-containing isotopomers of a given metabolite. Only fragmented analytes were detectable for G6P and R5P. Note that our experimental design of metabolomics had to make a reasonable trade-off between obtaining a sufficient amount of purified tachyzoites for the metabolite measurements and maximum growth inhibition (a proxy for the enzyme activity). This trade-off was best met between days 3 and 4 of ATc treatment, on which the parasite growth is still nearly the half-maximum (see Fig. 5C). The obvious explanation for the residual labeling in the off-state mutant is that knockdown of PEPCK_mt is incomplete in the conditions used to prepare samples for metabolomic analysis. In particular, when we inferred enzymatic activity from reduction in PEP labeling (a direct product of PEPCK_mt), we observed a decline of ∼50%, which is consistent with the growth (half-maximum) at the time of sample collection. Statistical significance was determined separately for each group (with or without ATc) using Student's t test (n = 4 assays; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Error bars, S.E.