Fig. 8: Tachyzoites can synthesize as well as salvage selected PtdIns species from host cells.

a HFF labeling with [¹³C]-myo-inositol and intracellular propagation of tachyzoites in prelabeled host cells to test the salvage of [¹³C]-PtdIns. Briefly, host cells were grown to confluence in the presence of 0.5 mM [¹³C]-myo-inositol, and then infected with tachyzoites of the PIS mutant (MOI = 1) in the presence of 5 mM myo-inositol to minimize the import and usage of residual intracellular isotope by the parasite’s de novo synthesis. Purified extracellular tachyzoites were examined by lipidomic analysis. Note that this assay was technically (intricate data normalization) not feasible with the auxin-treated TgPIS mutant due to a need of higher MOI for initial infection (and thus more unlabeled PtdIns), and much slower growth in the presence of IAA (and thus prolonged incubation). The data plotted are from 5 independent assays (mean ± S.E.). b Proportion of [¹³C]-PtdIns species detected after stable isotope labeling of extracellular tachyzoites (left, see Fig. 4e), and in tachyzoites after propagation in prelabelled host cells (right, see a). Only primarily-labeled PtdIns species (≥1%) are named; the minor-labeled species are shown as “Others”.