FIGURE 7.

Growth impairment in the Δtggt1 mutant is restored by acetate supplementation. A, schematic illustration of epitope-tagging for expressing TgACS-HA under the control of its native promoter and TgGRA2–3′-UTR. The construct for 3′-HA tagging of the TgACS gene was transfected and drug-selected in the RHΔku80-hxgprt⁻ strain. B, immunoblot of transgenic (expressing TgACS-HA) and parental strains using α-HA and α-TgSag1 (loading control) antibodies. C, immunofluorescent localization of TgACS-HA protein in intracellular tachyzoites. Parasitized cells (24 h infection) were immunostained with α-HA and α-TgHsp90 (cytosolic marker) antibodies. D, plaque growth of the designated parasite strains cultured in standard culture medium with or without 2 mm acetate (7 days, 37 °C, 5% CO₂). Plaques were stained with crystal violet before ImageJ analysis (mean ± S.E., n = 3). Statistical significance (Student's t test) was measured separately for each group with respect to the parental strain (***, p < 0.001). E, replication rates of the three parasite strains cultured in the absence or presence of 2 mm acetate. Parasitized fibroblasts (24 h infection) were immunostained with αTgGap45, and the parasitophorous vacuoles were scored for the parasite numbers (mean ± S.E., n = 3). F, radiolabeling of total parasite lipids using [U-¹⁴C]acetate. Extracellular or intracellular tachyzoites were incubated with the radioisotope for 4 h (37 °C, 5% CO₂), followed by scintillation counting of lipid fractions isolated from purified parasites (mean ± S.E., n = 3). MPA, mycophenolic acid; R.C., resistance cassette.