FIGURE 1.

Metabolic labeling of intracellular tachyzoites with [U-¹³C]glucose and analysis of de novo fatty acid biosynthesis. A, T. gondii-infected fibroblasts were precultured in the presence or absence of ATc for 24 h and then labeled with [U-¹³C]glucose 24 h prior to parasite egress. After host cell lysis, extracellular tachyzoites were metabolically quenched in a dry ice/ethanol bath and separated from host cell debris by filtration prior to metabolite extraction. Examples of different labeling patterns (¹³C indicated in gray) and their interpretation are schematically shown for myristate. B, lipids from [U-¹³C]glucose-labeled tachyzoites were subjected to methanolysis and trimethylsilyl derivatization and analyzed by GC-MS. A portion of the mass spectrum containing the molecular ions of the methyl ester of myristate is shown. Unlabeled myristoyl methyl ester (M₀) has an m/z of 242 atomic mass units. Higher mass isotopomers (M_1–12) contain between 1 and 12 ¹³C atoms, corresponding to incorporation of ¹³C into the fatty acid (FA) biosynthetic pathways via [¹³C]acetyl-CoA. arb., arbitrary units. C, incorporation of ¹³C into the major fatty acids of intracellular wild type T. gondii tachyzoites (labeling is given as mol % of all labeled mass isotopomers (M_1–12) relative to M₀, after correction for natural abundance). The fatty acid notation Cn:m indicates the length of the fatty acid (n, carbon number) and degree of unsaturation (m, number of double bonds). Note that treatment of wild type parasites with ATc (black) does not result in any significant changes of the fatty acid labeling pattern when compared with untreated controls (white). D, intracellular tachyzoites of the T. gondii ΔACP/ACPi mutant carrying an inducible copy of FASII ACP were labeled with [U-¹³C]glucose in the presence or absence of ATc. Total lipids were extracted from the purified tachyzoites and released fatty acid methyl esters analyzed by GC-MS. The mass spectrum (molecular ion region from m/z 240 to 250) of the myristate fatty acid methyl esters from unlabeled parasites, and from [¹³C]glucose-labeled parasites cultured in the absence or presence of ATc is shown. Incorporation of multiple ¹³C₂H₄ units into myristate is observed in the absence of ATc (ACP active) and largely inhibited in the presence of ATc (ACP repressed). The spectrum is representative of three individual experiments. E, total ¹³C incorporation into fatty acids in ΔACP/ACPi tachyzoites labeled in the presence (black) or absence (white) of ATc. Error bars represent standard deviation where n = 3. Fatty acids for which significant changes in labeling were observed in the presence and absence of ATc using the Wilcoxon rank sum test (p values less than 0.05) are indicated with an asterisk. The absolute amount of incorporation can vary from experiment to experiment depending on the stage of the culture (no ATc in C and E). We therefore always directly compare ATc-treated samples with an untreated control from the same culture batch.