Lipidomic analysis

The triple mutant strain was transformed with vector pD0170-CLIP or pDO1050 (lacking the HAD domain) and the cells were grown in YNB glucose medium at 30 °C until OD₆₀₀ reached 1–3. Cultures were centrifuged for 5 min at 1000 × g, washed with PBS thrice, and normalized by cell weight.

Total lipid analysis: Total lipids were extracted in chloroform/methanol/water (1:3:1, v/v/v) containing FFA (free fatty acids C13:0, 10 nmol) and PC (21:0/21:0, 10 nmol) as internal standards for extraction. Next, the polar and apolar metabolites were separated by phase partitioning by adding chloroform and water to give the ratio of chloroform/methanol/water as 2:1:0.8 (v/v/v). For lipid analysis, 50 µl of the extract was directly dried and dissolved in 2:1 choloform:methanol and trimethylsulfonium hydroxide (TMSH, Machenery Nagel) for total fatty acid content. Resultant FAMEs were then analyzed by GC-MS as previously described⁴⁴. All FAMEs were identified by comparison of retention time and mass spectra from GC-MS with authentic chemical standards. The concentration of FAMEs was quantified after initial normalization to different internal standards.

Phospholipid, DAG/TAG analyses: The extracted total lipid extracted (as above) was separated with 5 nmol DAG/PA(C16:0/C18:1) (Avanti Polar lipids) by one-dimensional silica gel high-performance thin-layer chromatography (HPTLC, Merck). The 1st and 2nd solvent system used were chloroform/methanol/water/ acetic acid, 25:15:2:4 (v/v/v/v) and hexane/MTBE/acetic acid, 35:15:0.5 (v/v/v), respectively. For DAG, TAG, FFA, and CE analysis, total lipid fraction was separated by 1D-HPTLC using hexane/diethyl ether/formic acid, 80:20:2 (v/v/v) as solvent system. The spots correlating to TAG, DAG, PA, and PC on the HPTLC plate were scraped off, and lipids were methanolized with 200 μl 0.5 M methanolic HCl in the presence of 1 nmol pentadecanoic acid (C15:0) as internal standard at 85 °C for 4 h. The resulting FAMEs were extracted with hexane and analyzed by GC-MS (Agilent).

The parasites were grown for 24 h ad 48 h in + /− ATc conditions within a confluent monolayer of HFF in flasks (175 cm²). At each time point, parasites were harvested as intracellular tachyzoites (1 × 10⁷ cell equivalents per replicate) after syringe filtration with 3-μm pore size membrane. These parasites were metabolically quenched by rapid chilling in a dry ice-ethanol slurry bath and then centrifuged down at 4 °C. The parasite pellet was washed with ice-cold PBS thrice, before transferring the final pellet to a microcentrifuge tube. Then total lipids were extracted in chloroform/methanol/water (1:3:1, v/v/v) containing PC (C13:0/C13:0), 10 nmol and C21:0 (10 nmol) as internal standards for extraction. Polar and apolar metabolites were separated by phase partitioning by adding chloroform and water to give the ratio of chloroform/methanol/water as 2:1:0.8 (v/v/v). For lipid analysis, the organic phase was dried under N₂ gas and dissolved in 1-butanol to obtain 1 µl butanol/10⁷ parasites.

Total lipid analysis: The extracted total lipid sample was then added with 1 nmol pentadecanoic acid (C15:0) as an internal standard as stated before using TMSH for total fatty acid content. Resultant FAMEs were analyzed by GC-MS as previously described⁴⁴. All FAMEs were identified by comparison of retention time and mass spectra from GC-MS with authentic chemical standards. The concentration of FAMEs was quantified after initial normalization to different internal standards and finally to parasite number.

Free fatty acid and cholesterol analysis: Total lipid samples were dried and derivatized with BSTFA + TMCS, 99:1 (Sigma) to generate trimethylsilyl (TMS-) fatty acids and TMS-cholesterol. These TMS derivatives were analyzed by GCMS as described above.

Phospholipid and neutral lipid analysis: For phospholipid analysis, the extracted total lipid extracted (as above) was separated with 1 nmol PA(C17:0/C17:0) (Avanti Polar lipids) by two-dimensional silica gel high-performance thin-layer chromatography (HPTLC, Merck). The solvent system used for the 1st and 2nd dimension was chloroform/methanol/28% ammonium hydroxide,12:7:1.6 (v/v) and chloroform/acetone/methanol/acetic acid/water, 10:4:2:2.6:1 (v/v/v/v/v), respectively. For DAG, TAG, Free fatty acids (FFA) and cholesteryl ester (CE) analysis, total lipid fraction was separated by 1D-HPTLC using hexane/diethyl ether/formic acid, 80:20:2 (v/v/v) as solvent system. Then each lipid spot on the HPTLC plate was scraped off, and lipids were methanolized with 200 μl 0.5 M methanolic HCl in the presence of 1 nmol pentadecanoic acid (C15:0) as internal standard at 85 °C for 3 h. The resulting FAMEs were extracted with hexane and analyzed by GC-MS (Agilent).