Protein kinase TgCDPK7 regulates vesicular trafficking and phospholipid synthesis in Toxoplasma gondii
Fig 5
TgCDPK7 is involved in phospholipid metabolism.
A. Total lipid isolated from TgCDPK7-iKD tachyzoites grown in the presence or absence of ATc and separated by HPTLC. After quantification by GC-MS and normalization with respect to cell number content relative to total fatty acids was determined. Phospholipid profiling showed the abundance of major phospholipid classes and points at the significant reduction of PE upon TgCDPK7 depletion (mean ± SE, *, n = 4, p<0.05, t-test). B. Metabolic labeling to monitor the synthesis of PC and PE. Extracellular TgCDPK7-iKD tachyzoites were incubated with 14C-Eth or 14C-Cho in the presence or absence of ATc. Subsequently, lipids were extracted and radiolabeled lipids were detected by phosphoimaging of TLC (Left panel). Right Panel, Radiolabeled spots corresponding to PE and PC (left panel) were quantitated by densitometry (right panel) and % change in PC and PE formation in ATc-treated parasites with respect to untreated parasites was calculated (mean ± SE, *n = 3, p<0.001, ANOVA). C. Metabolic pathways depicting the synthesis of PC and PE in P. falciparum and T. gondii. The enzymes conserved in two parasites are indicated in black. PMT is solely encoded by P. falciparum, is indicated in red and PTS/PSS involved in conversion of PE to PT or PS in T. gondii is shown in green [29]. D. TgCDPK7-iKD parasites were pre-incubated for 48h with ATc and were subsequently allowed to invade fresh HFFs in the presence or absence of ATc. In one case, 200 μM Eth was added to cultures prior to the addition of ATc. The number of parasites per vacuole was determined after 24h. Data represent mean ± SE, n = 3 and at least 200 vacuoles were counted for each condition (ANOVA, *, p<0.001 for 8/16 parasites/vacuole).