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Fig. 6 | BMC Biology

Fig. 6

From: Multi-omics analysis delineates the distinct functions of sub-cellular acetyl-CoA pools in Toxoplasma gondii

Fig. 6

Loss of BCKDH causes extensive hypo-acetylation of TCA cycle enzymes and the gluconeogenic enzyme PEPCK-1. a Proteins identified as differentially acetylated in ΔBCKDH parasites were analysed using the GO enrichment R-package topGo to identify enrichment in biological processes against the total T. gondii genome/proteome. The bubble sizes are proportional to the fold enrichment of the respective pathway, ranging from 36-fold (GO:0072350—tricarboxylic acid metabolic process) to 4-fold (GO:0044281—small molecule metabolic process). Statistically significant enrichment was assessed by Fisher’s exact test (p value < 0.001). b Scheme highlighting the distribution of differentially acetylated sites on metabolic enzymes of ΔBCKDH parasites. Displayed pathways include the glycolysis/gluconeogenesis, the TCA cycle and the apicoplast resident FASII. Coloured circles on the enzymes represent sites which are either hyper- (red) or hypo-acetylated (blue) in ΔBCKDH. c Endogenous PEPCK-1 presents a nucleo-cytosolic localisation by immunofluorescence assay (IFA) after C-terminal tagging by knock-in of the endogenous PEPCK-1 locus both in RH parasites. PEPCK-1-3Ty was detected using α-Ty (green) while α-GAP45 (red) was used as a pellicle marker and 4′,6 diamidin-2-phenylindol (DAPI, blue) to stain the nuclei (scale bars in b and c, 5 μm). d Schematic representation of PEPCK and its identified acetylation sites. Lysine at position K223 was changed to glutamine (K223Q acetylation mimetics) or arginine (K223R, de-acetylation mimetics). e The ability of different PEPCK-1 acetylation mimetics to grow in a medium lacking glucose was tested in an intracellular growth assay. Error bars represent the standard deviation between 3 independent infections. Per infection, > 100 vacuoles were counted. f Constitutive activation of gluconeogenesis was assessed in PEPCK-1 acetylation mimetics by growing cells in a medium containing U-13C-glutamine in the presence of unlabelled glucose and measuring 13C-labelling in glycolytic intermediates using GC-MS (shown here, glucose-6-phosphate). Uptake and utilisation of U-13C-glutamine were confirmed by measuring 13C-labelling in the TCA cycle by-product aspartate. Error bars represent the standard deviation between replicates (n = 3). Throughout the figure, PEPCK refers to PEPCK-1, the active enzyme in tachyzoites [42]. GO, Gene Ontology; TCA, tricarboxylic acid; FASII, fatty acid synthase II; PEPCK, phosphoenolpyruvate carboxykinase-1; BCKDH, branched-chain α-keto acid dehydrogenase-complex; GC-MS, gas chromatography-mass spectrometry; TCA, tricarboxylic acid; other abbreviations, see Fig. 5

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