Additional file 8 of Multi-omics analysis delineates the distinct functions of sub-cellular acetyl-CoA pools in Toxoplasma gondii
Creators: name: Kloehn, Joachim
name: Oppenheim, Rebecca D.
name: Ghizal Siddiqui
name: Pieter-Jan De Bock
name: Dogga, Sunil Kumar
name: Yohann Coute
name: Mohamed-Ali Hakimi
name: Creek, Darren J.
name: Soldati-Favre, Dominique
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 8 : Figure S5. Generation and analysis of PEPCK-1-3Ty knock-in, ΔPEPCK-1 strains and PEPCK-1 acetylation mimetics in T. gondii. PDF file showing the (a) schematic of the putative mechanism of gluconeogenesis-activation through PEPCK-1 de-acetylation. Gluconeogenesis is inactive in wild-type parasites (RH) under glucose replete conditions (left panel), during which PEPCK is acetylated in position K223. Inactivity of the gluconeogenesis pathway if graphically represented by the faint dashed arrow and PEPCK-1 acetylation is graphically represented by an orange circle. We identified PEPCK-1 to be hypo-acetylated in ΔBCKDH parasites (right panel) and a previous study highlighted the constitutive activation of gluconeogenesis in parasites lacking BCKDH [13]. Activation of gluconeogenesis is represented by the thick arrow. These findings prompted us to hypothesize that PEPCK-1 acetylation in lysine 223 may be responsible for the activation of the pathway and to assess the role of gluconeogenesis in parasites lacking BCKDH. (b) Schematic representation of the pepck-1 locus. Two alternative translational starts are represented by inverted triangles (blue) while the translational stop is represented by a red triangle. (c) Secondary expression of the long isoform of PEPCK-1 (PEPCK-1-3Ty-L, mitochondrial) and the short PEPCK-1 (PEPCK-1-3Ty-S, cytosolic). Expression of the second PEPCK-1 copy was detected using α-Ty (green) while α-gliding associated protein 45 (α-GAP45, red) was used as a pellicle marker. (d) Graphical representation of the knock-in strategy used to introduce a 3Ty tag at the C-terminus of the endogenous pepck-1 locus in RH parasites. (e) PCRs performed on genomic DNA extracted from clones showing correct integration of the construct. (f) Schematic representation of the double homologous recombination strategy used to KO the endogenous pepck-1 locus in RH and ΔBCKDH parasites. (g) PCRs performed on genomic DNA using primers listed in Additional file 14: Table S8 show correct integration of the construct. (h) Expression of the acetylation mimetics of PEPCK-1 was tested by IFA using an α-Ty antibody. (i) pepck-1 was replaced in ΔBCKDH parasites by a cat resistance cassette as shown in panel f. PCRs were performed on genomic DNA extracted from clones showing correct integration of the construct. (j) Plaque assays of RH, ΔBCKDH, ΔPEPCK-1 or double KO ΔBCKDH/ΔPEPCK-1 parasites. (k,l) Intracellular growth assays at 24 hours post infection of RH, ΔBCKDH, ΔPEPCK-1 and ΔBCKDH/ΔPEPCK-1 parasites in complete medium (k) or in medium lacking glucose (l). Error bars represent the standard deviation between 3 independent infections. Per infection &gt;100 vacuoles were counted. Throughout the figure PEPCK refers to PEPCK-1, the active enzyme in tachyzoites [42]. Abbreviations: GNG: gluconeogenesis; PEP: phosphoenolpyruvate; Gln: glutamine; Cit: citrate; OAA: oxaloacetate; PM: plasma membrane; PEPCK: phosphoenolpyruvate carboxykinase-1; BCKDH: branched-chain α-keto acid dehydrogenase-complex; DHFR-TS: dihydrofolate reductase-thymidylate synthase; CAT: chloramphenicol acetyltransferase; FS: flanking sequence; PCR: polymerase chain reaction; DNA: deoxyribonucleic acid.