BCKDH: The Missing Link in Apicomplexan Mitochondrial Metabolism Is Required for Full Virulence of Toxoplasma gondii and Plasmodium berghei
Figure 2
BCKDH is required for conversion of pyruvate to acetyl-CoA and catabolism of glucose in the mitochondrion.
Freshly egressed RH and Tge1a_ko tachyzoites were labelled with 13C-U-glucose or 13C-U-glutamine for 4 h. Abundance and label incorporation were assessed by GC-MS and LC-MS. (A) Relative (%) abundance of selected metabolites in the TgE1a_ko mutant parasites. Bars represent abundance of metabolites in Tge1a_ko cells compared with a parental (RH) control. The dashed line refers the abundance of the metabolite in the parental control (‘100%’). 2HE-TPP refers to 2-hydroxyethyl-thiamine pyrophosphate, the stable intermediate specifically generated by pyruvate dehydrogenase activity. 2HE-TPP and acetyl-CoA were measured by LC-MS while other metabolites were measured by GC-MS (B) 13C-glucose and (C) 13C-glutamine incorporation into central carbon metabolites in RH (blue) and Tge1a_ko (red) tachyzoites, where label incorporation is the fraction of molecules of that metabolite containing one or more 13C carbons (after correction for natural abundance). In A, B and C metabolites are colour-coded by metabolic pathway; central carbon metabolism, green; TCA cycle and associated amino acids, orange; PPP, purple; other, black. Error bars represent standard deviation (n = 3–6). Significance as determined by t-test is shown (corrected for multiple comparisons using the Holm-Sidak method), with significant (p-values of 0.05) differences indicated by an asterisk. † indicates metabolite not detected. (D) Mass isotopologue abundances of citrate generated in 13C-glucose and 13C-glutamine-fed RH and Tge1a_ko tachyzoites. ‘M’ indicates the monoisotopic mass containing no 13C atoms. Error bars indicate standard deviation (n = 3). (E) Intracellular growth of RH (blue) and Tge1a_ko (red) was assessed after 24 h in medium depleted of glucose complemented or not with 5 mM exogenous acetate. Data are represented as means ± SD from three independent biological replicates.