BCKDH: The Missing Link in Apicomplexan Mitochondrial Metabolism Is Required for Full Virulence of Toxoplasma gondii and Plasmodium berghei
Figure 3
(A) In vitro enzyme activity indicates a low level of classical branched chain keto-acid dehydrogenase activity and extensive pyruvate dehydrogenase activity in RH T. gondii cell lysates. Concentrations (mean ± SD) of acetyl-CoA (black), 2-methylpropanoyl-CoA (grey), and 3-methylbutanoyl-CoA (white columns), were measured following incubation of RH lysates with 2 mM pyruvate (black), 3-methyl-2-oxobutanoate (grey) or 4-methyl-2-oxopentanoate (white columns), respectively. (B) concentration of acetyl-CoA following in vitro incubation of RH or Tge1a_ko extracts with 0.5 mM pyruvate (C–D) Relative abundance of acyl-CoA products following in vitro incubation of RH or Tge1a_ko extracts with 0.5 mM (C) 3-methyl-2-oxobutanoate or (D) 4-methyl-2-oxopentanoate. (E–G) Relative abundance of hydroxyalkyl-TPP intermediates following incubation of RH or Tge1a_ko lysates with 0.5 mM (E) pyruvate, (F) 3-methyl-2-oxobutanoate or (G) 4-methyl-2-oxopentanoate. Metabolite intensity (y-axis) is measured by LC-MS peak area (mean ± SD; n = 2). Significance as determined by t-test is shown, with p-values of <0.05 and <0.01 indicated by an asterisk and double asterisk, respectively.