Figure 2.

Trypanosoma brucei produce and secrete aromatic ketoacids which are non-toxic and induce HO-1 expression in mixed glia and BMDM. Primary murine mixed glia and BMDM were incubated with indole pyruvate, hydroxyl-phenyl pyruvate, or phenylpyruvate (0.25–1 mM) for 24 h. (A) Viability of mixed glia was determined by alamarBlue reduction, and is expressed as a percentage of the untreated control. Results shown are mean ± SEM of the percentage viabilities of mixed glia from three independent experiments. (B) Expression of HO-1 in mixed glia was determined by Western blot. Blots shown are representative of three independent experiments. (C) Viability of BMDM was determined by alamarBlue reduction, and is expressed as a percentage of the untreated control. Results shown are mean ± SEM of the percentage viabilities of BMDM from four independent experiments. (D) Expression of HO-1 in BMDM was measured by Western blot. Blots shown are representative of two independent experiments. (E) Schematic depicting metabolism of aromatic ketoacids to aromatic hydroxyacids by AHADH. (F) Bloodstream form AHADHⁱⁿ cells were found to grow at similar rates to wild type MITat 1.2 T. brucei (▴), whether induced (°) or non-induced (•). (G) Secreted aromatic ketoacid concentrations in HMI-9 culture media, as measured via AHADH assay after 48 h. AHADH cells were either non-induced or induced with 2 mg/ml tetracycline. Results shown are mean ± SD of triplicate measurements, and are representative of three independent experiments. (H) Supernatants from WT and AHADHⁱⁿ T. brucei were added to mixed glia for 24 h. HO-1 expression was measured by Western blot. Representative blot of three independent experiments is shown. Full length blots are presented in Supplementary Figure 5.