Fig. 4.

Impaired GAPDH activity at acidic pH. (A) Pools sizes and ¹³C labeling of triose-3P (glyceraldehyde-3-phosphate [G3P] and dihydroxyacetone phosphate [DHAP]) and phosphoenolpyruvate (PEP) were determined in M. tuberculosis lysates after 8-h incubation with 1.2 mM [U-¹³C]Gly, 200 μM [U-¹³C]OA, [U-¹³C]Gly + OA, and [U-¹³C]OA + Gly at pH 7.0 or pH 5.0. Gray bars represent unlabeled fraction of the pools, and colored bars (red: OA, blue: Gly) represent the ¹³C- labeled fractions of the pools. (B) Measurement of NADH formation over time due to GAPDH enzyme activity in whole-cell lysates from M. tuberculosis cultured at pH 7.0 or pH 5.0 in the presence of 0.2% glycerol (Gly) or 200 μM OA. (C) Measurement of GAPDH activity at time T = 45 min from data shown in B (left panel) and percentage of GAPDH activity relative to Gly pH 7.0 (right panel). (D) Measurement of GAPDH activity at time T = 45 min in whole-cell lysates from M. tuberculosis cultured at pH 7.0 or pH 5.0 in the presence of 0.2% glycerol or 200 μM OA. When indicated, lysates were incubated 30 min at room temperature in the presence of DTT (1 mM) prior to measurements. (E) Measurements of intracellular ROS in M. tuberculosis in media at pH 5.0 or pH 7.0 in the presence of 0.2% glycerol or 200 μM OA or with no carbon source added. ROS were measured 2 h after the addition of 5 μM Cellrox Green reagent and were normalized to OD. Data in A are means ± SD of triplicates and are representative of two independent experiments. Data in B and C are means ± SD of triplicates and are representative of three independent experiments. Data in D are means ± SD of three independent experiments. Statistical significance of data (A–D) was determined using unpaired two-tailed t test. Data in E are means ± SD of duplicates and are representative of at least two independent experiments. Statistical significance in C (% GAPDH activity) and E were assessed by one-way ANOVA using the Dunnett’s multiple comparisons test. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001; ns = not significant. Gly = glycerol; OA = oleic acid; CTRL = Control; DTT = dithiothreitol.