Fig. 5: MEC-mediated glycolytic activation diminishes antibiotic tolerance in infected macrophages.

A Bar plot showing the mitochondrial respiratory parameters determined by the modified mitostress test at 24 h p.i. upon treatment with 20 µM MEC or 0.2% DMSO (vehicle control, labeled “DMSO”). BR- basal respiration, ATP- ATP production, H + -leak- proton leak; B Glycolytic stress test to assess the glycolytic levels in infected macrophages 24 h p.i. upon treatment with 20 µM MEC or vehicle. Gly- glycolysis, GC- glycolytic capacity, NGA- non-glycolytic acidification; C Mitochondrial ROS measured upon treatment with 20 µM MEC at 24 h p.i.; D Mitochondrial membrane polarization measured by JC-1 dye at 24 h p.i., 50 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) treatment used as positive control; UI: uninfected macrophages; E Representative pseudocoloured 3D views of uninfected and mCherry expressing Mtb-infected BMDMs at 24 h p.i., with and without exposure to 20 μM MEC. Mitochondria (amber), Mtb H37Rv (green), and the nucleus (purple) are shown as surfaces. Plots show the surface area (µm²) and volume (µm³) of mitochondria in uninfected and infected BMDMs (n > 500 from 6-8 cells per condition). Data are median with interquartile range. p-values calculated using a one-way ANOVA with Dunn’s multiple comparisons test. UI: uninfected BMDMs, Inf: infected BMDMs; F, G RNA-seq of BMDMs infected with Mtb and treated with 20 µM MEC at 24 h p.i. F OXPHOS gene expression between vehicle-treated and MEC-treated BMDMs; and (G) net enrichment score (NES) calculated by gene set enrichment analysis (GSEA) for the DEGs between MEC- and vehicle-treated infected macrophages; H Redox profile of the intracellular Mtb in the presence of indicated concentrations of MEC at 24 h p.i.; I Antibiotic tolerance in Mtb-infected BMDMs treated with 20 µM MEC and 0.5 mM 2-DG in the presence of 3X MIC of INH (0.375 µg/ml) or MOXI (0.75 µg/ml). Data are expressed as mean ± S.D. of three biological replicates done in triplicate. p-value determined using a Student’s two-tailed t-test with Welch’s correction for comparison between two groups or one-way ANOVA with Dunnett’s T3 test for multiple comparisons. Source data are provided as a Source Data file.