Fig. 5: Dysregulation of ROS defense genes presents a vulnerability to H2O2 in ketogenetic persisters.

a Starved BCG shows elevated levels of superoxide as detected by CellRox dye. Data represent the percentage of cells with elevated steady-state ROS production (CellROXhi) at different starvation time points. N ≥ 3; *P < 0.05; one-way ANOVA with Dunnett's test vs Log. b Heatmaps depicting transcriptional profiles and protein expressions for putative antioxidant response genes, showing a general lack of observable coordination between transcription and translation for these genes during starvation. c Catalase activity in Log, S4, S10, S20, and R6 cultures (n ≥ 6; *P > 0.05; one-way ANOVA with Dunnett's test vs Log). d Survival of BCG after 48 h of exposure to 0.5 mM H2O2. BD, below detection (1000 CFU). N = 6; *P < 0.05; one-way ANOVA with Bonferroni post test. e Killing of S20 BCG when exposed to H2O2. S20 cultures (25 mL of ~108 CFU/mL) were treated in 50 mL conical tubes at indicated H2O2 concentrations. N = 3; *P < 0.05, unpaired two-tailed t test with Welch’s correction. f Percentage of cells with elevated steady-state ROS levels (CellROXhi) following 4 h of mock or 0.5 mM H2O2 exposure. N = 8; *P < 0.05; unpaired, two-tailed t test of treatment groups with Welch’s correction. g Intracellular β-hydroxybutyrate levels in Log, S20, and R6 cells after 4 and 48 h of H2O2 exposure. N ≥ 3; *P < 0.05; two-way ANOVA with Bonferroni post tests comparing cell state and H2O2 dose. h Model of starvation-induced ketosis depicting conserved vulnerability of starved persisters to H2O2 blocking ketone body metabolism and thus carbon cycling for NRP survival. See Supplementary Data 4 for source data used in these graphs.