Fig 3. Perturbation of PrrA globally alters Mtb response to NO and hypoxia.

(A) prrA overexpression dampens hspX’::GFP reporter response to NO and hypoxia. Mtb(P₆₀₆’::prrA-FLAG-tetON, hspX’::GFP) was grown in 7H9, pH 7.0 media and treated with 0.1% ETOH (“control”) or 200 ng/ml ATC (“prrA OE”) for 1 day before exposure to 100 μM DETA NONOate for an additional 1 day (left panel), or to 1% oxygen for 2 days (right panel). EtOH or ATC was maintained as appropriate throughout the exposure. Reporter GFP signal was analyzed by flow cytometry, and fold induction is in comparison to control conditions (no added DETA NONOate and atmospheric O₂ respectively). Data are shown as means ± SD from 3 experiments. p-values were obtained with an unpaired t-test. (B-E) Overexpression of prrA globally modulates Mtb response to NO. Log-phase Mtb(P₁’::prrA-FLAG-tetON, rv2390c’::luciferase) was grown in 7H9, pH 7.0 media and treated with 0.1% EtOH or 200 ng/ml ATC for 2 hours, before exposure to 7H9, pH 7.0 ± 100 μM DETA NONOate for 4 hours in the continued presence of EtOH or ATC as appropriate, and samples extracted for RNA sequencing analysis (B) or qRT-PCR (C-E). In (B), log₂-fold change compares gene expression in the 7H9, pH 7 + 100 μM DETA NONOate condition versus the 7H9, pH 7 control condition for each of the EtOH (“cont”) or ATC (“prrA OE”) treatment sets. Genes marked in red had a log₂-fold change difference ≥0.25 between the ATC and EtOH treatment sets (p<0.05, FDR<0.01 in both sets, with log₂-fold change ≥1 in the EtOH set). In (C-E), fold change compares gene expression in the 7H9, pH 7 + 100 μM DETA-NONOate condition versus the 7H9, pH 7 control condition for each of the EtOH (“control”) or ATC (“prrA OE”) treatment sets. sigA was used as the control gene, and data are shown as means ± SD from 3 technical replicates. p-values were obtained with an unpaired t-test. N.S. not significant, * p<0.05, ** p<0.01, **** p<0.0001. (F-H) prrA overexpression inhibits induction of multiple hypoxia-responsive genes. qRT-PCR of Mtb(P₁’::prrA-FLAG-tetON, rv2390c’::luciferase) in 7H9 pH 7.0 media treated with 0.1% EtOH (“control”) or 200 ng/ml ATC (“prrA OE”) for 2 hours under aerated conditions before exposure to 1% oxygen for an additional 4 hours, in the continued presence of EtOH or ATC as appropriate. Fold change compares the 1% oxygen condition at 4 hours to the aerated 0 hour time point. sigA was used as the control gene, and data are shown as means ± SD from 3 technical replicates. p-values were obtained with an unpaired t-test. ** p<0.01, *** p<0.001, **** p<0.0001.