Figures and data
Starvation induces antibiotic persister cells across diverse mycobacteria.
(A) Msmeg, (B) Mtb or (C) Mabs were grown in 7H9 rich media or starved in PBS prior to the addition of the indicated antibiotics. Cells were allowed to adapt to starvation for a period of 48h for Msmeg and Mabs and 14-21d for Mtb prior to the addition of antibiotics in PBS where indicated. (D-F) As above, but with/without a period of adaptation in PBS prior to antibiotics as indicated. Antibiotic concentrations were: Msmeg- Isoniazid (INH) 32ug/ml (8 x MIC), rifampin (RIF) 32ug/ml (8 x MIC), ethambutol (EMB) 4ug/ml (8 x MIC), tigecycline (TIG) 1.25ug/ml (8 x MIC), linezolid (LZD) 2.5ug/ml (8 x MIC). Mtb – RIF 0.1ug/ml (4 x MIC), INH at 0.1ug/ml (4 x MIC), EMB at 8ug/ml (4 x MIC). Mabs – TIG (8 x MIC), LZD 100ug/ml (20 x MIC). Antibiotics with half-lives shorter than the duration of experiment were re-added at the following intervals: TIG, EMB every 3d; RIF, INH every 6d. Error bars represent SEM, statistical significance is calculated at each time point using student’s t test. ****: p<0.0001, ***: p<0.001, **: p<0.01, *: p<0.05, ns: p>0.05. Data are combined from 3 independent experiments.
Tn-Seq identifies genes required for antibiotic persistence in Mabs.
(A) Experimental design. (B-E) Tn-Seq analysis showing relative abundance of individual genes under the indicted conditions. All cultures were fully aerated throughout the experiment and cultures without antibiotics received and equal volume of DMSO. For (B-D) gene abundance in the indicated condition is measured relative the input log-phase population. In (E) an additional comparison is made relative to the PBS condition. Genes with significant decreases in abundance are shown in color (p-adj. < 0.05 and log2 fold-change > 0.5) using the Benjamini–Hochberg adjustment for multiple hypothesis testing. (F) Number of genes essential in each condition relative to the input population. (G) Pathway enrichment analysis of the essential genes in each condition using the DAVID knowledgebase (p <0.05). Screens were run as 3 independent experiments and the combined results analyzed. Antibiotic conditions were used as described in Figure 1.
Independent deletions of katG and pafA confirm Tn-Seq results.
(A-E) ORBIT recombineering was used to disrupt the indicated genes, or to generate a control strain with an intergenic region targeted distal to tRNA gene MAB_t5030c. Each mutant was either grown in 7H9 rich media or starved in PBS for 48 prior to the addition of antibiotics. Error bars represent SEM, statistical significance is calculated at each time point using student’s t test. ****: p<0.0001, ***: p<0.001, **: p<0.01, *: p<0.05, ns: p>0.05. Antibiotics were added as described above. Data are combined from 4 independent experiments.
Complementation analysis katG and pafA mutants confirms their role in persister survival.
(A) RT-qPCR analysis of katG expression in katG-(ΔkatG::pmv306), katG+ (ΔkatG::pmv306 katG), and control strain (ORBIT intergenic::pmv306). (B) CFU over time for katG+/katG- strains. (C) MICs for katG+/katG- strains. (D) Expression of pafA in pafA-(ΔpafA::pmv306), pafA+ (pafA::pmv306 pafA) and control strains. (E) CFU over time of pafA+/pafA- strains. (F) MICs for pafA+/pafA- strains. Antibiotic concentrations in (A-B, D-E) are as described above. Error bars represent SEM, statistical significance is calculated at each time point using student’s t test between katG+/katG- strains in (B) and between pafA+/pafA- strains in (E). ****: p<0.0001, ***: p<0.001, **: p<0.01, *: p<0.05, ns: p>0.05. Antibiotics were added as described above.
ROS-mediated toxicity following antibiotic exposure.
(A-D) Analysis of katG+/katG- cells challenged with different antibiotics. Cells were starved in PBS for 48h and then exposed to the indicated antibiotic. (E) Flow cytometry of cells katG+cells stained with DAPI and the ROS-sensitive dye cellROX after 72h in the indicated conditions. Percentage cellROX-positive cells are shown. (F) Persister survival over time for aerated and hypoxic and cultures of Mabs after exposure to TIG/LIN. Error bars represent SEM, statistical significance is calculated at each time point using student’s t test. ****: p<0.0001, ***: p<0.001, **: p<0.01, *: p<0.05, ns: p>0.05. (A-D) are combined data from 3 independent experiments. (E) are representative (median) data from 3 independent experiments. (F) are combined data from 4 independent experiments. Antibiotics were added as described above.
Incomplete penetrance of katG phenotype among Mabs strains.
ΔkatG strains and control strains targeting an intergenic region were constructed on the indicated Mabs backgrounds using ORBIT recombineering: (A) ATCC 19977, (B-C) clinical strains. Bacteria were cultured in 7H9 or starved for 48h in PBS and then treated with antibiotics where indicated. Cultures without antibiotics received an equal volume of DMSO as a control. Survival over time is shown. Error bars represent SEM, statistical significance is calculated at each time point using student’s t test. ****: p<0.0001, ***: p<0.001, **: p<0.01, *: p<0.05, ns: p>0.05.Combined data from 4 independent experiments are shown. Antibiotics were added as described above.
