Reactive oxygen detoxification contributes to Mycobacterium abscessus antibiotic survival
- Department of Internal Medicine, University of California, Davis, United States
- Graduate Group in Immunology, University of California, Davis, United States
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
- Department of Plant and Microbial Biology, University of California, Berkeley, United States
- Microbiology Graduate Group, University of California, Davis, United States
- Department of Medical Microbiology and Immunology, University of California, Davis, United States
Figures
Figure 1
Starvation induces antibiotic tolerance in diverse mycobacteria.
(A) M. smegmatis (Msmeg), (B) M. tuberculosis (Mtb), or (C) M. abscessus (Mabs) were grown in 7H9 rich media or starved in phosphate-buffered saline (PBS) prior to the addition of antibiotics, and surviving colony-forming units (CFUs) enumerated. For the rapidly growing mycobacteria, Msmeg and Mabs, cells were allowed to adapt for 48 hr prior to antibiotics; for slow-growing Mtb, cells were allowed to adapt for 14–21 days prior to antibiotics. Samples without pre-adaptation were washed and placed directly into PBS with antibiotics. Antibiotic concentrations were: Msmeg – Isoniazid (INH) 32 μg/ml (8× minimum inhibitory concentration [MIC]), rifampin (RIF) 32 μg/ml (8× MIC), ethambutol (EMB) 4 μg/ml (8× MIC), tigecycline (TIG) 1.25 μg/ml (8× MIC), linezolid (LZD) 2.5 μg/ml (8× MIC). Mtb – RIF 0.1 μg/ml (4× MIC), INH at 0.1 μg/ml (4× MIC), EMB at 8 μg/ml (4× MIC). Mabs – TIG 10 μg/ml (8× MIC), LZD 100 μg/ml (20× MIC). Antibiotics with half-lives shorter than the duration of the experiment were re-added at the following intervals: TIG, EMB every 3 days; RIF, INH every 6 days. 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.
Figure 2
Tn-Seq identifies genes required for antibiotic tolerance in M. abscessus (Mabs).
(A) Experimental design. (B–E) Tn-Seq analysis showing relative abundance of individual genes under the indicated conditions. For (B–D) gene abundance in each condition is measured relative to the input, with negative values for genes depleted in each experimental condition relative to the input. Log2 fold-change is on the x-axis with -log10 of the p-value on the y-axis. All cultures were fully aerated throughout the experiment, and cultures without antibiotics received an equal volume of DMSO. In (E), an additional comparison is made for phosphate-buffered saline (PBS) with antibiotics 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 as described above. Created with BioRender.com.
Figure 3 with 1 supplement
Validation of Tn-Seq results.
(A–E) ORBIT homologous recombination was used to delete the indicated genes or to generate a control strain targeting a distant intergenic region distal to the nonessential tRNA gene MAB_t5030c. Each strain was either grown in 7H9 rich media or starved in phosphate-buffered saline (PBS) for 48 hr prior to the addition of antibiotics as indicated. The conditions tested here correspond to the conditions in the Tn-Seq analysis where a phenotype was observed. Comparisons in panels A–C are made to the same control strain but plotted independently for clarity. 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 representative of 4 independent experiments.
Figure 3—figure supplement 1
Additional analysis of mutants.
(A) blaR or (B) recR mutants were examined under conditions where Tn-Seq did not predict a phenotype. Cells were starved in phosphate-buffered saline (PBS) for 48 hr prior to treatment with tigecycline and linezolid (TIG/LZD) or DMSO as described in Figure 3. (C) Growth recovery of mutants after antibiotic exposure. After 6 days of TIG/LZD exposure, cells were washed twice in antibiotic-free media and inoculated into 7H9 media. Growth was monitored by OD 600 of the culture. 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, B) are representative data from 4 independent experiments. (C) is representative data from 2 independent experiments.
Figure 4
Complementation analysis of katG and pafA mutants confirms their role in antibiotic tolerance.
(A) RT-qPCR analysis of katG expression in katG- (ΔkatG::pmv306), katG+ (ΔkatG::pmv306 katG), and control strain (ORBIT intergenic::pmv306). (B) Colony-forming unit (CFU) over time for katG+/katG- strains. (C) Minimum inhibitory concentrations (MICs) for katG+/katG- strains. (D) Expression of pafA in pafA- (ΔpafA::pmv306), pafA+ (pafA::pmv306 pafA), and control strain. (E) CFU over time for 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.
Figure 5 with 1 supplement
Reactive oxygen species (ROS)-mediated toxicity following antibiotic exposure.
(A–D) Analysis of katG+/katG- cells challenged with different antibiotics. Cells were starved in phosphate-buffered saline (PBS) for 48 hr and then exposed to the indicated antibiotic. (E) Flow cytometry of control cells exposed to tigecycline and linezolid (TIG/LZD) (4 hr in 7H9 media or 72 hr in PBS) and then stained with DAPI and the ROS-sensitive dye CellROX green; percentage CellROX-positive cells are indicated. (F) Survival over time for aerated and hypoxic cultures of M. abscessus (Mabs) after exposure to TIG/LZD. For hypoxia, cultures were allowed to gradually deplete oxygen until methylene blue dye became colorless on day 5, with antibiotics added 48 hr later. (G) Survival over time for bipyridyl-treated cells after exposure to TIG/LZD. 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, F) display combined data from 3 independent experiments. (E, G) are representative data from 3 independent experiments.
Figure 5—figure supplement 1
Effect of reactive oxygen species (ROS) scavengers.
ΔkatG cells were starved in phosphate-buffered saline (PBS) for 48 hr, then treated with tigecycline and linezolid (TIG/LZD) and the indicated concentration of (A) thiourea or (B) 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), and surviving colony-forming unit (CFU) over time were measured. Representative data from 2 independent experiments are shown.
Figure 6
Conserved antibiotic-induced reactive oxygen species (ROS) production but variable protection by KatG among different M. abscessus (Mabs) strains.
(A) The indicated strains of Mabs were cultured in 7H9 media, exposed to tigecycline and linezolid (TIG/LZD) for 4 hr and then analyzed by CellROX staining. (B) Colony-forming unit (CFU) over time following TIG/LZD exposure. 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 for persister survival experiments. Representative data from 2 independent experiments are shown for flow cytometry experiments. Antibiotics were added as described above.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mycobacterium abscessus) | katG | GenBank Accession CU458896 | MAB_2470c | |
| Gene (M. abscessus) | pafA | GenBank Accession CU458896 | MAB_2183 | |
| Gene (M. abscessus) | MAB_1456c | GenBank Accession CU458896 | MAB_1456c | |
| Gene (M. abscessus) | blaR | GenBank Accession CU458896 | MAB_2414c | |
| Gene (M. abscessus) | recR | GenBank Accession CU458896 | MAB_0320 | |
| Strain, strain background (M. abscessus ATCC 19977) | Mabs | ATCC | 19977 | |
| Strain, strain background (M. abscessus) | Mabs clinical strains 1 and 2 | This paper | Obtained from the Sacramento County Department of Public Health Mycobacteriology Laboratory | |
| Strain, strain background (Mycobacterium smegmatis MC2 155) | Msmeg | ATCC | 700084 | |
| Strain, strain background (Mycobacterium tuberculosis Erdman) | Mtb | ATCC | 35801 | |
| Recombinant DNA reagent | pkm444 (plasmid) | Addgene | 108319 | ORBIT recombineering plasmid |
| Recombinant DNA reagent | pkm496 (plasmid) | Addgene | 109301 | ORBIT payload plasmid |
| Recombinant DNA reagent | pmv306 (plasmid) | Snapper et al., 1988 | Mycobacteria shuttle vector | |
| Chemical compound, drug | Tigecycline (TIG) | Chem Impex | 29737 | |
| Chemical compound, drug | Linezolid (LZD) | Chem Impex | 29723 | |
| Chemical compound, drug | Levofloxacin | Sigma-Aldrich | 28266 | |
| Chemical compound, drug | Cefoxitin | Chem Impex | 1490 | |
| Chemical compound, drug | Rifabutin | Cayman Chemical | 16468 | |
| Chemical compound, drug | Rifampin (RIF) | Sigma-Aldrich | R7382 | |
| Chemical compound, drug | Isoniazid (INH) | Supelco | I3377 | |
| Chemical compound, drug | Ethambutol (EMB) | Thermo Scientific | J6069506 | |
| Chemical compound, drug | CellROX green | Invitrogen | C10444 | |
| Chemical compound, drug | DAPI | Invitrogen | D9542 | |
| Chemical compound, drug | 2,2′-Bipyridyl-2,2′-Bipyridine (Bipyridyl) | Sigma-Aldrich | D216305 | |
| Chemical compound, drug | Thiourea | Sigma-Aldrich | T7875 | |
| Chemical compound, drug | 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) | Sigma-Aldrich | 176141 | |
| Software, algorithm | TRANSIT | TRANSIT | RRID:SCR_016492 | |
| Software, algorithm | DAVID | DAVID | RRID:SCR_001881 |
Additional files
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Supplementary file 1
Tn-Seq results.
- https://cdn.elifesciences.org/articles/104944/elife-104944-supp1-v1.xlsx
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Supplementary file 2
DAVID functional pathway analysis.
- https://cdn.elifesciences.org/articles/104944/elife-104944-supp2-v1.xlsx
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Supplementary file 3
Oligonucleotide sequences.
- https://cdn.elifesciences.org/articles/104944/elife-104944-supp3-v1.xlsx
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Supplementary file 4
16s sequencing data of clinical strains.
- https://cdn.elifesciences.org/articles/104944/elife-104944-supp4-v1.xlsx
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Supplementary file 5
Minimum inhibitory concentration (MIC) values.
- https://cdn.elifesciences.org/articles/104944/elife-104944-supp5-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/104944/elife-104944-mdarchecklist1-v1.docx
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Reactive oxygen detoxification contributes to Mycobacterium abscessus antibiotic survival
eLife 14:RP104944.
https://doi.org/10.7554/eLife.104944.4
