ATP burst is the dominant driver of antibiotic lethality in Mycobacterium smegmatis
- Department of Biology, Indian Institute of Science Education and Research (IISER), India
- Centre for Bacterial Resistance Biology, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, United Kingdom
- Department of Microbial Sciences, Faculty of Health and Medical Sciences, University of Surrey, United Kingdom
Figures
Figure 1 with 1 supplement
Exposure to sub-lethal doses of aminoglycoside and fluoroquinolone reveals physiological adaptation to antibiotics.
The growth curve of M. smegmatis challenged with either 1 x, ½x, or ¼x MBC99 of streptomycin (A) or with norfloxacin (B). Data points represent the mean of at least three independent replicates ± SD. 1 x MBC99 of streptomycin (250 ng/ml) and 1 x MBC99 of norfloxacin (2 µg/ml). Bacterial viability at the start was 600,000 CFU/ml, corresponding to an OD600nm of 0.0025 /ml.
Figure 1—figure supplement 1
MIC verification of drug treated and untreated M. smegmatis.
Graphs depict the MIC of M. smegmatis grown in the presence and absence of ¼x MBC99 of streptomycin (A) or norfloxacin (B) for 25 hr. Cells were harvested at 25 hr (recovery phase), and MIC was compared.
Figure 2 with 1 supplement
Differential expression analysis of total proteome upon norfloxacin and streptomycin treatment.
Volcano plot analysis of the proteins temporally quantified at 7.5 hr, 15 hr, and 25 hr time points for streptomycin (A) norfloxacin treatment (B). Each dot represents one protein. Fold change cut-off ±2 and a t-test significance cut-off of ≤0.05 were applied to identify differentially expressed proteins, shown in different colours.
Figure 2—figure supplement 1
Cytosolic proteins identified upon treatement with antibiotics.
The bar graph (A) shows the number of proteins identified from norfloxacin and streptomycin treatment at 7.5, 15, and 25 hr. Each dot represents the number of proteins identified per sample. Box plot (B) depicts the Pearson correlation analysis of the label-free quantitative (LFQ) intensities among all 10 replicates of each set.
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Figure 2—figure supplement 1—source data 1
Normalized intensities of the protein IDs.
- https://cdn.elifesciences.org/articles/99656/elife-99656-fig2-figsupp1-data1-v1.xlsx
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Figure 2—figure supplement 1—source data 2
Volcano plot analysis of proteomics data.
- https://cdn.elifesciences.org/articles/99656/elife-99656-fig2-figsupp1-data2-v1.xlsx
Figure 3 with 1 supplement
Norfloxacin and streptomycin have a common mechanism of action.
Heat map analysis represents the log2 fold changes of proteins involved in (A) DNA damage response and proteostasis network, (B) DNA replication and transcription, (C) translation, and (D) cell cycle and cell division processes, identified from proteomics data. Colours in the heat map depict the extent of differential expression, where EE demonstrates exclusively expressed, ER demonstrates exclusively repressed/absent in response to antibiotics, and Ab represents proteins that were not identified or quantified with high significance. (E) Scatter plot of the Pearson correlation analysis of common proteins identified upon norfloxacin (NOR) and streptomycin (STR) treatment. Each dot represents a single protein with its log2 fold difference for NOR (x-axis) and STR (y-axis) treatment at t=15 hr time point. (F) Heat map depicting the log2 fold changes of proteins involved in central carbon metabolism (CCM) of M. smegmatis. Panel (G) illustrates the CCM pathway of M. smegmatis and denotes the expression status of indicated enzymes with colour coding similar to (F).
Figure 3—figure supplement 1
Commonalities in altered proteome upon antibiotic treatment.
The scatter plot (A) depicts the Pearson correlation analysis of proteins commonly identified upon norfloxacin and streptomycin treatment. Each dot represents a single protein with its log2 fold change for norfloxacin (NOR) (x-axis) and streptomycin (STR) (y-axis) treatment at t=25 hr time point. Heatmap (B) depicts the KEGG pathway enrichment analysis of the proteins upregulated and exclusively expressed in response to NOR and STR at 15 hr.
Figure 4 with 1 supplement
Norfloxacin and streptomycin generate reactive oxygen species as part of their lethality.
The heat map (A) depicts the log2 fold differences of proteins involved in antioxidant response. EE demonstrates proteins that are exclusively expressed in response to norfloxacin (NOR) or streptomycin (STR). Temporal (B, C) and dose-dependent (D, G) ratiometric response of M. smegmatis expressing Mrx1-roGFP2 redox biosensor upon STR and NOR treatment at 3 hr. The biosensor fluorescence measurements were recorded at the excitation of 405 nm and 488 nm, for a common emission at 520 nm. Panels demonstrate the effect of 10 mM glutathione on the survival of M. smegmatis treated with lethal dose (10 x MBC99) of STR (E) and NOR (H). Panels (F) and (I) present the effect of bipyridyl on the survival of M. smegmatis treated with lethal doses of STR and NOR, respectively. Data points represent the mean of at least three independent replicates ± SD. Statistical significance was calculated by Student’ t-test (unpaired), *p<0.05, **p<0.01, ***p<0.001.
Figure 4—figure supplement 1
Ratio metric response of M. smegmatis expressing Mrx1-roGFP2.
(A) Time-course ratio metric response of M. smegmatis expressing Mrx1-roGFP2 redox biosensor, at increasing concentrations of menadione (MD) and glutathione (GSH). Mean fluorescence intensities of CellROX deep red dye (B) upon 3 hr of treatment with varying concentrations of norfloxacin and streptomycin; 5 mM cumene hydroperoxide (CHP) was used as a positive control. Ratiometric response of M. smegmatis in response to increasing concentrations of streptomycin (C) and norfloxacin (D) for 3 hr, with and without co-treatment with 10 mM glutathione. (E) Effects of GSH alone on the survival of M. smegmatis. *p<0.05, **p<0.01, ***p<0.001 were calculated by Student’s t-test (unpaired).
Figure 5 with 1 supplement
Norfloxacin and streptomycin treatment induces a lethal burst in ATP levels.
Time course of relative luminescence measurements representing ATP levels in M. smegmatis in response to ¼x (A, D), ½x (B, E), 1 x MBC99 (C, F) of streptomycin and norfloxacin, respectively. The right y-axis represents the viability of cells measured just before the measurement of ATP. Bar graph (G) depicting PHR/mCherry ratios (ATP/ADP) of the ATP biosensor in M. smegmatis exposed to increasing concentrations of NOR and STR for 3 hr; 50 micromolar CCCP was used as a control. 1 x MBC99 for streptomycin (STR) and norfloxacin (NOR) are 1 µg/ml and 16 µg/ml, respectively for OD600=0.8. All data points represent the mean of at least three independent replicates ± SD. Statistical significance was calculated by Student’ t-test (unpaired), *p<0.05, **p<0.01, ***p<0.001, ****p<0.
Figure 5—figure supplement 1
Norfloxacin and streptomycin induce metabolic activity in M. smegmatis.
Panels represent the resazurin fluorescence (at 530 nm excitation and 590 nm emission) on the left y-axis and viability of cells on the right y-axis, in M. smegmatis exposed to either ¼x, ½x, or 1x-MBC99 of streptomycin (STR) and norfloxacin (NOR), respectively.
Figure 6 with 3 supplements
Inhibition of excess ATP production mitigates antibiotic lethality.
Bar graphs report the dose-dependent reduction in ATP levels upon carbonyl cyanide 3-chlorophenylhydrazone (CCCP) co-treatment with streptomycin (STR) (A, B) and norfloxacin (NOR) (D, E). The effect of increasing concentrations of CCCP on survival of M. smegmatis challenged with lethal doses of STR (C) and NOR (F), respectively. Bar graphs represent the relative luminescence measurements indicating ATP levels in wild-type and ΔatpD in response to 1 x MBC99 of STR and NOR for 3 hr (G). Time-kill curves demonstrate the survival differences of wild-type and ΔatpD in response to 1 x MBC99 of STR (H) and NOR (I). All data points represent the mean of at least three independent replicates ± SD. Statistical significance was calculated by Student’ t-test (unpaired), *p<0.05, **p<0.01, ***p<0.001, ****p<0.
Figure 6—figure supplement 1
The effect of CCCP uncoupler on ATP levels and survival of M. smegmatis.
Bar graph depicts the effect of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment alone on relative luminescence (RLU) measurements indicating intracellular ATP levels (A, B), and mycobacterial viability (C), at indicated time points. Data represent the mean of three biological replicates.
Figure 6—figure supplement 2
Suppression of ATP burst by Bedaquiline and Nigericin mitigates antibiotic lethality.
Bar graphs depict relative luminescence (RLU) measurements indicating intracellular ATP levels (A, B) in response to 5 µM nigericin (Nig) alone and in co-treatment with 1 x MBC99 norfloxacin (NOR) or streptomycin (STR) for indicated time points. The effect of nigericin co-treatment on the survival of M. smegmatis for up to 6 hr (C). Panels (D, E) depict the time-kill curve of M. smegmatis pre-treated with BDQ for 24 hr and subsequently challenged with STR and NOR, respectively. 1 x MIC99 of BDQ used in the study was 0.0075 µg/ml. All data points represent the mean of at least three independent replicates ± SD. Statistical significance was calculated by Student’ t-test (unpaired), *p<0.05, **p<0.01, ***p<0.001, ****p<0.
Figure 6—figure supplement 3
The growth curve of wild-type and ΔatpD strain of M. smegmatis.
Data represent the mean of three biological replicates.
Figure 7 with 1 supplement
Norfloxacin and streptomycin-induced reactive oxygen species (ROS) is insufficient to mediate cell death.
Ratiometric response of M. smegmatis expressing Mrx1-roGFP2 redox biosensor (left y-axis) and the cell viability (right y-axis) in response to streptomycin (STR) (A, B) and norfloxacin (NOR) (C, D) with and without the co-supplementation with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) post 3 hr and 6 hr of treatments. Data points represent the mean of at least three independent replicates ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Student’ t-test (unpaired).
Figure 7—figure supplement 1
ROS is not the dominant driver of antibiotic lethality.
Line graph (A) represents the ratio metric response of M. smegmatis expressing Mrx1-roGFP2 redox biosensor for 3 hr in response to 5 µM nigericin alone and in co-treatment with 1 x MBC99 norfloxacin (NOR) or streptomycin (STR). Panel (B) represents the ratio metric response of wild-type and ΔatpD (HygR) strain of M. smegmatis expressing Mrx1-roGFP2 redox biosensor (kanR) in the presence and absence of 1 x MBC99 NOR. Bar graph (C) illustrates the effect of co-treatment of 10 mM GSH on antibiotic-induced ATP levels - the panel depicts the relative luminescence (RLU) measurements indicating intracellular ATP as well as ratiometric response of M. smegmatis expressing Mrx1-roGFP2 redox biosensor in response to 1 x MBC99 of NOR or STR alone, 10 mM GSH alone or in combination, after 3 and 6 hrs of treatments. Bar graphs depict the ratio metric response of M. smegmatis expressing Mrx1-roGFP2 redox biosensor in response to increasing concentrations of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) alone after 3 hr (D) and 6 hr (E). All data points represent the mean of at least three independent replicates ± SD. Statistical significance was calculated by Student’ t-test (unpaired), *p<0.05, **p<0.01, ***p<0.001, ****p<0.
Figure 8 with 1 supplement
Antibiotic-induced ATP levels chelate divalent metal ions.
(A) Effect of increasing concentrations of bipyridyl (BP) for 6 hr on the survival of M. smegmatis (0.8 OD/ml). Bar graphs indicate the effect of increasing concentrations of MgSO4 (B), MnCl2 (C), ZnSO4 (D), and FeSO4 (E) on the survival of M. smegmatis (0.8 OD/ml) challenged with lethal doses of the antibiotics for 6 hr. Growth curve (F) showing the effect of 5 mM of FeSO4, MgSO4, MnCl2, ZnSO4 on the survival of M. smegmatis. Data points represent the mean of at least three independent replicates ± SD. Statistical significance was calculated using Student’ t-test (unpaired). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 8—figure supplement 1
Effect of 5 mM potassium chloride on the survival of M. smegmatis challenged with 1 X MBC99 of streptomycin (STR) and norfloxacin (NOR) for 6 hr.
Co-treatment with monovalent metal ions (which is not sequestered by ATP) do not provide rescue against STR and NOR lethality.
Figure 9 with 1 supplement
13C metabolomics suggests increased metabolic flexibility and bifurcation of TCA cycle flux as bacterial adaptive mechanism.
(A) Percent 13C enrichment in central carbon metabolism (CCM) metabolites of M. smegmatis challenged with ¼x MBC99 of antibiotics at 25 hr time point (recovery phase). ‘M’ denotes the molecular mass of the metabolites, where M+1(n) denotes the incorporation of 13C labelled carbon in the metabolites, leading to increase in the mass by that extent. Data for each isotopologue are represented as mean ± SD from independent triplicates. PEP, phosphoenolpyruvate carboxykinase; OXG, alpha-ketoglutarate; Glu, glutamate, Gln, glutamine, OAA, oxaloacetate; Asp, aspartate; GABA, γ-Aminobutyric acid, SSA, succinic semialdehyde. Line graphs represent the ratios of NADH/NAD+ and NADPH/NADP+ with time (B, D) and dose-dependent manner (C, E), respectively, after antibiotic treatments. Data points represent the mean of at least three independent replicates ± SD. *p<0.05, **p<0.01, ***p<0.001 were calculated by Student’s t-test (unpaired).
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Figure 9—source data 1
Normalised abundance of metabolites identified.
- https://cdn.elifesciences.org/articles/99656/elife-99656-fig9-data1-v1.xlsx
Figure 9—figure supplement 1
Antibiotic-induced metabolic changes in the central carbon metabolism of M. smegmatis.
Bar graph (A) depicts the Peredox/mCherry ratio (NADH/NAD+) of the NADH biosensor in M. smegmatis exposed to increasing concentrations of streptomycin (STR) and norfloxacin (NOR); 50 micromolar carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a control. Panels depict the % 13C enrichment of M+3 (B) and M+5 (C) species of citrate. Panel (D) illustrates the % 13C enrichment in metabolites synthesised from central carbon metabolism of M. smegmatis challenged with ¼ xMBC99 of NOR and STR at 25 hr time point (recovery phase). All data represent the mean ± SD from independent triplicates. *p<0.05, **p<0.01, ***p<0.001 were calculated by Student’s t-test (unpaired).
Figure 10
Sub-lethal antibiotic exposure can potentiate the development of antibiotic resistance.
Heat map (A) showing the log2 fold differences of protein Eis in response to antibiotic treatment. Panel (B) depicts the mutational frequency of M. smegmatis grown in ¼ x MBC99 of the antibiotics for 25 hours; each dot represents the mutational frequency of one replicate (n=10). *p<0.05, **p<0.01, ***p<0.001 were calculated by Student’s t-test (unpaired). (C) Proposed extended model of antibiotic lethality in Mycobacteria. Panel on left explains the interaction among the central dogma processes and central carbon metabolism (CCM), where, depending on the need for energy, flux through CCM is modulated leading to optimal growth and division. Panel on right explains the effect of inhibition of essential cellular processes by antibiotics, resulting in activation of CCM and respiration, leading to reactive oxygen species (ROS) generation and ATP burst. An increase in ATP levels is also contributed by the inhibition of ATP-consuming central dogma and cell division processes by antibiotics. Elevated ATP levels in part chelate essential divalent metal ions and can deny them as co-factors for various proteins, eventually leading to cell death. The thickness of arrows indicates the impact of the processes.
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ATP burst is the dominant driver of antibiotic lethality in Mycobacterium smegmatis
eLife 13:RP99656.
https://doi.org/10.7554/eLife.99656.4
