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A Minor Subpopulation of Mycobacteria Inherently Produces High Levels of Reactive Oxygen Species That Generate Antibiotic Resisters at High Frequency From Itself and Enhance Resister Generation From Its Major Kin Subpopulation

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A Minor Subpopulation of Mycobacteria Inherently Produces High Levels of Reactive Oxygen Species That Generate Antibiotic Resisters at High Frequency From Itself and Enhance Resister Generation From Its Major Kin Subpopulation

Rashmi Ravindran Nair et al. Front Microbiol.

Abstract

Antibiotic-exposed bacteria produce elevated levels of reactive oxygen species (ROS), to which either they succumb or get mutated genome-wide to generate antibiotic resisters. We recently showed that mycobacterial cultures contained two subpopulations, short-sized cells (SCs; ∼10%) and normal/long-sized cells (NCs; ∼90%). The SCs were significantly more antibiotic-susceptible than the NCs. It implied that the SCs might naturally be predisposed to generate significantly higher levels of ROS than the NCs. This in turn could make the SCs more susceptible to antibiotics or generate more resisters as compared to the NCs. Investigation into this possibility showed that the SCs in the actively growing mid-log phase culture naturally generated significantly high levels of superoxide, as compared to the equivalent NCs, due to the naturally high expression of a specific NADH oxidase in the SCs. This caused labile Fe2+ leaching from 4Fe-4S proteins and elevated H2O2 formation through superoxide dismutation. Thus, the SCs of both Mycobacterium smegmatis and Mycobacterium tuberculosis inherently contained significantly higher levels of H2O2 and labile Fe2+ than the NCs. This in turn produced significantly higher levels of hydroxyl radical through Fenton reaction, promoting enhanced antibiotic resister generation from the SCs than from the NCs. The SCs, when mixed back with the NCs, at their natural proportion in the actively growing mid-log phase culture, enhanced antibiotic resister generation from the NCs, to a level equivalent to that from the unfractionated whole culture. The enhanced antibiotic resister generation from the NCs in the reconstituted SCs-NCs natural mixture was found to be due to the high levels of H2O2 secreted by the SCs. Thus, the present work unveils and documents the metabolic designs of two mycobacterial subpopulations where one subpopulation produces high ROS levels, despite higher susceptibility, to generate significantly higher number of antibiotic resisters from itself and to enhance resister generation from its kin subpopulation. These findings show the existence of an inherent natural mechanism in both the non-pathogenic and pathogenic mycobacteria to generate antibiotic resisters. The presence of the SCs and the NCs in the pulmonary tuberculosis patients' sputum, reported by us earlier, alludes to the clinical significance of the study.

Keywords: NADH oxidase; ROS level heterogeneity; antibiotic resisters; labile iron; mycobacterial subpopulations.

Figures

FIGURE 1
FIGURE 1
Determination of the oxidative status and ROS (hydroxyl radical and H2O2) levels in the unexposed freshly prepared SCF/Mrx1-roGFP2 and NCF/Mrx1-roGFP2 cells. Flow cytometry profile of roGFP2 fluorescence of: (A) SCF/Mrx1-roGFP2 cells, (D) NCF/Mrx1-roGFP2 cells. (B,E) Scatter plot of roGFP2 fluorescence of the respective cells from the data in (A,D), respectively. (C,F) Percentages of the respective cells in each quadrant with their V500 and FITC median fluorescence values and their ratios. The major proportion of the cells amongst the four quadrants and their fluorescence ratio are given in bold red color. (G) Quantitation of V500:FITC fluorescence ratio in the SCF/Mrx1-roGFP2 and NCF/Mrx1-roGFP2 cells (n = 3). Statistical significance was calculated using paired t-test where *Indicates p ≤ 0.05.
FIGURE 2
FIGURE 2
The HPF fluorescence flow cytometry profile of Msm SCF and NCF cells and the ROS levels in the Msm and Mtb SCF and NCF cells. (A) Histogram overlay of the median fluorescence of HPF-stained Msm SCF and NCF cells. (B,E) and (C,F) Density plots of HPF-stained: Msm SCF and NCF cells in the (B,E) absence and (C,F) presence of non-toxic concentration of thiourea (5 μM, Nair et al., 2019), respectively. (D,G) Histogram overlay of median fluorescence in the absence and presence of thiourea for the HPF-stained Msm (D) SCF and (G) NCF cells. (H) Quantitation of the median fluorescence, indicating hydroxyl radical levels, in the HPF-stained Msm SCF and NCF cells after normalization with their respective autofluorescence samples from (I–O) (n = 3). Statistical significance was calculated using paired t-test where *Indicates p ≤ 0.05. (I,J) Flow cytometry profile of autofluorescence of unstained 104 cells/ml of Msm (I) SCF, and (J) NCF cells. (K) Histogram overlay of the unstained SCF and NCF cells. (L,M) Autofluorescence of unstained control cells of: (L) SCF and (M) NCF in the presence of thiourea (TU). (N,O) Histogram overlay of unstained (N) SCF cells (from I and L) and (O) NCF cells (from J and M) in the absence and presence of TU, respectively. (P) Quantitation of the levels of H2O2 per cell in the unexposed and DMTU-exposed non-toxic concentration of DMTU (1 mM; Supplementary Figures S1A–C) Msm SCF and NCF cells using Amplex Red assay (n = 3). (Q) Quantitation of the levels of H2O2 per μg protein in the Mtb SCF and NCF cells using Amplex Red assay (n = 3). The values of statistical significance in (P) and (Q) were calculated using paired t-test where * and ∗∗ indicate p ≤ 0.05 and p ≤ 0.01, respectively.
FIGURE 3
FIGURE 3
Superoxide and H2O2 levels in SCF and NCF cells and analysis of NADH oxidase expression and activity. (A) Relative levels of 2-OH-ethidium fluorescence in SCF and NCF cells from unexposed and DPI-exposed conditions (n = 3). (B) NADH oxidase activity in SCF and NCF cells from unexposed and DPI-exposed conditions (n = 3). (C) Quantitation of H2O2 levels in SCF and NCF cells from unexposed and DPI-exposed conditions, using Amplex Red assay (n = 3 technical triplicates). (D) Superoxide dismutase (SOD) activity in SCF and NCF cells in the absence and presence of SOD inhibitor sodium azide (n = 6). (E) Fold change in the expression of NADH oxidase genes of SCF cells in normalized with the expression in NCF cells by qPCR (n = 2). (F) Fluorescence microscopy images of Msm/pAKMN2-PMSMEG_6603-ugfpm2+ MLP cells. Arrows indicate SCs. (G) Single cell fluorescence intensity analysis of SCs and NCs in MLP [calculated from (F), n = 85 cells]. Statistical significance was calculated using Students’ t-test for (A), (B), and (E) and paired t-test for (C), (D), and (G) where *, ∗∗, and ∗∗∗ indicate p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively.
FIGURE 4
FIGURE 4
Analysis of iron levels in Msm and Mtb SCF and NCF cells. (A) Estimation of labile and total iron levels (μmoles/cell) in the SCF and NCF cells using FeRhoNox-1 assay (n = 3). (B) Levels of Fe2+ (μmoles/cell) in Msm SCF and NCF cell lysates in the presence of 100 μM bipyridyl (n = 3). (C) Fluorescence microscopy images of FeRhoNox-1 stained MLP cells. Arrows indicate SCs. (D) Single cell analysis of the fluorescence intensities of FeRhoNox-1 stained SCs and NCs [calculated from (C), n = 29 cells]. (E) Estimation of labile iron levels in the Mtb SCF and NCF cells using FeRhoNox-1 assay (n = 3). (F) Estimation of labile iron levels in the unexposed, DMTU-exposed and DPI-exposed Msm SCF, and NCF cells using FeRhoNox-1 assay (n = 3). (G) Determination of aconitase activity in the unexposed, DMTU-exposed and DPI-exposed Msm SCF and NCF cells (n = 3). Statistical significance was calculated using paired t-test for (A), (D), and (G), and Students’ t-test for (E), and (F) where *, ∗∗, ∗∗∗ indicate p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively.
FIGURE 5
FIGURE 5
Resister generation frequencies of Msm SCF and NCF cells against antibiotics. (A,B) Resister generation frequencies of SCF and NCF cells when exposed to: (A) RIF and (B) MXF (n = 9 in each case). (C–E) Resister generation frequencies of SCF and NCF cells against RIF when exposed to: (C) DMTU (n = 7); (D) DPI (n = 3); (E) TU (n = 3). (F) Resister generation frequency of SCF, NCF, and NLP against RIF (n = 17). (G) Resister generation frequency of NLP and MLP against RIF (n = 3). S + N represents NLP constituted of unexposed SCF and NCF cells at 1:9 ratio. Statistical significance was calculated using paired t-test, where *, ∗∗∗ indicate p ≤ 0.05, p ≤ 0.001, respectively.
FIGURE 6
FIGURE 6
Contribution of the SCF and NCF cell to the RIF resister generation from NLP. (A) Upper panels: Colonies formed from the NLP cross-mixture 1 and 2 constituted with: (left panel) SCF1-WT, SCF2-WT, and NCF/pAKMN2-ugfpm2+-hygr integrant cells (NLP Cross-mixture 1); (right panel) SCF1/pAKMN2-ugfpm2+-hygr, SCF2/pAKMN2-ugfpm2+-hygr, and NCF-WT cells (NLP Cross-mixture 2). Lower panels in (A): PCR amplification products of ugfpm2+ from the genomic DNA of the RIF resisters from the NLP cross mixtures 1 and 2. (B) Table showing the number of observed and expected RIF resisters from the NLP cross-mixtures obtained from (A). (C) Quantitation of H2O2 levels released by the SCF and NCF cells during unexposed and 1 mM DMTU-exposed conditions, measured using Amplex Red assay. Average of technical triplicates are represented in the graph. (D) Resister generation frequency of NCF in the presence of unexposed and DMTU-exposed (represented as subscript D) SCF cells. SD + N indicates NLP mixture comprising of DMTU-exposed SCF cells and unexposed NCF cells at 1:9 ratio (n = 3). Statistical significance was calculated using paired t-test where *, ∗∗ indicates p ≤ 0.05, p ≤ 0.01, respectively.
FIGURE 7
FIGURE 7
Functional model depicting the metabolic status of SCF and NCF cells that promote higher resister generation frequency and enhanced resister generation from the population. Higher expression of NADH oxidase resulting in high superoxide levels, which gets dismutated to generate H2O2. The superoxide and H2O2 levels result in the higher extent of Fe2+ leaching, which along with H2O2 undergo Fenton reaction to generate hydroxyl radical. Higher ROS levels eventually inflict higher rate of mutation and thereby increase resister generation from SCF cells. The NCF cells display lesser extent of such processes and thereby lesser resister generation.

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