1887

Abstract

Phenotypically heterogeneous but genetically identical mycobacterial subpopulations exist in in vitro cultures, in vitro-infected macrophages, infected animal models and tuberculosis patients. In this regard, we recently reported the presence of two subpopulations of cells, which are phenotypically different in length and buoyant density, in mycobacterial cultures. These are the low-buoyant-density short-sized cells (SCs), which constitute ~10–20 % of the population, and the high-buoyant-density normal/long-sized cells (NCs), which form ~80–90 % of the population. The SCs were found to be significantly more susceptible to rifampicin (RIF), isoniazid (INH), H2O2 and acidified nitrite than the NCs. Here we report that the RIF-/INH-/H2O2-exposed SCs showed significantly higher levels of oxidative stress and therefore higher susceptibility than the equivalent number of exposed NCs. Significantly higher levels of hydroxyl radical and superoxide were found in the antibiotic-exposed SCs than in the equivalently exposed NCs. Different proportions of the subpopulation of SCs were found to have different levels of reactive oxygen species (ROS). The hydroxyl radical quencher, thiourea, and the superoxide dismutase mimic, TEMPOL, significantly reduced hydroxyl radical and superoxide levels, respectively, in the antibiotic-exposed SCs and NCs and thereby decreased their differential susceptibility to antibiotics. Thus, the present study shows that the heterogeneity of the reactive oxygen species (ROS) levels in these mycobacterial subpopulations confers differential susceptibility to antibiotics. We have discussed the possible mechanisms that can generate differential ROS levels in the antibiotic-exposed SCs and NCs. The present study advances our current understanding of the molecular mechanisms underlying antibiotic tolerance in mycobacteria.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000797
2019-05-15
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/165/6/668.html?itemId=/content/journal/micro/10.1099/mic.0.000797&mimeType=html&fmt=ahah

References

  1. Hallez R, Bellefontaine AF, Letesson JJ, de Bolle X. Morphological and functional asymmetry in alpha-proteobacteria. Trends Microbiol 2004; 12:361–365 [View Article][PubMed]
    [Google Scholar]
  2. Aertsen A, Michiels CW. Diversify or die: generation of diversity in response to stress. Crit Rev Microbiol 2005; 31:69–78 [View Article][PubMed]
    [Google Scholar]
  3. Zgur-Bertok D. Phenotypic heterogeneity in bacterial populations. Acta Agriculturae Slovenica 2007; 90:17–24
    [Google Scholar]
  4. Davidson CJ, Surette MG. Individuality in bacteria. Annu Rev Genet 2008; 42:253–268 [View Article][PubMed]
    [Google Scholar]
  5. Diard M, Garcia V, Maier L, Remus-Emsermann MN, Regoes RR et al. Stabilization of cooperative virulence by the expression of an avirulent phenotype. Nature 2013; 494:353–356 [View Article][PubMed]
    [Google Scholar]
  6. Nyka W. Studies on the effect of starvation on mycobacteria. Infect Immun 1974; 9:843–850[PubMed]
    [Google Scholar]
  7. Smeulders MJ, Keer J, Speight RA, Williams HD. Adaptation of Mycobacterium smegmatis to stationary phase. J Bacteriol 1999; 181:270–280[PubMed]
    [Google Scholar]
  8. Thanky NR, Young DB, Robertson BD. Unusual features of the cell cycle in mycobacteria: polar-restricted growth and the snapping-model of cell division. Tuberculosis 2007; 87:231–236 [View Article][PubMed]
    [Google Scholar]
  9. Anuchin AM, Mulyukin AL, Suzina NE, Duda VI, El-Registan GI et al. Dormant forms of Mycobacterium smegmatis with distinct morphology. Microbiology 2009; 155:1071–1079 [View Article][PubMed]
    [Google Scholar]
  10. Markova N, Slavchev G, Michailova L. Unique biological properties of Mycobacterium tuberculosis L-form variants: impact for survival under stress. Int Microbiol 2012; 15:61–68 [View Article][PubMed]
    [Google Scholar]
  11. Khomenko AG. The variability of Mycobacterium tuberculosis in patients with cavitary pulmonary tuberculosis in the course of chemotherapy. Tubercle 1987; 68:243–253 [View Article][PubMed]
    [Google Scholar]
  12. Seiler P, Ulrichs T, Bandermann S, Pradl L, Jörg S et al. Cell-wall alterations as an attribute of Mycobacterium tuberculosis in latent infection. J Infect Dis 2003; 188:1326–1331 [View Article][PubMed]
    [Google Scholar]
  13. Ryan GJ, Hoff DR, Driver ER, Voskuil MI, Gonzalez-Juarrero M et al. Multiple M. tuberculosis phenotypes in mouse and guinea pig lung tissue revealed by a dual-staining approach. PLoS One 2010; 5:e11108 [View Article][PubMed]
    [Google Scholar]
  14. Daniel J, Maamar H, Deb C, Sirakova TD, Kolattukudy PE. Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages. PLoS Pathog 2011; 7:e1002093 [View Article][PubMed]
    [Google Scholar]
  15. Manina G, Dhar N, McKinney JD. Stress and host immunity amplify Mycobacterium tuberculosis phenotypic heterogeneity and induce nongrowing metabolically active forms. Cell Host Microbe 2015; 17:32–46 [View Article][PubMed]
    [Google Scholar]
  16. Vijay S, Nagaraja M, Sebastian J, Ajitkumar P. Asymmetric cell division in Mycobacterium tuberculosis and its unique features. Arch Microbiol 2014a; 196:157–168 [View Article][PubMed]
    [Google Scholar]
  17. Vijay S, Mukkayyan N, Ajitkumar P. Highly deviated asymmetric division in very low proportion of mycobacterial mid-log phase cells. Open Microbiol J 2014b; 8:40–50 [View Article][PubMed]
    [Google Scholar]
  18. Vijay S, Nair RR, Sharan D, Jakkala K, Mukkayyan N et al. Mycobacterial cultures contain cell size and density specific sub-populations of cells with significant differential susceptibility to antibiotics, oxidative and nitrite stress. Front Microbiol 2017; 8:463 [View Article]
    [Google Scholar]
  19. Hartmann GR, Heinrich P, Kollenda MC, Skrobranek B, Tropschug M et al. Molecular Mechanism of Action of the Antibiotic Rifampicin. Angewandte Chemie International Edition in English 1985; 24:1009–1014 [View Article]
    [Google Scholar]
  20. Campbell EA, Korzheva N, Mustaev A, Murakami K, Nair S et al. Structural mechanism for rifampicin inhibition of bacterial rna polymerase. Cell 2001; 104:901–912 [View Article][PubMed]
    [Google Scholar]
  21. Takayama K, Wang L, David HL. Effect of isoniazid on the in vivo mycolic acid synthesis, cell growth and viability of Mycobacterium tuberculosis. Antimicrob Agents Chemother 1972; 2:29–35 [View Article][PubMed]
    [Google Scholar]
  22. Banerjee A, Dubnau E, Quemard A, Balasubramanian V, Um KS et al. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 1994; 263:227–230 [View Article][PubMed]
    [Google Scholar]
  23. Dwyer DJ, Kohanski MA, Hayete B, Collins JJ. Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli. Mol Syst Biol 2007; 3:91 [View Article][PubMed]
    [Google Scholar]
  24. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 2007; 130:797–810 [View Article][PubMed]
    [Google Scholar]
  25. van Acker H, Gielis J, Acke M, Cools F, Cos P et al. The role of reactive oxygen species in antibiotic-induced cell death in burkholderia cepacia complex bacteria. PLoS One 2016; 11:e0159837 [View Article][PubMed]
    [Google Scholar]
  26. Snapper SB, Melton RE, Mustafa S, Kieser T, Jacobs WR. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 1990; 4:1911–1919 [View Article][PubMed]
    [Google Scholar]
  27. Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 1985; 33:103–119 [View Article][PubMed]
    [Google Scholar]
  28. Bhaskar A, Chawla M, Mehta M, Parikh P, Chandra P et al. Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection. PLoS Pathog 2014; 10:e1003902 [View Article][PubMed]
    [Google Scholar]
  29. Alting-Mees MA, Short JM. pBluescript II: gene mapping vectors. Nucleic Acids Res 1989; 17:9494 [View Article][PubMed]
    [Google Scholar]
  30. Roy S, Narayana Y, Balaji KN, Ajitkumar P. Highly fluorescent GFPm 2+ -based genome integration-proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages. Microb Biotechnol 2012; 5:98–105 [View Article][PubMed]
    [Google Scholar]
  31. Setsukinai K, Urano Y, Kakinuma K, Majima HJ, Nagano T. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J Biol Chem 2003; 278:3170–3175 [View Article][PubMed]
    [Google Scholar]
  32. Mukherjee P, Sureka K, Datta P, Hossain T, Barik S et al. Novel role of Wag31 in protection of mycobacteria under oxidative stress. Mol Microbiol 2009; 73:103–119 [View Article][PubMed]
    [Google Scholar]
  33. Nazarewicz RR, Bikineyeva A, Dikalov SI. Rapid and specific measurements of superoxide using fluorescence spectroscopy. J Biomol Screen 2013; 18:498–503 [View Article][PubMed]
    [Google Scholar]
  34. Yeware AM, Shurpali KD, Athalye MC, Sarkar D. Superoxide generation and its involvement in the growth of Mycobacterium smegmatis. Front Microbiol 2017; 8:105 [View Article][PubMed]
    [Google Scholar]
  35. Peshavariya HM, Dusting GJ, Selemidis S. Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase. Free Radic Res 2007; 41:699–712 [View Article][PubMed]
    [Google Scholar]
  36. Wilcox CS, Pearlman A. Chemistry and antihypertensive effects of tempol and other nitroxides. Pharmacol Rev 2008; 60:418–469 [View Article][PubMed]
    [Google Scholar]
  37. Fisher AE, Maxwell SC, Naughton DP. Superoxide and hydrogen peroxide suppression by metal ions and their EDTA complexes. Biochem Biophys Res Commun 2004; 316:48–51 [View Article][PubMed]
    [Google Scholar]
  38. Piccaro G, Pietraforte D, Giannoni F, Mustazzolu A, Fattorini L. Rifampin induces hydroxyl radical formation in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2014; 58:7527–7533 [View Article][PubMed]
    [Google Scholar]
  39. Grant SS, Kaufmann BB, Chand NS, Haseley N, Hung DT. Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals. Proc Natl Acad Sci USA 2012; 109:12147–12152 [View Article][PubMed]
    [Google Scholar]
  40. Nandakumar M, Nathan C, Rhee KY. Isocitrate lyase mediates broad antibiotic tolerance in Mycobacterium tuberculosis. Nat Commun 2014; 5:4306 [View Article][PubMed]
    [Google Scholar]
  41. Vattanaviboon P, Mongkolsuk S. Evaluation of the role hydroxyl radicals and iron play in hydrogen peroxide killing of Xanthomonas campestris pv. phaseoli. FEMS Microbiol Lett 1998; 169:255–260 [View Article]
    [Google Scholar]
  42. Grossman JN, Kahan TF. Hydroxyl radical formation from bacteria-assisted Fenton chemistry at neutral pH under environmentally relevant conditions. Environ Chem 2016; 13:757–766 [View Article]
    [Google Scholar]
  43. McBee ME, Chionh YH, Sharaf ML, Ho P, Cai MW et al. Production of superoxide in bacteria is stress- and cell state-dependent: a gating-optimized flow cytometry method that minimizes ros measurement artifacts with fluorescent dyes. Front Microbiol 2017; 8:459 [View Article][PubMed]
    [Google Scholar]
  44. Hassan HM, Fridovich I. Intracellular production of superoxide radical and of hydrogen peroxide by redox active compounds. Arch Biochem Biophys 1979; 196:385–395 [View Article][PubMed]
    [Google Scholar]
  45. Liu X, Marrakchi M, Jahne M, Rogers S, Andreescu S. Real-time investigation of antibiotics-induced oxidative stress and superoxide release in bacteria using an electrochemical biosensor. Free Radic Biol Med 2016; 91:25–33 [View Article][PubMed]
    [Google Scholar]
  46. Winterbourn CC. Toxicity of iron and hydrogen peroxide: the Fenton reaction. Toxicol Lett 1995; 82-83:969–974 [View Article][PubMed]
    [Google Scholar]
  47. Korshunov S, Imlay JA. Two sources of endogeneous H2O2 in Escherichia coli. Mol Microbiol 2010; 75:1389–1401
    [Google Scholar]
  48. McCord JM, Fridovich I, Dismutase S. An enzymatic function for erythrocuprein (hemocuprein). J Biol Chem 1969; 244:6049–6055
    [Google Scholar]
  49. McCord JM, Fridovich I. The reduction of cytochrome c by milk xanthine oxidase. J Biol Chem 1968; 243:5753–5760[PubMed]
    [Google Scholar]
  50. González-Flecha B, Demple B. Metabolic sources of hydrogen peroxide in aerobically growing Escherichia coli. J Biol Chem 1995; 270:13681–13687 [View Article][PubMed]
    [Google Scholar]
  51. Imlay JA. Cellular defenses against superoxide and hydrogen peroxide. Annu Rev Biochem 2008; 77:755–776 [View Article][PubMed]
    [Google Scholar]
  52. Diaz JM, Hansel CM, Voelker BM, Mendes CM, Andeer PF et al. Widespread production of extracellular superoxide by heterotrophic bacteria. Science 2013; 340:1223–1226 [View Article][PubMed]
    [Google Scholar]
  53. Yang X, Ma K. Characterization of an exceedingly active NADH oxidase from the anaerobic hyperthermophilic bacterium Thermotoga maritima. J Bacteriol 2007; 189:3312–3317 [View Article][PubMed]
    [Google Scholar]
  54. Nishiyama Y, Massey V, Takeda K, Kawasaki S, Sato J et al. Hydrogen peroxide-forming NADH oxidase belonging to the peroxiredoxin oxidoreductase family: existence and physiological role in bacteria. J Bacteriol 2001; 183:2431–2438 [View Article][PubMed]
    [Google Scholar]
  55. Esterházy D, King MS, Yakovlev G, Hirst J. Production of reactive oxygen species by complex I (NADH:ubiquinone oxidoreductase) from Escherichia coli and comparison to the enzyme from mitochondria. Biochemistry 2008; 47:3964–3971 [View Article][PubMed]
    [Google Scholar]
  56. Vinogradov AD, Grivennikova VG. Oxidation of NADH and ROS production by respiratory complex I. Biochim Biophys Acta 1857; 2016:863–871
    [Google Scholar]
  57. Imlay JA. The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat Rev Microbiol 2013; 11:443–454 [View Article][PubMed]
    [Google Scholar]
  58. Reddy PV, Puri RV, Khera A, Tyagi AK. Iron storage proteins are essential for the survival and pathogenesis of Mycobacterium tuberculosis in THP-1 macrophages and the guinea pig model of infection. J Bacteriol 2012; 194:567–575 [View Article][PubMed]
    [Google Scholar]
  59. Pandey R, Rodriguez GM. IdeR is required for iron homeostasis and virulence in Mycobacterium tuberculosis. Mol Microbiol 2014; 91:98–109 [View Article][PubMed]
    [Google Scholar]
  60. Sritharan M. Iron homeostasis in Mycobacterium tuberculosis: mechanistic insights into siderophore-mediated iron uptake. J Bacteriol 2016; 198:2399–2409 [View Article][PubMed]
    [Google Scholar]
  61. Flint DH, Tuminello JF, Emptage MH. The inactivation of Fe-S cluster containing hydro-lyases by superoxide. J Biol Chem 1993; 268:22369–22376[PubMed]
    [Google Scholar]
  62. Jang S, Imlay JA. Hydrogen peroxide inactivates the Escherichia coli Isc iron-sulphur assembly system, and OxyR induces the suf system to compensate. Mol Microbiol 2010; 78:1448–1467 [View Article][PubMed]
    [Google Scholar]
  63. Hoff DR, Ryan GJ, Driver ER, Ssemakulu CC, de Groote MA et al. Location of intra- and extracellular M. tuberculosis populations in lungs of mice and guinea pigs during disease progression and after drug treatment. PLoS One 2011; 6:e17550 [View Article][PubMed]
    [Google Scholar]
  64. Lenaerts AJ, Hoff D, Aly S, Ehlers S, Andries K et al. Location of persisting mycobacteria in a Guinea pig model of tuberculosis revealed by r207910. Antimicrob Agents Chemother 2007; 51:3338–3345 [View Article][PubMed]
    [Google Scholar]
  65. Farr SB, Natvig DO, Kogoma T. Toxicity and mutagenicity of plumbagin and the induction of a possible new DNA repair pathway in Escherichia coli. J Bacteriol 1985; 164:1309–1316[PubMed]
    [Google Scholar]
  66. Liou JW, Hung YJ, Yang CH, Chen YC. The antimicrobial activity of gramicidin A is associated with hydroxyl radical formation. PLoS One 2015; 10:e0117065 [View Article][PubMed]
    [Google Scholar]
  67. Duan X, Huang X, Wang X, Yan S, Guo S et al. l-Serine potentiates fluoroquinolone activity against Escherichia coli by enhancing endogenous reactive oxygen species production. J Antimicrob Chemother 2016; 71:2192–2199 [View Article][PubMed]
    [Google Scholar]
  68. Zhao X, Drlica K. Reactive oxygen species and the bacterial response to lethal stress. Curr Opin Microbiol 2014; 21:1–6 [View Article][PubMed]
    [Google Scholar]
  69. van Acker H, Coenye T. The role of reactive oxygen species in antibiotic-mediated killing of bacteria. Trends Microbiol 2017; 25:456–466 [View Article][PubMed]
    [Google Scholar]
  70. Vaubourgeix J, Lin G, Dhar N, Chenouard N, Jiang X et al. Stressed mycobacteria use the chaperone ClpB to sequester irreversibly oxidized proteins asymmetrically within and between cells. Cell Host Microbe 2015; 17:178–190 [View Article][PubMed]
    [Google Scholar]
  71. Bergmiller T, Andersson AMC, Tomasek K, Balleza E, Kiviet DJ et al. Biased partitioning of the multidrug efflux pump AcrAB-TolC underlies long-lived phenotypic heterogeneity. Science 2017; 356:311–315 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000797
Loading
/content/journal/micro/10.1099/mic.0.000797
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error