Skip to main content

In Vitro Models That Utilize Hypoxia to Induce Non-replicating Persistence in Mycobacteria

  • Protocol
  • First Online:
Mycobacteria Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1285))

Abstract

The Wayne model and Rapid Anaerobic Dormancy model are widely used methods to analyze the response of Mycobacterium tuberculosis to hypoxia and anaerobiosis. In these models tubercle bacilli are grown in sealed tubes in which bacilli aerobic respiration produces a temporal oxygen gradient. The gradual depletion of oxygen results in a non-replicating persistent culture capable of extended microaerobic and anaerobic survival. Here we describe both models used to induce hypoxic non-replicating persistence in M. tuberculosis. Additional techniques such as the isolation of RNA, the detection of nitrate reductase activity and ATP levels, and the determination of the NAD+/NADH ratio are described.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Wayne LG, Sohaskey CD (2001) Non-replicating persistence of Mycobacterium tuberculosis. Annu Rev Microbiol 55:139–163

    Article  CAS  PubMed  Google Scholar 

  2. Via LE, Lin PL, Ray SM, Carrillo J, Allen SS, Eum SY, Taylor K, Klein E, Manjunatha U, Gonzales J, Lee EG, Park SK, Raleigh JA, Cho SN, McMurray DN, Flynn JL, Barry CEI (2008) Tuberculous granulomas are hypoxic in guinea pigs, rabbits, and nonhuman primates. Infect Immun 76:2332–2340

    Article  Google Scholar 

  3. Wayne LG, Hayes LG (1996) An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of non-replicating persistence. Infect Immun 64:2062–2069

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Watanabe S, Zimmermann M, Goodwin MB, Sauer U, Barry CEI, Boshoff HI (2011) Fumarate reductase activity maintains an energized membrane in anaerobic Mycobacterium tuberculosis. PLoS Pathog 7:1–15

    Google Scholar 

  5. Voskuil MI, Schnappinger D, Harrell MI, Visconti KC, Dolganov GM, Sherman DR, Schoolnik GK (2003) Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis persistence program. J Exp Med 198:705–713

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sohaskey CD, Wayne LG (2003) Role of narK2X and narGHJI in hypoxic upregulation of nitrate reduction by Mycobacterium tuberculosis. J Bacteriol 185:7247–7256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wayne LG, Hayes LG (1999) Nitrate reduction as a marker for hypoxic shiftdown of Mycobacterium tuberculosis. Tuber Lung Dis 79:127–132

    Article  Google Scholar 

  8. Leistikow RL, Morton RA, Bartek IL, Frimpong I, Wagner K, Voskuil MI (2010) The Mycobacterium tuberculosis DosR regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dormancy. J Bacteriol 192:1662–1670

    Article  CAS  PubMed  Google Scholar 

  9. Boshoff HIM, Myers TG, Copp BR, McNeil MR, Wilson MA, Barry CEI (2004) The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism. J Biol Chem 279:40174–40184

    Article  CAS  PubMed  Google Scholar 

  10. Wayne LG (1977) Synchronized replication of Mycobacterium tuberculosis. Infect Immun 17:528–530

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Bartek IL, Woolhiser LK, Baughn AD, Basaraba RJ, Jacobs WRJ, Lenaerts AJ, Voskuil MI (2014) Mycobacterium tuberculosis Lsr2 is a global transcriptional regulator required for adaptation to changing oxygen levels and virulence. MBio 5:e01106–e01114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Shi L, Sohaskey CD, Pfeiffer C, Parks M, McFadden J, North RJ, Gennaro ML (2010) Carbon flux rerouting during Mycobacterium tuberculosis growth arrest. Mol Microbiol 78:1199–1215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sohaskey CD, Modesti L (2009) Differences in nitrate reduction between Mycobacterium tuberculosis and Mycobacterium bovis are due to differential expression of both narGHJI and narK2. FEMS Microbiol Lett 290:129–134

    Article  CAS  PubMed  Google Scholar 

  14. Honaker RW, Stewart A, Schittone S, Izzo A, Klein MR, Voskuil MI (2008) BCG vaccine strains lack narK2 and narX induction and exhibit altered phenotypes during dormancy. Infect Immun 76:2587–2593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lim A, Eleuterio M, Hutter B, Murugasu-Oei B, Dick T (1999) Oxygen depletion induced dormancy in Mycobacterium bovis BCG. J Bacteriol 181:2252–2256

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Dick T, Lee BH, Murugasu-Oei B (1998) Oxygen depletion induced dormancy in Mycobacterium smegmatis. FEMS Microbiol Lett 163:159–164

    Article  CAS  PubMed  Google Scholar 

  17. London J, Knight M (1966) Concentrations of nicotinamide nucleotide coenzymes in micro-organisms. J Gen Microbiol 44:254

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Charles D. Sohaskey .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Sohaskey, C.D., Voskuil, M.I. (2015). In Vitro Models That Utilize Hypoxia to Induce Non-replicating Persistence in Mycobacteria. In: Parish, T., Roberts, D. (eds) Mycobacteria Protocols. Methods in Molecular Biology, vol 1285. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2450-9_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2450-9_11

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2449-3

  • Online ISBN: 978-1-4939-2450-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics