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.
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References
Wayne LG, Sohaskey CD (2001) Non-replicating persistence of Mycobacterium tuberculosis. Annu Rev Microbiol 55:139–163
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
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
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
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
Sohaskey CD, Wayne LG (2003) Role of narK2X and narGHJI in hypoxic upregulation of nitrate reduction by Mycobacterium tuberculosis. J Bacteriol 185:7247–7256
Wayne LG, Hayes LG (1999) Nitrate reduction as a marker for hypoxic shiftdown of Mycobacterium tuberculosis. Tuber Lung Dis 79:127–132
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
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
Wayne LG (1977) Synchronized replication of Mycobacterium tuberculosis. Infect Immun 17:528–530
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
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
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
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
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
Dick T, Lee BH, Murugasu-Oei B (1998) Oxygen depletion induced dormancy in Mycobacterium smegmatis. FEMS Microbiol Lett 163:159–164
London J, Knight M (1966) Concentrations of nicotinamide nucleotide coenzymes in micro-organisms. J Gen Microbiol 44:254
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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
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DOI: https://doi.org/10.1007/978-1-4939-2450-9_11
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