Abstract
The physiology of dormant Mycobacterium tuberculosis was studied in detail by examining the gene expression of 51 genes using quantitative Reverse-Transcription Polymerase Chain Reaction. A forty-day period of dormancy in the Wayne culture model depicted four major transcription patterns. Some sigma factors and many metabolic genes were constant, whereas genes belonging to the dormancy regulon were activated on day 9. In particular, alpha-crystallin mRNA showed more than a 1,000-fold increase compared to replicating bacilli. Genes belonging to the enduring hypoxic response were up-regulated at day 16, notably, transcription factors sigma B and E. Early genes typical of log-phase bacilli, esat-6 and fbpB, were uniformly down-regulated during dormancy. Late stages of dormancy showed a drop in gene expression likely due to a lack of substrates in anaerobic respiration as demonstrated by the transcriptional activation observed following nitrates addition. Among genes involved in nitrate metabolism, narG was strongly up-regulated by nitrates addition. Dormant bacilli responded very rapidly when exposed to oxygen and fresh medium, showing a transcriptional activation of many genes, including resuscitation-promoting factors, within one hour. Our observations extend the current knowledge on dormant M. tuberculosis gene expression and its response to nutrients and to aerobic and anaerobic respiration.
Similar content being viewed by others
References
Bacon, J., Alderwick, L.J., Allnutt, J.A., Gabasova, E., Watson, R., Hatch, K.A., Clark, S.O., Jeeves, R.E., Marriott, A., Rayner, E., et al. 2014. Non-replicating Mycobacterium tuberculosis elicits a reduced infectivity profile with corresponding modifications to the cell wall and extracellular matrix. PLoS One 9, e87329.
Betts, J.C., Lukey, P.T., Robb, L.C., McAdam, R.A., and Duncan, K. 2002. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol. Microbiol. 43, 717–731.
Biketov, S., Mukamolova, G.V., Potapov, V., Gilenkov, E., Vostroknutova, G., Kell, D.B., Young, M., and Kaprelyants, A.S. 2000. Culturability of Mycobacterium tuberculosis cells isolated from murine macrophages: a bacterial growth factor promotes recovery. FEMS Immunol. Med. Microbiol. 29, 233–240.
Daniel, J., Maamar, H., Deb, C., Sirakova, T.D., and Kolattukudy, P.E. 2011. Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages. PLoS Pathog. 7, e1002093.
Deb, C., Lee, C.M., Dubey, V.S., Daniel, J., Abomoelak, B., Sirakova, T.D., Pawar, S., Rogers, L., and Kolattukudy, P.E. 2009. A novel in vitro multiple-stress dormancy model for Mycobacterium tuberculosis generates a lipid-loaded, drug-tolerant, dormant pathogen. PLoS One 4, e6077.
Downing, K.J., Mischenko, V.V., Shleeva, M.O., Young, D.I., Young, M., Kaprelyants, A.S., Apt, A.S., and Mizrahi, V. 2005. Mutants of Mycobacterium tuberculosis lacking three of the five rpf-like genes are defective for growth in vivo and for resuscitation in vitro. Infect. Immun. 73, 3038–3043.
Fattorini, L., Piccaro, G., Mustazzolu, A., and Giannoni, F. 2013. Targeting dormant bacilli to fight tuberculosis. Mediterr. J. Hematol. Infect. Dis. 5, e2013072.
Filippini, P., Iona, E., Piccaro, G., Peyron, P., Neyrolles, O., and Fattorini, L. 2010. Activity of drug combinations against dormant Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 54, 2712–2715.
Garton, N.J., Waddell, S.J., Sherratt, A.L., Lee, S.M., Smith, R.J., Senner, C., Hinds, J., Rajakumar, K., Adegbola, R.A., Besra, G.S., et al. 2008. Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum. PLoS Med. 5, e75.
Hong, W., Deng, W., and Xie, J. 2013. The structure, function, and regulation of Mycobacterium FtsZ. Cell Biochem. Biophys. 65, 97–105.
Iona, E., Pardini, M., Gagliardi, M.C., Colone, M., Stringaro, A.R., Teloni, R., Brunori, L., Nisini, R., Fattorini, L., and Giannoni, F. 2012. Infection of human THP-1 cells with dormant Mycobacterium tuberculosis. Microbes Infect. 14, 959–967.
Lenaerts, A., Barry, C.E., and Dartois, V. 2015. Heterogeneity in tuberculosis pathology, microenvironments and therapeutic responses. Immunol. Rev. 264, 288–307.
Madiraju, M.V., Moomey, M., Neuenschwander, P.F., Muniruzzaman, S., Yamamoto, K., Grimwade, J.E., and Rajagopalan, M. 2006. The intrinsic ATPase activity of Mycobacterium tuberculosis DnaA promotes rapid oligomerization of DnaA on oriC. Mol. Microbiol. 59, 1876–1890.
Manganelli, R., Voskuil, M.I., Schoolnik, G.K., and Smith, I. 2001. The Mycobacterium tuberculosis ECF sigma factor σE : role in global gene expression and survival in macrophages. Mol. Microbiol. 41, 423–437.
Mariotti, S., Pardini, M., Gagliardi, M.C., Teloni, R., Giannoni, F., Fraziano, M., Lozupone, F., Meschini, S., and Nisini, R. 2013. Dormant Mycobacterium tuberculosis fails to block phagosome maturation and shows unexpected capacity to stimulate specific human T lymphocytes. J. Immunol. 191, 274–282.
Muttucumaru, D.G., Roberts, G., Hinds, J., Stabler, R.A., and Parish, T. 2004. Gene expression profile of Mycobacterium tuberculosis in a non-replicating state. Tuberculosis (Edinb) 84, 239–246.
Park, H.D., Guinn, K.M., Harrell, M.I., Liao, R., Voskuil, M.I., Tompa, M., Schoolnik, G.K., and Sherman, D.R. 2003. Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol. Microbiol. 48, 833–843.
Piccaro, G., Giannoni, F., Filippini, P., Mustazzolu, A., and Fattorini, L. 2013. Activities of drug combinations against Mycobacterium tuberculosis grown in aerobic and hypoxic acidic conditions. Antimicrob. Agents Chemother. 57, 1428–1433.
Rodríguez, J.G., Hernández, A.C., Helguera-Repetto, C., Aguilar Ayala, D., Guadarrama-Medina, R., Anzóla, J.M., Bustos, J.R., Zambrano, M.M., González-Y-Merchand, J., García, M.J., et al. 2014. Global adaptation to a lipid environment triggers the dormancy-related phenotype of Mycobacterium tuberculosis. mBio 5, e01125–14.
Rustad, T.R., Harrell, M.I., Liao, R., and Sherman, D.R. 2008. The enduring hypoxic response of Mycobacterium tuberculosis. PLoS One 3, e1502.
Serra-Vidal, M., Latorre, I., Franken, K., Dìaz, J., de Souza-Galvão, M., Casas, I., Maldonado, J., Milà, C., Solsona, J., Jimenez-Fuentes, M.A., et al. 2014. Immunogenicity of 60 novel latency-related antigens of Mycobacterium tuberculosis. Front. Microbiol. 5, 1–13.
Sherman, D.R., Voskuil, M., Schnappinger, D., Liao, R., Harrell, M.I., and Schoolnik, G.K. 2001. Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding α-crystallin. Proc. Natl. Acad. Sci. USA 98, 7534–7539.
Sohaskey, C.D. and 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.
Sohaskey, C.D. and Wayne, L.G. 2003. Role of narK2X and narGHJI in hypoxic up-regulation of nitrate reduction by Mycobacterium tuberculosis. J. Bacteriol. 185, 7247–7256.
Tufariello, J.M., Mi, K., Xu, J., Manabe, Y.C., Kesavan, A.K., Drumm, J., Tanaka, K., Jacobs, W.R., and Chan, J. 2006. Deletion of the Mycobacterium tuberculosis resuscitation promoting factor Rv1009 gene results in delayed reactivation from chronic tuberculosis. Infect. Immun. 74, 2985–2995.
Voskuil, M.I., Schnappinger, D., Visconti, K.C., Harrell, M.I., Dolganov, G.M., Sherman, D.R., and Schoolnik, G.K. 2003. Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J. Exp. Med. 198, 705–713.
Voskuil, M.I., Visconti, K.C., and Schoolnik, G.K. 2004. Mycobacterium tuberculosis gene expression during adaptation to stationary phase and low-oxygen dormancy. Tuberculosis (Edinb) 84, 218–227.
Wayne, L.G. and Hayes, L.G. 1996. An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence. Infect. Immun. 64, 2062–2069.
Weinrich Olsen, A., van Pinxteren, L.A., Meng Okkels, L., Birk Rasmussen, P., and Andersen, P. 2001. Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85b and esat-6. Infect. Immun. 69, 2773–2778.
WHO. Global Tuberculosis Report. 2015, World Health Organization. WHO/HTM/TB 2015.22.
Author information
Authors and Affiliations
Corresponding author
Additional information
These authors contributed equally to this work.
Supplemental material for this article may be found at http://www.springerlink.com/content/120956
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Iona, E., Pardini, M., Mustazzolu, A. et al. Mycobacterium tuberculosis gene expression at different stages of hypoxia-induced dormancy and upon resuscitation. J Microbiol. 54, 565–572 (2016). https://doi.org/10.1007/s12275-016-6150-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12275-016-6150-4