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A replication clock for Mycobacterium tuberculosis

Nature Medicine volume 15, pages 211–214 (2009)Cite this article

Abstract

Few tools exist to assess replication of chronic pathogens during infection. This has been a considerable barrier to understanding latent tuberculosis, and efforts to develop new therapies generally assume that the bacteria are very slowly replicating or nonreplicating during latency1,2,3. To monitor Mycobacterium tuberculosis replication within hosts, we exploit an unstable plasmid that is lost at a steady, quantifiable rate from dividing cells in the absence of antibiotic selection. By applying a mathematical model, we calculate bacterial growth and death rates during infection of mice. We show that during chronic infection, the cumulative bacterial burden—enumerating total live, dead and removed organisms encountered by the mouse lung—is substantially higher than estimates from colony-forming units. Our data show that M. tuberculosis replicates throughout the course of chronic infection of mice and is restrained by the host immune system. This approach may also shed light on the replication dynamics of other chronic pathogens.

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Figure 1: In vitro stability of pBP10 in M. smegmatis and Mtb in the absence of antibiotic selection.
Figure 2: Stability of pBP10 in Mtb in the absence of bacterial replication.
Figure 3: Plasmid loss, bacterial replication and cumulative bacterial burden in mouse lungs.

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References

  1. Warner, D.F. & Mizrahi, V. Tuberculosis chemotherapy: the influence of bacillary stress and damage response pathways on drug efficacy. Clin. Microbiol. Rev. 19, 558–570 (2006).

    Article  CAS  Google Scholar 

  2. Cho, S.H. et al. Low-oxygen-recovery assay for high-throughput screening of compounds against nonreplicating Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 51, 1380–1385 (2007).

    Article  CAS  Google Scholar 

  3. Lenaerts, A.J. et al. Preclinical testing of the nitroimidazopyran PA-824 for activity against Mycobacterium tuberculosis in a series of in vitro and in vivo models. Antimicrob. Agents Chemother. 49, 2294–2301 (2005).

    Article  CAS  Google Scholar 

  4. Frieden, T.R., Sterling, T.R., Munsiff, S.S., Watt, C.J. & Dye, C. Tuberculosis. Lancet 362, 887–899 (2003).

    Article  Google Scholar 

  5. Wayne, L.G. & Sohaskey, C.D. Nonreplicating persistence of Mycobacterium tuberculosis. Annu. Rev. Microbiol. 55, 139–163 (2001).

    Article  CAS  Google Scholar 

  6. Harries, A.D. & Dye, C. Tuberculosis. Ann. Trop. Med. Parasitol. 100, 415–431 (2006).

    CAS  PubMed  Google Scholar 

  7. Lewis, K.N. et al. Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guerin attenuation. J. Infect. Dis. 187, 117–123 (2003).

    Article  Google Scholar 

  8. Lazarevic, V., Nolt, D. & Flynn, J.L. Long-term control of Mycobacterium tuberculosis infection is mediated by dynamic immune responses. J. Immunol. 175, 1107–1117 (2005).

    Article  CAS  Google Scholar 

  9. Rees, R.J.M. & Hart, P.D. Analysis of the host-parasite equilibrium in chronic murine tuberculosis by total and viable bacillary counts. Br. J. Exp. Pathol. 42, 83–88 (1961).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Munoz-Elias, E.J. et al. Replication dynamics of Mycobacterium tuberculosis in chronically infected mice. Infect. Immun. 73, 546–551 (2005).

    Article  CAS  Google Scholar 

  11. Cosma, C.L., Humbert, O. & Ramakrishnan, L. Superinfecting mycobacteria home to established tuberculous granulomas. Nat. Immunol. 5, 828–835 (2004).

    Article  CAS  Google Scholar 

  12. Meijer, A.H. et al. Identification and real-time imaging of a myc-expressing neutrophil population involved in inflammation and mycobacterial granuloma formation in zebrafish. Dev. Comp. Immunol. 32, 36–49 (2008).

    Article  CAS  Google Scholar 

  13. Swaim, L.E. et al. Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity. Infect. Immun. 74, 6108–6117 (2006).

    Article  CAS  Google Scholar 

  14. Comstock, G.W., Baum, C. & Snider, D.E. Isoniazid prophylaxis among alaskan eskimos: a final report of the Bethel isoniazid studies. Am. Rev. Respir. Dis. 119, 827–830 (1979).

    CAS  PubMed  Google Scholar 

  15. American Thoracic Society & Centers for Disease Control and Prevention. Supplement—American Thoracic Society Centers for Disease Control and Prevention Targeted tuberculin testing and treatment of latent tuberculosis infection. Am. J. Respir. Crit. Care Med. 161, S221–S247 (2000).

  16. Stover, C.K. et al. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature 405, 962–966 (2000).

    Article  CAS  Google Scholar 

  17. Kanai, K. Experimental studies on host-parasite equilibrium in tuberculous infection, in relation to vaccination and chemotherapy. Jpn. J. Med. Sci. Biol. 19, 181–199 (1966).

    Article  CAS  Google Scholar 

  18. Keane, J. TNF-blocking agents and tuberculosis: new drugs illuminate an old topic. Rheumatology (Oxford) 44, 714–720 (2005).

    Article  CAS  Google Scholar 

  19. Bachrach, G. et al. A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon. Microbiology 146, 297–303 (2000).

    Article  CAS  Google Scholar 

  20. Zambrano, M.M., Siegele, D.A., Almiron, M., Tormo, A. & Kolter, R. Microbial competition: Escherichia coli mutants that take over stationary phase cultures. Science 259, 1757–1760 (1993).

    Article  CAS  Google Scholar 

  21. Smeulders, M.J., Keer, J., Speight, R.A. & Williams, H.D. Adaptation of Mycobacterium smegmatis to stationary phase. J. Bacteriol. 181, 270–283 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Davison, A.C. & Hinkley, D.V. Bootstrap Methods and Their Application, Chs. 2 and 4 (Cambridge University Press, Cambridge, 1997).

    Book  Google Scholar 

  23. Rhoades, E.R., Frank, A.A. & Orme, I.M. Progression of chronic pulmonary tuberculosis in mice aerogenically infected with virulent Mycobacterium tuberculosis. Tuber. Lung Dis. 78, 57–66 (1997).

    Article  CAS  Google Scholar 

  24. Mogues, T., Goodrich, M.E., Ryan, L., LaCourse, R. & North, R.J. The relative importance of T cell subsets in immunity and immunopathology of airborne Mycobacterium tuberculosis infection in mice. J. Exp. Med. 193, 271–280 (2001).

    Article  CAS  Google Scholar 

  25. Rivas-Santiago, B. et al. β-defensin gene expression during the course of experimental tuberculosis infection. J. Infect. Dis. 194, 697–701 (2006).

    Article  CAS  Google Scholar 

  26. Nuermberger, E. et al. Rifapentine, moxifloxacin or DNA vaccine improves treatment of latent tuberculosis in a mouse model. Am. J. Respir. Crit. Care Med. 172, 1452–1456 (2005).

    Article  Google Scholar 

  27. Aly, S. et al. Oxygen status of lung granulomas in Mycobacterium tuberculosis–infected mice. J. Pathol. 210, 298–305 (2006).

    Article  CAS  Google Scholar 

  28. Tsai, M.C. et al. Characterization of the tuberculous granuloma in murine and human lungs: cellular composition and relative tissue oxygen tension. Cell. Microbiol. 8, 218–232 (2006).

    Article  CAS  Google Scholar 

  29. Via, L.E. et al. Tuberculous granulomas are hypoxic in guinea pigs, rabbits and nonhuman primates. Infect. Immun. 76, 2333–2340 (2008).

    Article  CAS  Google Scholar 

  30. Wards, B.J. & Collins, D.M. Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria. FEMS Microbiol. Lett. 145, 101–105 (1996).

    Article  CAS  Google Scholar 

  31. Park, H.D. et al. Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol. Microbiol. 48, 833–843 (2003).

    Article  CAS  Google Scholar 

  32. Rustad, T.R., Harrell, M.I., Liao, R. & Sherman, D.R. The enduring hypoxic response of Mycobacterium tuberculosis. PLoS ONE 3, e1502 (2008).

    Article  Google Scholar 

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Acknowledgements

We would like to thank L. Ramakrishnan, J. Chang, K. Guinn, T. Walker and members of the Sherman lab for review of and contributions to this work, C. DeMille for expert help with manuscript preparation and K.G. Papavinasasundaram (University of Massachusetts Medical School) for providing pBP10. This research was supported by grants from the US National Institutes of Health and the Paul G. Allen Family Foundation.

Author information

Author notes

  1. Wendy P Gill

    Present address: Current address: Infectious Disease Consultants, 1601 East Nineteenth Avenue, Suite 3650, Denver, Colorado 80218, USA.,

  2. Wendy P Gill and Nada S Harik: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Allergy and Infectious Diseases, University of Washington Medical Center, 1959 Northeast Pacific Street, Box 356523, Seattle, 98195, Washington, USA

    Wendy P Gill

  2. Division of Pediatric Infectious Diseases, University of Arkansas for Medical Sciences, 800 Marshall Street, Slot 512-11, Little Rock, 72202, Arkansas, USA

    Nada S Harik

  3. Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, 98109, Washington, USA

    Molly R Whiddon, Reiling P Liao & David R Sherman

  4. Department of Global Health, Interdisciplinary Program of Pathobiology, University of Washington, Health Sciences Building H-660, Box 357660, Seattle, 98195, Washington, USA

    Molly R Whiddon & David R Sherman

  5. Department of Microbiology, University of Washington, Box 357242, Seattle, 98195, Washington, USA

    John E Mittler

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Contributions

W.P.G., N.S.H., M.R.W., R.P.L. and D.R.S. designed all experiments. W.P.G., N.S.H., R.P.L. and M.R.W. performed all experiments. W.P.G., N.S.H., M.R.W., J.E.M. and D.R.S. analyzed the data. J.E.M. derived the equations in consultation with W.P.G., and D.R.S. W.P.G. and D.R.S. wrote the paper, which was edited by all authors.

Corresponding author

Correspondence to David R Sherman.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Fig.1 and Supplementary Equations 1 and 2 (PDF 364 kb)

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Gill, W., Harik, N., Whiddon, M. et al. A replication clock for Mycobacterium tuberculosis. Nat Med 15, 211–214 (2009). https://doi.org/10.1038/nm.1915

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