Dormant tubercle bacilli: the key to more effective TB chemotherapy?

T. Dick

Mycobacterium Laboratory, Institute of Molecular & Cell Biology, 30 Medical Drive, Singapore 117609, Singapore

Sir,

A key problem in tuberculosis control is the persistence of Mycobacterium tuberculosis despite prolonged chemotherapy: treatment for 6–12 months is required to cure acute disease or eliminate latent infection. Why does it take so long to eradicate the bacilli once they reside within the human host? Development of physiological (as opposed to genetic) drug resistance could play a role. Mycobacteria are obligate aerobes. However, it has been known for years that tubercle bacilli encounter hypoxic environments in acute disease as well as in latent infection.1,2 Wayne established a link between starvation for oxygen and drug resistance. The author demonstrated that upon depletion of oxygen in culture the bacillus terminates growth and develops into a defined non-replicating or dormant form (FigureGo). Dormant bacilli are adapted to anaerobiosis and maintain viability for extended periods of time. A key feature of the dormant culture is that it is synchronized, i.e. the bacilli are arrested at a uniform stage of the cell cycle. Importantly, the dormant form of the bacterium was found to be resistant to conventional anti-mycobacterials.2 Recent genetic evidence suggests that the capability of tubercle bacilli to adapt to hypoxic conditions indeed plays a role in vivo. Bacilli that were disrupted for their respiratory nitrate reductase3 and isocitrate lyase4 (an enzyme of the glyoxylate pathway and implicated in the metabolic adaptation to anaerobiosis2) showed reduced virulence in mice. Taking these in vitro and in vivo findings together, the following working model to explain the persistence of genetically drug-sensitive bacilli during chemotherapy can be proposed: current tuberculosis drugs kill growing bacilli rapidly. However, subpopulations of the bacterium encounter (self-generate?) severely hypoxic micro-environments within the patient and develop into a dormant form that is phenotypically drug resistant. The micro-environments containing dormant bacilli might change over time and oxygen could become available again, the bacilli resume growth and are killed by standard drugs. After long-term chemotherapy the acute disease is cured, and the latent infection is eliminated. Evidently, this model is speculative and oversimplifies the situation in vivo. However, if it turns out that it does reflect some aspects of the life of the bacterium in its host, the development of drugs that specifically target dormant bacilli could have a profound impact on tuberculosis therapy: administration of conventional anti-mycobacterials together with ‘anti-dormancy' drugs might considerably shorten the time necessary for treatment. As a result, treatment of 16 million patients suffering from acute tuberculosis could become more efficient and treatment of 2 billion latently infected people might become feasible.



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Figure. Oxygen starvation-induced dormancy. Wayne developed the first culture system for mycobacterial dormancy, which is now known as the ‘Wayne model'.2 Wayne's system is based on gradual, self-generated oxygen withdrawal due to growth of the bacilli in sealed stirred tubes. Schematic diagrams of the culture tube and growth curve are shown.2,5

 
The development of a culture model for dormancy in M. tuberculosis by Wayne has two important implications. First, the model can be employed as a cell-based assay system for anti-dormancy drug screening. Secondly, it makes the molecular mechanisms of the dormancy response experimentally accessible. Thus it allows the identification of dormancy targets. In an effort to develop a model for dormancy that does not require work to be carried out in a level III biosafety facility we characterized the dormancy physiology and the expression of dormancy-specific markers in the closely related but attenuated bovine tubercle bacillus Mycobacterium bovis BCG. This work revealed that the dormancy response of BCG is virtually identical to that of virulent M. tuberculosis. Thus, BCG can be considered to be a useful model organism to study the dormancy response of tubercle bacilli.5 It is worth mentioning that a dormancy response similar to the one observed in tubercle bacilli was also demonstrated in the saprophytic Mycobacterium smegmatis.5 This could indicate that the response is to some extent conserved within parasitic and soil-dwelling mycobacteria. Hence, it is tempting to speculate that the ability to shift down to dormancy evolved early in the evolution of mycobacteria, perhaps as a strategy to survive changing oxygen availability encountered in the soil.

Nitroimidazoles (metronidazole) and nitroimidazopyrans (PA-824) are the first compounds that show activity against dormant tubercle bacilli.2,6 However, the bactericidal activity of these nitroheterocycles is low. High concentrations and long exposure times are required to achieve substantial killing in vitro.2,5,6 Hence, it is remarkable that some, albeit limited, antimicrobial effect of metronidazole was detectable in a mouse model for tuberculosis7 (metronidazole's cidal activity is strictly specific to dormant bacilli, growing bacilli are not affected by the drug).2,5 Considering the potential clinical importance of mycobacterial dormancy, exciting new anti-dormancy compounds and mechanistic insights into the molecular machinery controlling proliferation in mycobacteria are to be expected within the next few years.

Acknowledgments

Work in the author' s laboratory is supported by the Institute of Molecular and Cell Biology (IMCB).

Notes

J Antimicrob Chemother 2001; 47: 117–118

Tel: +65-874-8606; Fax: +65-779-1117; E-mail: mcbtd{at}imcb.nus.edu.sg

References

1 . Segal, W. (1984). Growth dynamics of in vivo and in vitro grown mycobacterial pathogens. In The Mycobacteria – A Source Book, (Kubica, G. P. & Wayne, L. G., Eds), pp. 547–73. Marcel Dekker Inc., New York.

2 . Wayne, L. G. & Hayes, L. G. (1996). An in vitro model for sequential study of down shift of Mycobacterium tuberculosis through two stages of non-replicating persistence. Infection and Immunity 64, 2062–9.[Abstract]

3 . Weber, I., Fritz, C., Ruttkowski, S., Kreft, A. & Bange, F. C. (2000). Anaerobic nitrate reductase (narGHJI) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice. Molecular Microbiology 35, 1017–25.[ISI][Medline]

4 . McKinney, J. D., zu Bentrup, K. H., Munoz-Elias, E. J., Miczak, A., Chen, B., Chan, W. T. et al. (2000). Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406, 735–8.[ISI][Medline]

5 . Lim, A., Eleuterio, M., Hutter, B., Murugasu-Oei, B. & Dick, T. (1999). Oxygen depletion-induced dormancy in Mycobacterium bovis BCG. Journal of Bacteriology 181, 2252–6.[Abstract/Free Full Text]

6 . Stover, C. K., Warrener, P., VanDevanter, D. R., Sherman, D. R., Arain, T. M., Langhorne, M. H. et al. (2000). A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature 405, 962–6.[ISI][Medline]

7 . Brooks, J. V., Furney, S. K. & Orme, I. M. (1999). Metronidazole therapy in mice infected with tuberculosis. Antimicrobial Agents and Chemotherapy 43, 1285–8.[Abstract/Free Full Text]