Abstract
Mycobacterium tuberculosis infects a third of the world's population. Primary tuberculosis involving active fast bacterial replication is often followed by asymptomatic latent tuberculosis, which is characterised by slow or non-replicating bacteria. Reactivation of the latent infection involving a switch back to active bacterial replication can lead to post-primary transmissible tuberculosis. Mycobacterial mechanisms involved in slow growth or switching growth rate provide rational targets for the development of new drugs against persistent mycobacterial infection. Using chemostat culture to control growth rate, we screened a transposon mutant library by Transposon site hybridization (TraSH) selection to define the genetic requirements for slow and fast growth of Mycobacterium bovis (BCG) and for the requirements of switching growth rate. We identified 84 genes that are exclusively required for slow growth (69 hours doubling time) and 256 genes required for switching from slow to fast growth. To validate these findings we performed experiments using individual M. tuberculosis and M. bovis BCG knock out mutants. We have demonstrated that growth rate control is a carefully orchestrated process which requires a distinct set of genes encoding several virulence determinants, gene regulators, and metabolic enzymes. The mce1 locus appears to be a component of the switch to slow growth rate, which is consistent with the proposed role in virulence of M. tuberculosis. These results suggest novel perspectives for unravelling the mechanisms involved in the switch between acute and persistent TB infections and provide a means to study aspects of this important phenomenon in vitro.
Highlights
With the average daily death toll from tuberculosis at 4,500 the worldwide burden of this disease is overwhelming [1]
In the experiments described by Sassetti et al (2003) transposon libraries of M. tuberculosis and M. bovis BCG were constructed and recovered on 7H10 agar and Transposon site hybridization (TraSH) analysis was used to identify genes which were essential for the survival of both species on this media
The experiments described here used just the BCG transposon library generated by Sassetti et al (2003) for TraSH analysis to investigate the genetic basis of growth control in a chemostat model
Summary
With the average daily death toll from tuberculosis at 4,500 the worldwide burden of this disease is overwhelming [1]. The identification of genes which provide essential but unique functions in M. tuberculosis greatly facilitates drug discovery programs and provides further information about the complex biology of this highly successful pathogen. Tuberculosis is characterized by two distinct phases an acute phase where the bacteria are actively growing and a persistent phase where the bacteria are in a slow growing or non-growing state [2]. This ability to persist for decades in a state refractory to immune clearance but primed for reactivation is key to the success of M. tuberculosis and represents an important barrier to the control of tuberculosis as presently used chemotherapies are largely inactive against non-dividing cells. The identification of pathways and genes essential to establish and maintain persistence would facilitate the development of drugs which target this phase of infection
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