Abstract
A unique approach, combining defined and reproducible in vitro models with DNA microarrays, has been developed to study environmental modulation of mycobacterial gene expression. The gene expression profiles of samples of Mycobacterium tuberculosis, from independent chemostat cultures grown under defined and reproducible conditions, were found to be highly correlated. This approach is now being used to study the effect of relevant stimuli, such as limited oxygen availability, on mycobacterial gene expression. A modification of the chemostat culture system, enabling largevolume controlled batch culture, has been developed to study starvation survival. Cultures of M. tuberculosis have been maintained under nutrient-starved conditions for extended periods, with 106 – 107 bacilli surviving in a culturable state after 100 days. The design of the culture system has made it possible to control the environment and collect multiple time-course samples to study patterns of gene expression. These studies demonstrate that it is possible to perform long-term studies and obtain reproducible expression data using controlled and defined in vitro models.
Highlights
During infection, bacteria such as Mycobacterium tuberculosis encounter a dynamic host environment and modulate gene expression in order to adapt, survive and replicate
Of particular interest is oxygen availability, as it is essential for the growth of M. tuberculosis and in vitro studies suggest that oxygen deprivation triggers the bacillus to enter a non-replicating persistent state, which is analogous to latency [3,9,10]
To investigate gene expression during two distinct stages of infection, viz. the active and latent stages, we developed two specialist culture systems; a chemostat to study replicating bacilli and a controlled batch culture system to study starvation survival
Summary
Bacteria such as Mycobacterium tuberculosis encounter a dynamic host environment and modulate gene expression in order to adapt, survive and replicate. It is still unclear how M. tuberculosis responds to the stimuli it encounters in vivo, but post-genomic technologies have provided the tools and opportunity to study this interaction at the molecular and biochemical level [1,4,5,11]. To gain maximum benefit from microarray experiments it is essential to study organisms from relevant and/or defined environments This can be achieved by recovering tubercle bacilli from macrophages or from infected tissues; progress is restricted by poor recovery of bacteria. It was necessary to modify the chemostat and develop a large-scale batch culture system which would allow us to monitor and control environmental conditions and collect multiple samples over time
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