It is now generally accepted that new drugs are needed to make global tuberculosis control a reality. The completion of the sequence of the M. tuberculosis genome has laid the foundation for greatly accelerating new drug discovery. While a limited number of studies of the TB transcriptome and proteome have been reported, these global macromolecular analyses are still not able to contribute directly to drug discovery except where gene/protein function are known or protein crystal structures are available. Elucidating the full complement of low molecular weight compounds in an organism -the metabolome - is becoming recognized as a complementary and perhaps the most tractable approach to a comprehensive understanding of any pathogen including the insight necessary for rational drug discovery. This R-21 proposal seeks to define the metabolome of the tubercle bacillus in the stage of non-replicating persistence (NRP), the physiological state considered to be responsible for the required long treatment duration for TB. The metabolome will be elucidated by high-resolution chromatography (CCC, LC) and -spectroscopy (MS, NMR). Initial studies will utilize a high biomass of BCG to evaluate several separation schemes in optimizing the resolution of (secondary) metabolites. This protocol will then be used to define the metabolome of M. tuberculosis in NRP. Subsequent experiments will focus on understanding the growth-phase specificity of unique metabolites produced under NRP. The information obtained will not only complement the extensive knowledge of the chemistry of the cell wall but is also expected to I) identify novel secondary metabolites possibly related to those recently described for other mycobacteria, 2) help to clarify events and identify low MW markers during the metabolic shift to a low oxygen environment especially considering that >70% of genes up-regulated during this adaptation are of unknown function, 3) provide leads for drug development through analoging around TB-specific compounds not known to occur in mammalian cells and 4) assist in the understanding of the mechanism of action of newly discovered anti-TB agents. The operating hypothesis for this study is that elucidating the metabolome of Mycobacterium tuberculosis will overcome limitations inherent in global macromolecular analyses with respect to gaining key insights into dormancy and drug discovery.
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