Our understanding of the physiology of the tubercle bacillus is limited by our artificial in vitro axenic growth conditions. The completion of the genomic DNA sequence of a virulent strain of the bacterium (H37Rv) offered a glimpse into the varied metabolic potential of this complex Actinomycete. Far from the textbook description of an obligate aerobe, the tubercle bacillus has preserved all of the machinery necessary for life without oxygen. Likewise, far from growth on glucose in a laboratory flask the bacillus appears to be supremely adapted for living off of abundant host lipid constituents. The studies encompassed within this project all have in common the aim of discerning the nature of the intracellular physiology of Mycobacterium tuberculosis and modulation of the host's response to infection during actual disease. In addition the variation among circulating strains of Mycobacterium tuberculosis with respect to these properties has been studied. ? ? Within this broad project area there were significant advances in three major areas this reporting year:? ? First, in the area of genetic variability amongst extant clinical strains of tuberculosis we described, together with our collaborators at the Russian Academy of Sciences, a new technique for comparative genomic analysis and its application to epidemic strains in the Former Soviet Union. ? ? Second we have continued to characterize the hypervariable polyketide-derived lipid known as the phenolic glycolipid which is present among a subset of circulating strains of the Beijing/W family. A significant question was the extent of the immunosuppressive effect of this molecule during infection. Using a mixed infection model we were ale to show that this molecule exerts only a local effect, conferring on strains expressing it a survival advantage even in competition with strains that lack this molecule. In addition we continue to explore the downstream immunomodulatory effects of this molecule in collaboration with scientists at the Public Health Research Institute. These studies have resulted in significant progress in understanding the molecular mechanism of this important virulence determinant of TB.? ? Third we have made significant progress in understanding the biochemical target and activation process of bicyclic nitroimidazoles, including PA-824. The careful selection of mutants of biochemically defined properties, assisted by the chemical synthesis of analogs to refine the mutant phenotypes studied allowed the identification of the major protein involved in nitroreduction. The final identification of the gene involved employed a novel whole-genome resequencing technique based upon tiled arrays of the known genomic sequence of MTb.
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