Characterization of Virulence Metabolites Produced by Mycobacterium tuberculosis: A Mass Spectrometric Approach

A number of unique metabolites contribute to the virulence of the human pathogen Mycobacterium tuberculosis, the primary causative agent of tuberculosis. Mycobacterium tuberculosis synthesizes a formidable, highly lipid-rich cell envelope, which non-covalently associates with cell surface-exposed lipids specific to pathogenic mycobacteria. Many of these lipids have been linked to the virulence and pathogenesis of the bacterium. However detailing the precise biochemical functions of these metabolites remains an active area of investigation. Determining the detailed chemical structures of these metabolites and elucidating the biosynthetic pathways leading to their production are essential to further our understanding of their specific functions. Therefore, the focus of the research presented in this thesis is to determine the detailed chemical structures and biosynthetic pathways of key Mycobacterium tuberculosis metabolites, as well as to improve the current methodology in the analysis of these compounds.;Chapter 1 describes the biology of Mycobacterium tuberculosis and provides a historical background of the investigations into the metabolites further studied in this thesis. This chapter also provides a detailed review of the mass spectrometry techniques essential to the research performed in these studies. Chapter 2 describes the detailed structural characterization of the novel sulfated menaquinone S881, a negative regulator of Mycobacterium tuberculosis virulence. An investigation into the biosynthesis of another cell-surface lipid, polyacyltrehalose, is detailed in Chapter 3. Chapter 4 describes the development of a rapid mass spectrometry-based method to relatively quantitate the mycobacterium-specific redox metabolite mycothiol across different strains of Mycobacterium smegmatis. Finally, Chapter 5 investigates the high-resolution MS analysis of M. tuberculosis cell-surface metabolites, with a focus on the results obtained between two different high-resolution instruments. This study also describes the analysis of S881 levels across five strains of Mycobacterium tuberculosis with differing levels of virulence, as well as the vaccine strain Mycobacterium bovis BCG.