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The widespread availability of antimicrobial chemotherapeutics over the last half of the twentieth century has offered dramatic increases in life expectancy. Unfortunately, many pathogenic agents are exhibiting ever increasing resistance to many frontline antibiotics. New chemotherapeutic agents are urgently needed to combat this threat; this work seeks to illustrate applications in which mass spectrometric techniques may be applied to the investigation of novel, small molecule chemotherapeutics for the treatment of bacterial infections. Chapter 1 contains an introduction to mass spectrometry, as well as an overview of relevant chromatographic techniques. Chapter 2 introduces the bacterium F tularensis and M ulcerans and details the MS based investigation of their secreted virulence factors. Chapter 3 introduces a human pathogen of particular concern, M tuberculosis, and contains a detailed reporting on the MS based investigation of internally synthesized compounds for the treatment of latent Tb.

The mycobacterial cell wall metabolites have always imposed great challenges to researchers due to their unusual complexity and structural diversity. A lot of research efforts have been directed towards the evaluation of these metabolites and the role they play in the pathogenesis and virulence of different serious human pathogens including Mycobacterium tuberculosis the causative agent of tuberculosis (TB). In the genomic era, it is crucial to develop new methodologies to analyze these components from a global perspective in a comprehensive and well-validated manner. Towards this end, we developed a rapid NMR-based approach to produce metabolic profiles in the form of 2D1H-13C HSQC maps. Due to NMR low sensitivity, we used 13C-isotope enrichment strategy to improve the detection threshold of NMR. We developed and investigated the use of three NMR-based profiles. The first is for the cell wall associated lipids, the second is for covalently bound mycolic acids and the third deals with the complex cell wall polysaccharides. Key structural features and major lipid species were readily identifiable using this technique. This approach can be used for observing changes in the cell wall due to drug treatment, gene mutation and changes in the physiological environment, species characterization and screening for virulence factor expression. It also allows for rapid comparative analysis of several cell wall metabolites within the same sample. We have used this approach to evaluate the structural diversity displayed in the cell wall metabolites among different Mycobacteria from different clades. We have successfully used the same technique to monitor changes in the carbohydrate and lipid pools from Mycobacteria grown under laboratory conditions that simulate latency and resuscitation from latency. Moreover, we were also able to use such technique to screen for virulence factor in severalM. tuberculosis clinical isolates. Although we have successfully developed and used an NMR approach for metabolite analysis, NMR however remains less sensitive than other spectroscopic techniques like mass spectroscopy (MS). The high sensitivity of MS makes it one of the best candidates for metabolomic studies. However in case of cellular lipids, the low polarity and the variable ionization potential deter its routine use for lipid analysis. Certain metabolites that have low cellular abundance and good ionizability are better analyzed through an MS-based approach. Mycolactones of M. ulcerans and other closely related species are good examples for such metabolites and in this work we have demonstrated the utility of MS as a tool for screening for mycolactones from both bacterial culture and tissue samples. Metabolomics is currently a rapidly growing field and many researchers have directed their attention in developing the right research tools to explore it. Spectroscopic methods along with chromatographic methods have been in the front of this vigorous research effort. The work presented here represents an innovative attempt to utilize such techniques to investigate the metabolome of some of the most serious human pathogens and discusses the potential area where the application of these techniques can be the most useful.