Whole Cell NMR Studies of MYcobacteria

University Of Tennessee Health Sci Ctr, Memphis TN

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Abstract

[unreadable] DESCRIPTION (provided by applicant): This proposal aims to use live, whole-cell, high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS-NMR) as a novel, nondestructive technique to study carbohydrate and carbonaceous metabolism in mycobacteria. There is currently intense research in genomic and proteomic studies of mycobacteria. However, complementary methodologies to study globally, the carbonaceous metabolic products from genes or proteins has been lacking and it is this deficit in knowledge that this proposal aims to address. Firstly, it is proposed to assign the complex, live, whole-cell HRMAS-NMR spectra of M. bovis BCG using purified standard metabolites and multi-dimensional NMR techniques. Secondly, it is proposed that mycobacteria can adapt their carbonaceous metabolism in response to different physiological conditions. Thus HRMAS-NMR, will be used to study changes in mycobacterial carbonaceous metabolites, including the cell wall, under different physiological conditions: (i) physical conditions of growth such as different media, temperature and pH; (ii) growth phase; (iii) exposure to anti-mycobacterial agents, including those which target the mycobacterial cell wall; (iv) the effects of conditions which may mimic growth in vivo; (v) differences in the HRMAS-NMR spectra between different mycobacterial species. The central hypothesis is that whole cell HRMAS-NMR will be an excellent way to study these changes. Thirdly, using whole cell NMR, a novel study will be performed of the mycobacterial cell wall tertiary structure. Initially, it is hoped to locate the position of metabolites identified in this study in the cell ultra structure. Finally, we plan to initiate 3D-NMR studies and studies of 3-bond torsional coupling constants that will provide 3-dimensional, conformational information on the macromolecules identified. The findings from these studies will lead to a better understanding of mycobacterial carbohydrate and lipid physiology and hence, their role in the virulence and pathogenesis of the tubercle bacilli. [unreadable] [unreadable]