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Glycobiology Advance Access published online on September 15, 2004
Glycobiology, doi:10.1093/glycob/cwh150
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Received July 21, 2004
Revised September 13, 2004
Accepted September 14, 2004

ORIGINAL ARTICLES

Rapid structural characterization of the arabinogalactan and lipoarabinomannan in live mycobacterial cells using 2D and 3D HR-MAS NMR

Robin E. B. Lee 1, Wei Li 1, Delphi Chatterjee 2, and Richard E. Lee 1*

1 Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, 847 Monroe Ave Rm327, Tennessee 38163
2 Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, 80523

* To whom correspondence should be addressed. E-mail: relee{at}utmem.edu.


  Abstract

Mycobacteria possess a unique, highly evolved, carbohydrate and lipid-rich cell wall that is believed to be important for their survival in hostile environments. Until now, our understanding of mycobacterial cell wall structure has been based upon destructive isolation and fragmentation of individual cell wall components. This study describes the observation of the major cell wall structures in live, intact mycobacteria using two-dimensional (2D) and three-dimensional (3D) High-Resolution Magic-Angle Spinning (HR-MAS) NMR. As little as 20mg (wet weight) of [13C] enriched cells were required to produce a whole-cell spectra in which discrete cross-peaks corresponding to specific cell wall components could be identified. The most abundant signals of the arabinogalactan (AG) and lipoarabinomannan (LAM) were assigned in the HR-MAS NMR spectra by comparing the 2D and 3D NMR whole-cell spectra with the spectra of purified cellular components. This study confirmed that the structures of the AG and LAM moieties in the cell wall of live mycobacteria are consistent with structural reports in the literature, which were obtained via degradative analysis. Most importantly, using intact cells, it was possible to directly demonstrate the effects of Ethambutol on the mycobacterial cell wall polysaccharides, characterize the effects of embB gene knockout in the M. smegmatis {Delta}embB mutant, and observe differences in the cell wall structures of two mycobacterial species (M. bovis BCG and M. smegmatis.) Herein, we show that HR-MAS NMR is a powerful, rapid, non-destructive technique to monitor changes in the complex, carbohydrate rich cell wall of live mycobacterial cells.

Keywords: arabinogalactan; HCCH-TOCSY; HR-MAS NMR; lipoarabinomannan; mycobacteria.
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