Important physiological and immunopathogenic features of mycobacteria including slow growth, acid fastness, resistance to drug permeation, and immunological persistence have been attributed to the presence of lipoarabinomannan (LAM) and arabinogalactan (AG) in the cell wall. This competitive renewal of Al 37139 is built on success in the initial funding period of determining structural characteristics of truncated LAM from laboratory induced ethambutol (Emb) resistant M. smegmatis and LAM and AG from clinical isolates of M. tuberculosis resistant to Emb. To take the next step in fully understanding the cell surface of M. tuberculosis and its manifestation in bacterial physiology and pathology, it is proposed to determine the entire structures of LAM and AG, to characterize LAM and AG in a panel of Emb resistant M. tuberculosis isolates, and, in collaboration, to determine and precise structures of LAM needed for CD1 T-cell and macrophage mannose receptor recognition. Although some structural features of LAM and AG have been delineated, key issues, including the structure of the arabinan regions of both polymers, and the site of the arabinan attachments to the hexose backbones of each polymer remain unknown. To obtain the complete primary structure of these two 100 glycosyl residues glycans, novel cell wall degrading enzymes will be used to prepare fragments whose structures will be determined by state-of-the-art nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. The enzymes include seven novel arabinanases and galactofuranases secreted by a Cellulomonas species and an endo alpha-1,6 mannosidase from Bacillus circulans. NMR will focus on the analysis of fully 1 3C labeled polysaccharides and polysaccharide fragments by 2-D and 3-D techniques. Mass Spectrometry will feature a novel Q-TOF-tandem electrospray mass spectrometer to obtain sequence information. Techniques based on SDS-PAGE, endo-arabinanase cleavage, 1-D-NMR, and MS will be used to efficiently characterize LAM and AG from our panel of Emb resistant M. tuberculosis. The structural studies will directly aid the immunological studies by providing a wealth of LAM related molecules needed to decipher the structural characteristics necessary for recognition by T-cells and macrophages.
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