Two of the pioneers in linkage region and arabinogalactan (AG) synthesis, I.C. Hancock (University of Newcastle, UK) and K. Takayama (University of Wisconsin, Madison) combine with the host laboratory to: 1) Apply knowledge of the biosynthesis of the Gram-positive teichoic acid linkage unit to determine; (i) if the mycobacterial linkage unit Rha-G1cNAc-1-P is synthesized on a C55-35-polyprenol-P which then acts as the fulcrum for galactose polymerization and initiates mycolyl-ASG synthesis; (ii) the relationship of arabinose to galactose polymerization (ie. independent or dependent on galactan acceptor?) And its association to underlying peptidoglycan. Methods previously successfully employed in the teichoic acid context (use of subcellular fractions; continuous culture; pulse- chase; permeabilized cells; a range of antibiotics) will be extrapolated to mycobacteria. 2) Characterize the extramembranous glycosyltransferases and synthetases responsible for the final elaboration of AG and lipoarabinomannan (LAM) through: (i) synthesis and application of the key nucleotide arabinose donor; (ii) synthesis of the presumed oligosaccharide acceptors;a nd (iii) isolation and characterization for ultimate study of structure activity relationships of the appropriate enzymes. 3) The host laboratory will conduct specific tasks [synthesis of nucleotide and polyprenol sugars; enzyme purification and sequencing; special chemical analysis; use of virulent M. tuberculosis; pursuit of ongoing studies on the mode of action of ethambutol; biosynthesis of lipomannan (LM) ] and ensure interaction between labs, projects, and industrial partners for purposes of exploiting emerging information for drug development.
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