The re-emergence of tuberculosis as a public health problem has been complicated by the lack of effective chemotherapeutic agents and the development of drug resistant strains. The cell wall of its causative agent, Mycobacterium tuberculosis, is known to be the target of some of the most effective antimycobacterial drugs including ethambutol which is known to inhibit the biosynthesis of the arabinan of the cell wall proper and the associated lipoarabinomannan (LAM). A diverse range of biological studies over the past few years has collectively provided compelling evidence implicating LAM as a key surface molecule in host-pathogen interactions. The production of truncated LAM variants by cell wall mutants defective in LAM biosynthesis as a consequence of ethambutol resistance provides invaluable model compounds for both structural and functional studies aiming at defining the relevance of LAM in pathogenesis. Specifically, the fine details of the arabinan assembly and its attachment to the phosphatidylinositol mannan core will be characterized and structural motifs positively correlating with particular biological attributes of clinical isolates will be identified. Tailor-made synthetic oligosaccharide probes and well defined monoclonal antibodies will be generated for structural, biosynthesis, and genetic studies of LAM, as well as to define its situation and orientation within the cell wall framework of viable bacilli. CD1 restricted recognition of LAM by T cells will be examined in the context of cell mediated immunity in both tuberculosis and leprosy and its concomitant induction of cytokine secretion. Chemically and/or enzymatically modified arabinan and mannan will be derived from LAM for detailed structural analysis and further used to delineate specific epitopes of LAM recognized by T cells. Thus, the unifying theme of this Research Proposal is the structural analysis and manipulation of LAM, supplemented by other synthetic probes and monoclonal antibodies to be generated, to define its primary structure, conformation and ultracellular location, in relation to its biosynthesis and biology.
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