Structure of LAM in Relation to Biology and Biosynthesis

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DESCRIPTION (provided by applicant): The re-emergence of tuberculosis as a public health problem has been complicated by the lack of effective chemotherapeutic agents and the development of new antibiotics. The cell wall of its causative agent, Mycobacterium tuberculosis, is known to be a target of some of the most effective antimycobacterial drugs including ethambutol which has been known to inhibit the biosynthesis of arabinan of the cell wall proper and associated lipoarabinomannan (LAM). A diverse range of biological studies over a decade has collectively provided compelling evidence implicating LAM as a key surface molecule in host-pathogen interactions. The finding of immature LAM or truncated LAM as a consequence of natural and laboratory induced resistance to ethambutol, and embC gene knockout events provide invaluable model compounds for both structural and functional studies aiming at defining the relevance of LAM in pathogenesis. Specifically, with the availability of new enzymes, advanced chemical, NrvIR and mass spectrometric tools can be combined to yield the fine details of the arabinan assembly and the mode and site(s) of arabinan attachment to the mannan core. Enzymatically modified structural arabinan motifs positively correlating with particular biological attributes of clinical isolates will be derived from LAM, and neoarabinolipids will be generated for functional studies. Efforts will be given in resolving the heterogeneity in LAM and relate it to biology. CD1 restricted recognition of LAM by T cells will be examined in the context of cell mediated immunity in tuberculosis and its concomitant induction of cytokine secretion. Finally, gene knock-out mutants, cell-free assays and synthetic arabinofuranosyl acceptors will be utilized to establish the metabolic events involved in the arabinan assembly of LAM about which almost nothing is known, followed by identification of proteins (transferases) involved. Thus, the unifying theme of this Research Proposal is the structural analysis and manipulation of LAM, supplemented by genetic and biosynthetic studies leading to a better understanding of its biology and biosynthesis.