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 dual problems of drug resistance and a limited drug repertoire have also compromised the treatment of disseminated M. avium disease among patients with AIDS. Thus a concerted effort has been undertaken by the research community to identify new antimycobacterial drugs and drug targets. The cell wall of mycobacteria is known to be the target of some of the most effective antimycobacterial drugs, and it provides an array of potential new drug targets. Within the Program's unifying theme of the biogenesis and targeting of the cell wall core of mycobacteria, the goal of Project 4 is to use a genetic means to identify enzymes involved in the synthesis of cell wall polysaccharides, and to interact closely with Project 5 wherein similar goals will be approached from a biochemical perspective. The research plan is to use a molecular genetic approach to identify enzymes involved in arabinogalactan (AG) or lipoarabinomannan (LAM) biosynthesis that could serve as targets for new drug development. AG is the core of the mycolyl-arabinogalactan complex that comprises most of the cell wall while LAM is a potent modulator of the immune response as well as a key cell wall component. Agents that interfere with the biogenesis of either AG or LAM would thus be expected to have serious consequences on cell viability and survival in the host. Specifically, we propose; (1) To continue to characterize athe sequenced ethambutol resistance region (e,b 1) of M. avium and M. tuberculosis in order to determine its role in arabinan biosynthesis; (2) To begin a study of the biosynthesis of the arabinan portion of LAM through the use of mutants; (3) To begin a study of the biosynthesis of the mannan portion of LAM by identifying the genes encoding the mycobacterial phosphomannomutase (PMM) and phosphomannose isomerase (PMI) through complementation of heterologous mutant hosts: (4) To use glycosyl transition state analogs that inhibit the biosynthesis of arabinogalactan and/or LAM as tools to clone the genes for the biosynthetic enzyme target(s) by selecting for a resistant phenotype consistent with overexpression of the wild-type enzyme, and to isolate resistant mutants that will complement or provide an alternative strategy to the above studies; and (5) To carry out preliminary studies on galactan biosynthesis by completing the DNA sequence of a partial open reading frame whose amino acid sequence similarity to lmb V suggests that it may be involved in galactan biosynthesis. The research plan is intended to provide basic information ont he biosynthesis of the cell wall such that key biosynthetic enzymes and the genes that encode them can ultimately be evaluated for their potential to serve as new molecular targets for selective antimycobacterial chemotherapy. The information and resources gained could contribute to a cell-free assay for drug screening or the information could be applied to computer-assisted drug discovery.
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