D-arabinose synthesis in TB using Azorhizobium as a tool

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DESCRIPTION (provided by applicant): Tuberculosis is a major opportunistic disease of AIDS which exacerbates the course of the illness. Furthermore, tuberculosis threatens AIDS caretakers and contacts and breeds drugs resistant strains of M. tuberculosis. Hence, new drugs, specific for M. tuberculosis are needed to help control the AIDS epidemic. D-arabinose formation is an ideal TB drug target as it is essential and specific for M. tuberculosis and D-arabinose is not found in humans. The pathway for formation of D-arabinose in M. tuberculosis has been determined by the PI; however, the genes have not been successfully identified. Thus, the purpose of this AIDS-FIRCA research is to identify the M. tuberculosis genes responsible for the synthesis of D-arabinose and determine the function of the encoded proteins. The breakthrough that has made both this research and the collaboration possible is the finding by the foreign collaborator, Dr. Holsters, of four genes in Azorhizobium caulinodans that synthesize D-arabinose (A. caulinodans is one of the very few other organisms in nature that synthesize D-arabinose besides mycobacteria). Furthermore, she discovered the existence of genes in M. tuberculosis homologous to three of the A. caulinodans genes. Therefore, we propose to identify the D-arabinose synthetic genes in M. tuberculosis by complementing specific gene knockout mutants of A. caulinodans with M. tuberculosis gene candidates. Further we propose to determine the function of each gene product by the identification of D-arabinose formation precursors in these specific gene knockout strains of A. caulinodans and by expression and enzymatic assay of the M. tuberculosis and A. caulinodans genes in E. coli. The genetics on A. caulinodans and the searches for D-arabinose precursors will be mostly performed at the foreign site while needed radioactive precursors and biochemical techniques and genetics in mycobacteria will come from the American site.