D-arabinose synthesis in TB using Azorhizobium as a tool

描述（由申请人提供）：结核病是艾滋病的一种主要机会性疾病，它会加剧病程。此外，结核病威胁着艾滋病看护者和接触者，并滋生结核分枝杆菌的耐药菌株。因此，需要针对结核分枝杆菌的新药物来帮助控制艾滋病的流行。 D-阿拉伯糖的形成是理想的结核病药物靶点，因为它对结核分枝杆菌至关重要且具有特异性，而人类中未发现 D-阿拉伯糖。 PI 已确定结核分枝杆菌中 D-阿拉伯糖的形成途径；然而，这些基因尚未被成功鉴定。因此，这项 AIDS-FIRCA 研究的目的是鉴定负责合成 D-阿拉伯糖的结核分枝杆菌基因并确定所编码蛋白质的功能。使这项研究和合作成为可能的突破是外国合作者Holsters博士在Azorhizobium caulinodans中发现了四个合成D-阿拉伯糖的基因（A. caulinodans是自然界中除分枝杆菌之外合成D-阿拉伯糖的极少数生物之一）。此外，她发现结核分枝杆菌中存在与三个 A. caulinodans 基因同源的基因。因此，我们建议通过用结核分枝杆菌候选基因补充 A. caulinodans 的特定基因敲除突变体来鉴定结核分枝杆菌中的 D-阿拉伯糖合成基因。此外，我们建议通过鉴定 A. caulinodans 的这些特定基因敲除菌株中的 D-阿拉伯糖形成前体以及通过在大肠杆菌中结核分枝杆菌和 A. caulinodans 基因的表达和酶测定来确定每个基因产物的功能。 A. caulinodans 的遗传学和 D-阿拉伯糖前体的搜索将主要在外国站点进行，而分枝杆菌所需的放射性前体和生化技术和遗传学将来自美国站点。

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.