D-arabinose synthesis in TB using Azorhizobium as a tool

描述（申请人提供）：结核病是艾滋病的一种主要机会性疾病，会加重病程。此外，结核病威胁到艾滋病护理人员和接触者，并滋生耐药结核分枝杆菌菌株。因此，需要针对结核分枝杆菌的新药来帮助控制艾滋病的流行。d -阿拉伯糖形成是一种理想的结核病药物靶点，因为它对结核分枝杆菌是必需的和特异性的，而d -阿拉伯糖在人类中没有发现。通过PI确定了结核分枝杆菌中d -阿拉伯糖的形成途径；然而，这些基因还没有被成功鉴定出来。因此，本AIDS-FIRCA研究的目的是鉴定结核分枝杆菌负责合成d -阿拉伯糖的基因，并确定编码蛋白的功能。使这项研究和这项合作成为可能的突破，是外国合作者霍尔斯特博士在花椰菜偶氮根瘤菌（Azorhizobium caulinodans）中发现了四个合成d -阿拉伯糖的基因（花椰菜根瘤菌是自然界中除分枝杆菌外，为数不多的几种合成d -阿拉伯糖的生物之一）。此外，她还发现结核分枝杆菌中存在与三个茎叶分枝杆菌基因同源的基因。因此，我们建议通过与结核分枝杆菌的候选基因互补来鉴定结核分枝杆菌中d -阿拉伯糖合成基因。进一步，我们建议通过在这些特定基因敲除菌株中鉴定d -阿拉伯糖形成前体，以及通过在大肠杆菌中表达和酶分析结核分枝杆菌和茎叶分枝杆菌基因来确定每个基因产物的功能。茎叶假单胞菌的遗传学和d -阿拉伯糖前体的寻找将主要在国外进行，而所需的放射性前体和分枝杆菌的生化技术和遗传学将在美国进行。

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.