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，它会加剧疾病的进程。此外，结核病威胁到艾滋病照顾者和接触者，并滋生耐药的结核分枝杆菌菌株。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-阿拉伯糖的形成是理想的结核病药物靶点，因为它对结核分枝杆菌是必要的和特异的，而D-阿拉伯糖在人类中找不到。结核分枝杆菌中D-阿拉伯糖的形成途径已经通过等电点确定，然而，基因还没有被成功鉴定。因此，这项艾滋病-FIRCA研究的目的是确定与D-阿拉伯糖合成有关的结核分枝杆菌基因，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性进展是，外国合作者霍尔斯特斯博士在偶氮菌中发现了四个合成D-阿拉伯糖的基因(除了分枝杆菌外，茎瘤菌是自然界中极少数合成D-阿拉伯糖的生物之一)。此外，她还发现在结核分枝杆菌中存在与三种丛枝杆菌基因同源的基因。因此，我们建议通过用结核分枝杆菌候选基因互补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，我们建议通过鉴定这些特定的基因敲除菌株中D-阿拉伯糖的形成前体，以及通过在大肠杆菌中表达和酶分析结核分枝杆菌和树枝杆菌基因，来确定每个基因产物的功能。对树枝杆菌的遗传学和对D-阿拉伯糖前体的搜索将主要在国外站点进行，而所需的放射性前体以及分枝杆菌的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.