A Reporter Transposon for Studying Exported Proteins of M. tuberculosis

描述（由申请人提供）：结核病仍然是一个严重的健康问题。导致这种疾病的结核分枝杆菌的一个主要毒力特性是在巨噬细胞内存活和生长的能力。结核分枝杆菌蛋白从细胞质出口到细菌表面或进一步分泌到环境中，这是与宿主相互作用并对抗巨噬细胞防御的理想位置。许多结核分枝杆菌蛋白在这种亚细胞输出类别中仍有待鉴定或研究。我们最近开发了一种基因报告系统，用于直接监测结核分枝杆菌的蛋白质输出。在这个系统中，截断的？-内酰胺酶本身不输出，它与蛋白质融合，报告它们输出到细胞质之外。因为?-内酰胺类抗生素靶向细胞壁合成酶，？-内酰胺酶报告蛋白必须出口，以保护细菌免受这些药物的侵害。我们演示了这个系统在？-内酰胺敏感结核分枝杆菌突变体。在这个R21应用中，我们建议使用一个携带？-内酰胺酶报告基因Tn' blem -1，以选择编码输出蛋白的结核分枝杆菌基因的框内转座子插入。这种遗传方法的优点是可以同时识别输出蛋白并在相应基因中产生结核分枝杆菌转座子插入突变体。由此产生的转座子突变文库将用于筛选巨噬细胞内生长缺陷和结核分枝杆菌对巨噬细胞反应的抑制缺陷。这项研究将补充现有的基因组方法，同时发现新的毒力因素，这应该有助于制定新的疾病干预策略。

DESCRIPTION (provided by applicant): Tuberculosis remains a serious health concern. A major virulence property of Mycobacterium tuberculosis, the bacterium responsible for this disease, is the ability to survive and grow within macrophages. M. tuberculosis proteins that are exported out from the cytoplasm to the bacterial surface or are further secreted into the environment are ideally positioned to interact with the host and combat macrophage defenses. Many of the M. tuberculosis proteins in this subcellular exported category remain to be identified or studied. We recently developed a genetic reporter system for monitoring protein export directly in M. tuberculosis. In this system a truncated ?-lactamase enzyme, which is not exported on its own, is fused to proteins to report on their export beyond the cytoplasm. Because ?- lactam antibiotics target cell wall synthesis enzymes, the ?-lactamase reporter must be exported in order to protect bacteria from these drugs. We demonstrated this system to function properly in a ?-lactam sensitive mutant of M. tuberculosis. In this R21 application we propose to use a modified Himar mariner transposon which carries a ?-lactamase reporter, Tn'blaTEM-1, to select for in-frame transposon insertions in M. tuberculosis genes encoding exported proteins. This genetic approach has the advantage of simultaneously identifying exported proteins and generating M. tuberculosis transposon insertion mutants in the corresponding genes. The resulting transposon mutant library will be screened for intracellular growth defects in macrophages and for defects in the M. tuberculosis inhibition of macrophage responses. This research will complement existing genomic approaches while uncovering new virulence factors, which should aid development of new disease intervention strategies. Tuberculosis is a severe world health problem. A virulence property of Mycobacterium tuberculosis, the causative agent of this disease, is the ability to survive and grow in macrophages. The proposed research will identify new virulence factors that enable M. tuberculosis to survive in macrophages and cause disease