GENETIC BASIS OF MYCOBACTERIAL PATHOGENICITY

描述：为了更好地了解心力衰竭的发病机制。 结核，一种生长更快的海洋分枝杆菌将被用作 分离和鉴定毒力因子的模型系统。最近 开发了用于转化、随机转座和 将利用同源重组来分离受影响的突变体 进入和生长巨噬细胞的毒性。A最近 开发的技术，签名标记转座子突变，将是 用于筛选无毒转座子突变体 青蛙物种尖吻林蛙的致病力及其在体内的复制 培养的巨噬细胞样细胞。作为识别身份的独立方法 编码毒力因子的基因，启动子具有增强的活性 宿主细胞环境将使用绿色荧光蛋白进行分离 (GFP)作为报告基因。转录融合将在 随机的海洋分枝杆菌染色体片段和一个无启动子的GFP基因。M. 用此融合文库转化的海参将用于感染培养的 宿主细胞与胞内细菌的荧光强度 用流式细胞仪检测。此外，宿主内荧光增强的细菌 细胞将通过流式细胞仪进行分类，并对负责的分子进行分析 将承接发起人。最后，海洋分枝杆菌贩运和 亚细胞定位将通过显微技术进行检查。M。 表达绿色荧光蛋白的海洋生物将用共聚焦显微镜进行研究。 免疫电子显微镜将用于补充这些研究。突变者 毒力受损的病毒也将通过这些技术进行检测。

DESCRIPTION: To better understand the mechanisms of the pathogenesis of tuberculosis, a more rapidly growing Mycobacterium marinum will be used as a model system to isolate and characterize virulence factors. Recently developed genetic systems for transformation, random transposition and homologous recombination will be exploited to isolate mutants compromised in virulence for entrance into and growth within macrophages. A recently developed technique, signature-tagged transposon mutagenesis, will be employed for the screening of avirulent transposon mutants compromised in virulence in the frog species Rana pipiens and in replication within cultured macrophage-like cells. As an independent approach to identifying genes encoding virulence factors, promoters with enhanced activity within the host cell milieu will be isolated using the green fluorescent protein (GFP) as a reporter gene. Transcriptional fusions will be made between random M. marinum chromosomal fragments and a promoter-less GFP gene. M. marinum transformed with this fusion library will be used to infect cultured host cells and the fluorescence intensity of the intracellular bacteria assayed by FACS. Also, the bacteria with enhanced fluorescence in host cells will be sorted by FACS, and a molecular analysis of the responsible promoters will be undertaken. Finally, M. marinum trafficking and subcellular localization will be examined by microscopic techniques. The M. marinum expressing the GFP will be studied with confocal microscopy. Immunoelectron microscopy will be used to complement these studies. Mutants compromised in virulence will be examined with these techniques as well.