GENETIC BASIS OF MYCOBACTERIAL INVASION

为了帮助理解结核病的基本发病机制，一个快速的 全球范围内死灰复燃的疾病，海洋分枝杆菌，一种快速增长的人类 病原菌将被用作阐明毒力因子的模型系统。 用于转化和转位的基因系统已经 在这种生物中建立，并将被利用来分离突变体 缺乏对真核细胞和细胞内的侵袭 复制。将尝试建立同源重组在 这种有机体使得推测的毒力因子的基因可以 插入失活以研究它们的影响。变种人会 也可以在淡水蛙的慢性病模型中进行测试，该模型已经 已经成立了。 一个含有海洋分枝杆菌基因的Cosmid库将被引入 不能持久和复制的非病原体污垢分枝杆菌 在真核细胞的体外和体内的持久性和疾病。泳池 转化的污垢分枝杆菌也将在体外进行持久性测试 就像在罗非鱼中一样。此外，表达的海洋分枝杆菌RNA 具体地说，在真核细胞环境中将通过差异分离 从宿主细胞中分离分枝杆菌，然后 分枝杆菌发酵液中cDNAs的消减杂交。 最后，分枝杆菌入侵、持续和持续的细胞生物学 将使用视频和视频的组合来检查细胞内的贩运， 免疫电子显微镜和共聚焦显微镜。补语和功能成分的作用 侵袭和细胞内转运的受体将由 使用转染了适当受体的上皮细胞。

To aid understanding of the basic pathogenesis of tuberculosis, a rapidly resurgent disease worldwide, Mycobacterium marinum, a rapidly growing human pathogen will be used as a model system to elucidate virulence factors. Genetic systems for transformation and transposition have already been established in this organism, and will be exploited to isolate mutants deficient in invasion into eukaryotic cells and in intracellular replication. Attempts will be made to establish homologous recombination in this organism so that genes for putative virulence factors can be insertionally inactivated in order to study their effects. Mutants will also be tested in the Rana pipiens model for chronic disease which has been established. A Cosmid bank containing M. marinum genes will be introduced into the nonpathogen, M. smegmatis which is incapable of persistence and replication in eukaryotic cells in vitro and of persistence and disease in vivo. Pools of transformed M. smegmatis will be tested for persistence in vitro as well as in R. pipiens. Additionally, M. marinum RNAs that are expressed specifically in the eukaryotic cell milieu will be isolated by differential centrifugation of the mycobacteria from the host cells followed by subtractive hybridization of cDNA from that of broth grown mycobacteria. Finally, the cell biology of mycobacterial invasion, persistence and intracellular trafficking will be examined using a combination of video, immunoelectron and confocal microscopy. The role of the complement and Fc receptors in invasion and intracellular trafficking will be examined by the use of epithelial cells transfected with the appropriate receptors.