GENETIC BASIS OF MYCOBACTERIAL INVASION

为了帮助了解结核病的基本发病机制， 世界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 病原体将被用作阐明毒力因子的模型系统。 用于转化和转座的遗传系统已经 在这种生物体中建立，并将被用于分离突变体 缺乏对真核细胞的侵袭和细胞内 复制的将尝试建立同源重组， 这种微生物，使基因推定的毒力因子可以 插入失活以研究它们的作用。突变株 也可以在蛙慢性疾病模型中进行测试， 确立了习 一个包含M. marinum基因将被引入 nonpathogen，M.无法持久和复制的smegalyptus 在体外真核细胞中，以及在体内的持久性和疾病。池 变形M.还将检测丝霉亲和素的体外持久性 如R。pipiens。此外，M。表达的海洋RNA 特别是在真核细胞环境中， 从宿主细胞中离心分枝杆菌，然后 来自肉汤培养分枝杆菌cDNA的消减杂交。 最后，分枝杆菌的细胞生物学的入侵，持久性和 将使用视频， 免疫电镜和共聚焦显微镜。补体和Fc的作用 受体的侵袭和细胞内运输将检查 使用用适当受体转染的上皮细胞。

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.