MOLECULAR AND CELL BIOLOGY OF PATHOGENIC MYCOBACTERIA

M.重新部署的目标 在这个项目中描述的是使用M。marinum作为一个模型， 鉴定M. 巨噬细胞内的结核病（MTB）以及 鉴定MTB介导的细胞毒性所需的基因 在前人的研究中，我们确定M. marinum与M. 结核病在一个独特的吞噬体区室中存活， 不与溶酶体融合以确定如何 分枝杆菌能够在巨噬细胞中存活，我们设计了一种 分离gfp与分枝杆菌基因融合的新策略 在吞噬体环境中差异表达。在这 方法，裂解感染细胞，释放吞噬体， FACS分选以获得含有与基因融合的细菌 在细胞内表达。然后检测这些细胞的表达 在固体培养基上，以获得差异融合的群体 在细胞内表达。使用这种方法，我们确定了12个 与巨噬细胞中差异表达的基因的融合。 与GFP相邻的DNA序列显示出与 M.肺结核对这些基因的进一步分析和鉴定 他们的产品应该提供深入了解分枝杆菌如何 在巨噬细胞中存活。在分离GFP的过程中， 融合，我们确定了一个构建体，GFP 13，其中含有一个 启动子组成型表达比HSP 60好至少5倍， 分枝杆菌热休克启动子。这个的鉴定 非常强的启动子可以提供非常有用的工具， 分枝杆菌基因的高水平表达。M.溃疡。感染 与M.溃疡，布鲁里溃疡的病原体，导致 重度坏死性皮肤病变伴极轻微急性炎症 反应为了了解M。溃疡引起疾病， 我们从M. 溃疡，MULT的结构研究表明，它是一个复杂的， 聚酮化合物，一种12元环大环内酯。复合聚酮 包括大量有效的生物活性分子，例如 抗生素（红霉素），免疫抑制剂（FK506）， 抗真菌药（阿替霉素）和细胞抑制素（巴弗洛霉素）。虽然 复合聚酮化合物在链霉菌属物种中是常见的， MULT是第一个复杂的聚酮化合物，也是第一个分离的大环内酯类化合物 来自分枝杆菌属。我们已经描述了 使用体外组织培养和体内的MULT活性 问题研究以pg量计，MULT导致小鼠成纤维细胞停滞， 细胞周期的G1期。更值得注意的是， 将MULT植入豚鼠体内产生的病变在病理学上与 布鲁里溃疡

M. marinum The objectives of the reasearch described in this project are to use M. marinum as a model for identifying genes required for survival and replication of M. tuberculosis (MTB) within the macrophage as well as for identifying genes required for MTB mediated cytotoxicity In previous studies, we established that M. marinum like M. tuberculosis survives in a unique phagosomal compartment which does not fuse with the lysosome. In order to determine how mycobacteria are able to survive in macrophages, we devised a novel strategy for isolating gfp fusions to mycobacterial genes expressed differentially in the phagosomal environment. In this method, infected cells were lysed and the released phagosomes FACS-sorted to obtain bacteria containing fusions to genes expressed intracellularly. These were then assayed for expression on solid media to obtain populations of fusions differentially expressed intracellularly. Using this methodology we identified 12 fusions to genes which are differentially expressed in macrophages. DNA sequence adjacent to GFP showed homology with genes in M. tuberculosis.. A further analysis of these genes and identification of their products should provide insight into how mycobacteria survive within the macrophage. During the process of isolating GFP fusions, we identified a construct , GFP13, which contained a promoter expressed constitutively at least 5 fold better than HSP60, the mycobacterial heat shock promoter.. The identification of this very strong promoter could provide a very useful tool for achieving high level expression of mycobacterial genes. M. ulcerans. Infection with M. ulcerans, the causative agent of Buruli ulcer , results in a severe necrotizing skin lesion with very little acute inflammatory response. In order to understand how M. ulcerans causes disease, we have purified and characterized a toxin, MULT, from M. ulcerans,. Structural studies of MULT reveal that it is a complex polyketide, a 12- membered ring macrolide. Complex polyketides include a large number of potent bioactive molecules such as antibiotics (erythromycin), immunosuppressants (FK506), antifungals (amphotericin) and cytostatins (Bafilomycin). Although complex polyketides are common among Streptomyces species, MULT is the first complex polyketide, and first macrolide isolated from Mycobacteria species. We have characterized the biological activities of MULT using both in vitro tissue culture and in vivo studies. In pg amounts, MULT causes mouse fibroblasts to arrest in G1 of the cell cycle. More remarkably, intradermal injection of MULT into a guinea pig produces lesions pathologically identical to those of Buruli ulcer.