Identification of novel M. tuberculosis secreted effector proteins

描述（申请人提供）：结核杆菌仍然是最具破坏性的人类传染病之一，每年导致200万人死亡，并潜伏感染世界三分之一的人口。作为一种适应长期生存的胞内病原菌，M.结核病已经进化出操纵宿主事件的机制，所述宿主事件依赖于动态膜过程，例如吞噬体成熟、吞噬体融合和自噬。其他病原菌通过分泌蛋白质毒力因子（称为“效应蛋白”或“效应物”）来实现类似的效果，这些毒力因子与宿主膜相关联以促进其活动。到目前为止，很少有这样的效应已确定在M。结核病和扩大这一知识基础可能为开发新药提供额外的途径。由于真核细胞膜是细胞的主要组织中心，我们假设膜是分枝杆菌效应蛋白的靶点。在初步实验中，我们测试了假定的分泌型分枝杆菌效应子的能力，结合宿主膜，使用高通量筛选试验在模型真核生物，酿酒酵母（酵母）。到目前为止筛选的40个基因中，有5个（12.5%）与膜相互作用，我们已经证明，在人类细胞系中，几个基因与细胞的主要蛋白质合成中心内质网相关。本文的目的是从根本上理解M.结核病能够利用膜靶向来操纵宿主。我们建议首先确定一个完整的补体膜结合蛋白分泌的M。结核病通过使用我们的高通量克隆和表达系统筛选总共400个基因。表征将进一步包括细胞生物学测定，以确定每种蛋白质在真核细胞内的缔合位置，并证明感染期间分枝杆菌效应物直接分泌到宿主细胞中。我们将选择几个命中进一步深入表征，包括在体外测定膜协会和鉴定宿主蛋白质的目标。最后，我们将利用获得的知识， 验证了M.结核病效应子对于分枝杆菌在人巨噬细胞内的存活是必需的。本文的工作将扩展现有的关于M.结核病操纵宿主膜动力学和揭示新的微生物生存策略的能力。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis remains one of the most devastating human infectious diseases, causing two million deaths annually and latently infecting a third of the world's population. As an intracellular pathogen adapted to long-term survival, M. tuberculosis has evolved mechanisms to manipulate host events that rely on dynamic membrane processes such as phagosome maturation, phagolysome fusion and autophagy. Other pathogenic bacteria achieve similar effects by secreting protein virulence factors (called "effector proteins" or "effectors") that associate with host membranes to facilitat their activities. To date, few such effectors have been identified in M. tuberculosis and expanding this knowledge base may provide additional avenues for the development of new drugs. Because eukaryotic cellular membranes are the major organizational centers of the cell, we hypothesize that membranes are targeted by mycobacterial effector proteins. In preliminary experiments, we tested putative secreted mycobacterial effectors for their ability to bind host membranes using a high-throughput screening assay in the model eukaryotic organism, Saccharomyces cerevisiae (yeast). Of the 40 genes screened to date, 5 (12.5%) interact with membranes, and we have demonstrated that in a human cell line several of the genes associate with the major protein synthesis machinery center of the cell, the endoplasmic reticulum. The aims of this proposal are to fundamentally understand how M. tuberculosis is able to use membrane targeting to manipulate the host. We propose to first identify a complete complement of membrane binding proteins secreted by M. tuberculosis by screening a total of 400 genes using our high- throughput cloning and expression system. Characterization will further include cell biologic assays to determine where each protein associates within eukaryotic cells and demonstration of direct secretion of mycobacterial effectors into host cells during infection. We will select several hits for further in-depth characterization including in vitro assays of membran association and identification of host protein targets. Lastly, we will use the knowledge gained to test the hypothesis that regulation of membrane and organelle dynamics by M. tuberculosis effectors is essential for mycobacterial survival within human macrophages. The proposed work will extend the current knowledge on M. tuberculosis's ability to manipulate host membrane dynamics and reveal novel microbial survival strategies.