Identification of novel M. tuberculosis secreted effector proteins

描述(申请人提供)：结核分枝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，并潜伏着感染世界三分之一的人口。作为一种适应长期生存的细胞内病原体，结核分枝杆菌已经进化出一种机制来操纵宿主事件，这些宿主事件依赖于动态的膜过程，如吞噬小体成熟、吞噬融合和自噬。其他病原菌通过分泌与宿主膜相关的蛋白质毒力因子(称为“效应物蛋白”或“效应物”)来实现类似的效果，以促进它们的活动。到目前为止，在结核分枝杆菌中发现的这种效应很少，扩大这一知识库可能会为开发新药提供更多的途径。因为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标。在初步实验中，我们使用高通量筛选试验在模式真核生物酿酒酵母(酵母)中测试了假定的分泌型分枝杆菌效应器与宿主膜结合的能力。到目前为止，在筛选的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，有几个基因与细胞的主要蛋白质合成机制中心-内质网相关。这项提议的目的是从根本上了解结核分枝杆菌是如何利用膜靶向来操纵宿主的。我们建议首先通过使用我们的高通量克隆和表达系统筛选总共400个基因来鉴定结核分枝杆菌分泌的完整的膜结合蛋白。鉴定将进一步包括细胞生物学分析，以确定每种蛋白质在真核细胞中的关联位置，以及在感染期间分枝杆菌效应物直接分泌到宿主细胞中的演示。我们将选择几个HITS进行进一步的深入表征，包括体外膜结合分析和宿主蛋白靶标的鉴定。最后，我们将利用所获得的知识来 验证结核分枝杆菌效应器对膜和细胞器动力学的调节对分枝杆菌在人巨噬细胞内的生存至关重要的假设。这项拟议的工作将扩展目前关于结核分枝杆菌操纵宿主膜动力学的能力的知识，并揭示新的微生物生存策略。

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.