Identification of novel M. tuberculosis secreted effector proteins

描述（由申请人提供）：结核分枝杆菌仍然是最具破坏性的人类传染病之一，每年造成200万人死亡，潜在感染世界三分之一的人口。作为适应长期生存的细胞内病原体，结核分枝杆菌已经进化出依赖于吞噬体成熟、吞噬体融合和自噬等动态膜过程的机制来操纵宿主事件。其他致病菌通过分泌与宿主膜相关的蛋白质毒力因子（称为“效应蛋白”或“效应器”）来促进其活动，从而达到类似的效果。迄今为止，在结核分枝杆菌中发现的此类效应物很少，扩大这一知识库可能为开发新药提供额外的途径。因为真核细胞的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目标。在初步实验中，我们在真核生物酵母（Saccharomyces cerevisiae，酵母）中使用高通量筛选法测试了假定分泌的分枝杆菌效应物结合宿主膜的能力。在迄今为止筛选的40个基因中，5个（12.5%）与细胞膜相互作用，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械中心内质网有关。这一建议的目的是从根本上了解结核分枝杆菌如何能够利用膜靶向来操纵宿主。我们建议首先利用我们的高通量克隆和表达系统，通过筛选总共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结合位置，并证明分枝杆菌效应物在感染期间直接分泌到宿主细胞中。我们将选择几个命中点进行进一步深入的表征，包括膜关联的体外测定和宿主蛋白靶点的鉴定。最后，我们将利用所获得的知识

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.