Identification of novel M. tuberculosis secreted effector proteins

新型结核分枝杆菌分泌效应蛋白的鉴定

基本信息

批准号：
8682011
负责人：
MICHAEL SHILOH
金额：
$ 19.53万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-02-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8682011
关键词：
Accounting Address Affect Alleles Autophagocytosis Bacterial Infections Binding Binding Proteins Biochemical Biochemistry Biological Assay Cell membrane Cells Cellular Membrane Cellular biology Cessation of life Chimeric Proteins Communicable Diseases Complement Coupling Data Development Drug Targeting Endoplasmic Reticulum Eukaryotic Cell Event Face Fluorescence Microscopy Genes Genetic Screening Goals Golgi Apparatus Growth Growth Factor Guanine Nucleotide Exchange Factors Health Human Human Cell Line Infection Intracellular Membranes Knowledge Lead Libraries Life Lipids Membrane Membrane Proteins Methodology Mitochondria Modeling Modification Molecular Morbidity - disease rate Mycobacterium tuberculosis Organelles Organism Outcome Pathogenesis Phagosomes Pharmaceutical Preparations Phospholipids Population Process Protein Binding Protein Biosynthesis Protein Secretion Proteins Proteome Regulation Research Saccharomyces cerevisiae System Temperature Testing Transmembrane Domain Tuberculosis Vesicle Virulence Factors Work Yeasts antimicrobial expression cloning high throughput screening in vitro Assay knowledge base macrophage microbial mortality mutant mycobacterial novel pathogen pathogenic bacteria prevent public health relevance ras Proteins research study screening tuberculosis treatment uptake

项目摘要

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.

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，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细胞内病原体，结核分枝杆菌已经进化出操纵宿主事件的机制，这些机制依赖于动态膜过程，如吞噬体成熟、吞噬体融合和自噬。其他病原细菌通过分泌与宿主膜结合以促进其活动的蛋白质毒力因子（称为“效应蛋白”或“效应子”）来实现类似的效果。迄今为止，在结核分枝杆菌中几乎没有发现这样的效应子，扩大这一知识库可能为新药的开发提供额外的途径。由于真核细胞膜是细胞的主要组织中心，我们假设细胞膜是分枝杆菌效应蛋白的目标。在初步实验中，我们在模型真核生物酿酒酵母（酵母）中使用高通量筛选试验测试了假定的分泌型分枝杆菌效应子结合宿主膜的能力。迄今为止筛选的 40 个基因中，有 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制中心（内质网）相关。该提案的目的是从根本上了解结核分枝杆菌如何利用膜靶向来操纵宿主。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膜结合蛋白的完整互补体。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关联位置，并证明分枝杆菌效应物在感染过程中直接分泌到宿主细胞中。我们将选择几个命中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标的鉴定。最后，我们将利用所学到的知识检验以下假设：结核分枝杆菌效应子对膜和细胞器动力学的调节对于人类巨噬细胞内分枝杆菌的生存至关重要。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能力的知识，并揭示新的微生物生存策略。