Inside out – The role of mycobacterial ESX secretion systems in phagosome escape

由内而外 â 分枝杆菌 ESX 分泌系统在吞噬体逃逸中的作用

• 批准号: 446371725
• 负责人: Professor Dr. Thomas Gutsmann
• 金额: --
• 依托单位: Forschungsgruppe Biophysik
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/446371725?language=en
• 关键词: Inside; out; role; mycobacterial; ESX

The aim of the project is to elucidate the role of type VII secretion systems (T7SS) in phagosome escape of pathogenic mycobacteria. In particular, our focus will be on the inter- and intraspecies genetic diversity of ESX secretion systems and on the structural and functional variability of T7SS substrates belonging to the WXG100 superfamily. Based on the results of a comprehensive in silico screening, a selection of WXG100 proteins representing the genetic variation across the genus will be recombinantly expressed to study the impact of structural changes on protein function. The role of ESX substrates in phagosome escape will be analyzed using human and bovine blood derived macrophages. To decipher the complex interaction of ESX substrates with biological membranes we will use high resolution microscopy (EM, AFM, STORM) on intracellular bacteria in macrophages and various biophysical techniques (spectroscopy, electrophysiology, binding assays) on natural and reconstituted well-defined lipid membrane systems. The specific role of the ESX molecules for the exit of mycobacteria will be mimicked by addition of the purified proteins, of proteins encapsulated in reconstituted liposomal containers, and inactivated mycobacteria into different cell culture systems. The structure of WXG100 monomers and heterodimers will be characterized, as well as the molecular basis of protein-membrane interactions between WXG100 proteins and biological membranes with a particular focus on pH-, lipid-, voltage-, and protein-structure-specificities.

该项目的目的是阐明VII型分泌系统（T7 SS）在致病性分枝杆菌吞噬体逃逸中的作用。特别是，我们的重点将是间和种内的ESX分泌系统的遗传多样性和属于WXG 100超家族的T7 SS底物的结构和功能的变异性。基于全面的计算机筛选结果，将重组表达代表该属遗传变异的WXG 100蛋白，以研究结构变化对蛋白功能的影响。将使用人和牛血衍生的巨噬细胞分析ESX底物在吞噬体逃逸中的作用。为了解读ESX基质与生物膜的复杂相互作用，我们将使用高分辨率显微镜（EM，AFM，STORM）对巨噬细胞中的细胞内细菌和各种生物物理技术（光谱学，电生理学，结合试验）对天然和重建的定义明确的脂质膜系统。将通过向不同的细胞培养系统中加入纯化的蛋白质、包封在复溶脂质体容器中的蛋白质和灭活的分枝杆菌来模拟ESX分子对分枝杆菌退出的特定作用。WXG 100单体和异源二聚体的结构将被表征，以及WXG 100蛋白和生物膜之间的蛋白质-膜相互作用的分子基础，特别关注pH值，脂质，电压和蛋白质结构特异性。