Clade specific virulence patterns of M. tuberculosis complex strains in human macrophages: Molecular mechanisms underlying phagosomal escape and induction of cell death.

结核病的结局由感染结核分枝杆菌复合体（MTC）菌株的毒力和宿主巨噬细胞的杀伤能力共同决定。在目前的研究中，我们将研究病原体变异性和宿主细胞信号转导变异性对MTC菌株携带室吞噬体分子组成的影响。该研究包括(i)比较分析来自人原代巨噬细胞的含有吞噬体的MTC分支i和分支II的蛋白质和脂质组成；（ii）鉴定控制巨噬细胞中结核分枝杆菌生长的关键宿主细胞因子；（iii）对从人类和小鼠巨噬细胞中分离出来的吞噬体进行差异分析，以确定存在于两种物种中并参与其中的“核心蛋白质组成分”和存在于两种物种中的吞噬体中或专门招募到其中一种物种的“个体蛋白质组成分”。一种新的基于脂质标记细菌表面的方法和一种快速免疫磁技术将用于从小鼠和人类原代细胞中分离完整的含细菌的区室，以便通过电子和原子力显微镜、流式细胞术、Western blot和质谱法进一步表征它们。新开发的方法促进了整个细胞内病原体王国的比较研究，并将提供给优先计划的其他成员。

The outcome of tuberculosis is determined by both the virulence of the infecting M. tuberculosis complex (MTC) strain and the killing capacity of the host macrophage. In the current study we will examine the impact of pathogen variability and host cell signaling variability on the molecular composition of the MTC strain harboring compartment, the phagosome. The study comprises (i) the comparative analysis of the protein and lipid composition of MTC clade I and clade II containing phagosomes from human primary macrophages, (ii) the identification of critical host cell factors that govern control of M. tuberculosis growth in macrophages and (iii) the differential analysis of phagosomes isolated from human and murine macrophages to define “core proteome components” which are present and engaged in both species and “individual proteome components” which are present or exclusively recruited to the phagosome in one of the two species. A new lipid-based procedure to label bacterial surfaces and a rapid immunomagnetic technique will be used to isolate intact bacteria-containing compartments from mouse and human primary cells in order to further characterize them by electron and atomic force microscopy, flow cytometry, Western blot, and mass spectrometry. The newly developed method facilitates comparative studies across the kingdom of intracellular pathogens and will be made available to other members of the priority program.