Welin Amanda, Weber Stephen, Hilbi Hubert
Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland.
Methods Mol Biol. 2019;1921:161-177. doi: 10.1007/978-1-4939-9048-1_10.
Legionella pneumophila enters and replicates within protozoan and mammalian phagocytes by forming through a conserved mechanism a specialized intracellular compartment termed the Legionella-containing vacuole (LCV). This compartment avoids fusion with bactericidal lysosomes but communicates extensively with different cellular vesicle trafficking pathways and ultimately interacts closely with the endoplasmic reticulum. In order to delineate the process of pathogen vacuole formation and to better understand L. pneumophila virulence, an analysis of markers of the different trafficking pathways on the pathogen vacuole is crucial. Here, we describe a method for rapid, objective and quantitative analysis of different fluorescently tagged proteins or probes on the LCV. To this end, we employ an imaging flow cytometry approach and use the D. discoideum -L. pneumophila infection model. Imaging flow cytometry enables quantification of many different parameters by fluorescence microscopy of cells in flow, rapidly producing statistically robust data from thousands of cells. We also describe the generation of D. discoideum strains simultaneously producing two different fluorescently tagged probes that enable visualization of compartments and processes in parallel. The quantitative imaging flow technique can be corroborated and enhanced by laser scanning confocal microscopy.
嗜肺军团菌通过一种保守机制在原生动物和哺乳动物吞噬细胞内进入并复制，形成一个特殊的细胞内区室，称为含军团菌液泡（LCV）。这个区室避免与杀菌性溶酶体融合，但与不同的细胞囊泡运输途径广泛交流，并最终与内质网密切相互作用。为了描绘病原体液泡形成的过程并更好地理解嗜肺军团菌的毒力，分析病原体液泡上不同运输途径的标记物至关重要。在此，我们描述一种对LCV上不同荧光标记蛋白或探针进行快速、客观和定量分析的方法。为此，我们采用成像流式细胞术方法，并使用盘基网柄菌-嗜肺军团菌感染模型。成像流式细胞术能够通过对流动中的细胞进行荧光显微镜检查来量化许多不同参数，从数千个细胞中快速产生具有统计学稳健性的数据。我们还描述了同时产生两种不同荧光标记探针的盘基网柄菌菌株的构建，这使得能够并行可视化区室和过程。定量成像流式技术可以通过激光扫描共聚焦显微镜得到证实和增强。
