Welin Amanda, Hüsler Dario, Hilbi Hubert
Division of Inflammation and Infection, Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden.
Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland.
Methods Mol Biol. 2023;2635:63-85. doi: 10.1007/978-1-0716-3020-4_4.
The causative agent of a severe pneumonia termed "Legionnaires' disease", Legionella pneumophila, replicates within protozoan and mammalian phagocytes in a specialized intracellular compartment called the Legionella-containing vacuole (LCV). This compartment does not fuse with bactericidal lysosomes but communicates extensively with several cellular vesicle trafficking pathways and eventually associates tightly with the endoplasmic reticulum. In order to comprehend in detail the complex process of LCV formation, the identification and kinetic analysis of cellular trafficking pathway markers on the pathogen vacuole are crucial. This chapter describes imaging flow cytometry (IFC)-based methods for the objective, quantitative and high-throughput analysis of different fluorescently tagged proteins or probes on the LCV. To this end, we use the haploid amoeba Dictyostelium discoideum as an infection model for L. pneumophila, to analyze either fixed intact infected host cells or LCVs from homogenized amoebae. Parental strains and isogenic mutant amoebae are compared in order to determine the contribution of a specific host factor to LCV formation. The amoebae simultaneously produce two different fluorescently tagged probes enabling tandem quantification of two LCV markers in intact amoebae or the identification of LCVs using one probe and quantification of the other probe in host cell homogenates. The IFC approach allows rapid generation of statistically robust data from thousands of pathogen vacuoles and can be applied to other infection models.
一种名为“军团病”的严重肺炎的病原体嗜肺军团菌，在原生动物和哺乳动物吞噬细胞内的一个特殊细胞内区室即含军团菌液泡（LCV）中进行复制。这个区室不与杀菌性溶酶体融合，但与多个细胞囊泡运输途径广泛沟通，最终与内质网紧密相连。为了详细理解LCV形成的复杂过程，对病原体液泡上的细胞运输途径标记物进行鉴定和动力学分析至关重要。本章描述了基于成像流式细胞术（IFC）的方法，用于对LCV上不同荧光标记的蛋白质或探针进行客观、定量和高通量分析。为此，我们使用单倍体变形虫盘基网柄菌作为嗜肺军团菌的感染模型，以分析固定的完整感染宿主细胞或来自匀浆变形虫的LCV。比较亲本菌株和同基因突变变形虫，以确定特定宿主因子对LCV形成的贡献。变形虫同时产生两种不同的荧光标记探针，能够对完整变形虫中的两种LCV标记物进行串联定量，或者在宿主细胞匀浆中使用一种探针鉴定LCV并对另一种探针进行定量。IFC方法能够从数千个病原体液泡中快速生成具有统计学稳健性的数据，并且可以应用于其他感染模型。
