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Imaging Flow Cytometry of Legionella-Containing Vacuoles in Intact and Homogenized Wild-Type and Mutant Dictyostelium

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Spectral and Imaging Cytometry

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2635))

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Abstract

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.

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Acknowledgments

Research in the laboratory of H.H. was supported by the Swiss National Science Foundation (SNF; 31003A_153200, 31003A_175557), the Novartis Foundation for Medical-Biological Research, the OPO foundation, and the Center of Microscopy and Image Analysis, University of Zürich (UZH). A.W. was supported by the Swedish Society of Medicine, the Linköping Society of Medicine, the Medical Inflammation and Infection Centre at Linköping University, and the Åke Wiberg Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Imaging flow cytometry was performed using equipment of the Flow Cytometry Unit, Core Facility, Medical Faculty, Linköping University.

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Authors and Affiliations

  1. Division of Inflammation and Infection, Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden

    Amanda Welin

  2. Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland

    Dario Hüsler & Hubert Hilbi

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  1. Amanda Welin
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  2. Dario Hüsler
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  3. Hubert Hilbi
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Correspondence to Hubert Hilbi .

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Editors and Affiliations

  1. Department of Biology, School of Sciences and Humanities, Nazarbayev University, Astana, Kazakhstan

    Natasha S. Barteneva

  2. Department of Biology, School of Sciences and Humanities, Nazarbayev University, Astana, Kazakhstan

    Ivan A. Vorobjev

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Welin, A., Hüsler, D., Hilbi, H. (2023). Imaging Flow Cytometry of Legionella-Containing Vacuoles in Intact and Homogenized Wild-Type and Mutant Dictyostelium. In: Barteneva, N.S., Vorobjev, I.A. (eds) Spectral and Imaging Cytometry. Methods in Molecular Biology, vol 2635. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3020-4_4

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  • DOI: https://doi.org/10.1007/978-1-0716-3020-4_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3019-8

  • Online ISBN: 978-1-0716-3020-4

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