Abstract
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.
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Abbreviations
- ACES:
-
N-(2-acetamido)-2-aminoethanesulfonic acid
- AYE:
-
ACES yeast extract
- Cam:
-
Chloramphenicol
- CYE:
-
Charcoal yeast extract
- DMSO:
-
Dimethyl sulfoxide
- DPBS:
-
Dulbecco's Phosphate-Buffered Saline
- ER:
-
Endoplasmic reticulum
- GFP:
-
Green fluorescent protein
- Icm/Dot:
-
Intracellular multiplication/defective organelle trafficking
- IFC:
-
Imaging flow cytometry
- LCV:
-
Legionella-containing vacuole
- MOI:
-
Multiplicity of infection
- PFA:
-
Paraformaldehyde
- RT:
-
Room temperature
- SSC:
-
Side scatter
- T4SS:
-
Type IV secretion system
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Acknowledgments
Research in the laboratory of H.H. was supported by the Swiss National Science Foundation (SNF; 31003A_153200), the Novartis Foundation for Medical-Biological Research, and the OPO foundation. A.W. was supported by a grant from the Swedish Research Council (2014-396). Imaging flow cytometry was performed using equipment of the Flow Cytometry Facility (University of Zürich) and microscopy using equipment of the Centre for Microscopy and Image Analysis (University of Zürich).
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Welin, A., Weber, S., Hilbi, H. (2019). Quantitative Imaging Flow Cytometry of Legionella-Containing Vacuoles in Dually Fluorescence-Labeled Dictyostelium. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 1921. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9048-1_10
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DOI: https://doi.org/10.1007/978-1-4939-9048-1_10
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