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Host-Bacteria Interactions pp 153–167Cite as

Live Cell Imaging of Phosphoinositide Dynamics During Legionella Infection

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1197))

Abstract

The “accidental” pathogen Legionella pneumophila replicates intracellularly in a distinct compartment, the Legionella-containing vacuole (LCV). To form this specific pathogen vacuole, the bacteria translocate via the Icm/Dot type IV secretion system approximately 300 different effector proteins into the host cell. Several of these secreted effectors anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. L. pneumophila thus largely controls the localization of secreted bacterial effectors and the recruitment of host factors to the LCV through the modulation of the vacuole membrane PI pattern. The LCV PI pattern and its dynamics can be studied in real-time using fluorescently labeled protein probes stably produced by the soil amoeba Dictyostelium discoideum. In this chapter, we describe a protocol to (1) construct and handle amoeba model systems as a tool for observing PIs in live cell imaging, (2) capture rapid changes in membrane PI patterning during uptake events, and (3) observe the dynamics of LCV PIs over the course of a Legionella infection.

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Abbreviations

ACES:

N-(2-acetamido)-2-aminoethane-sulfonic acid

HEPES:

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

icm/dot :

Intracellular multiplication/defective organelle trafficking

MOI:

Multiplicity of infection

PI:

Phosphoinositide

PtdIns(4)P :

Phosphatidylinositol-4-phosphate

T4SS:

Type IV secretion system

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Acknowledgments

This work was supported by the Max von Pettenkofer Institute, Ludwig-Maximilians University Munich, and the German Research Foundation (DFG; HI 1511/1-1, SPP1580, SFB914).

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Authors and Affiliations

  1. Max von Pettenkofer Institute, Ludwig-Maximilians University, Pettenkoferstraße 9a, 80336, Munich, Germany

    Stephen Weber & Hubert Hilbi

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  1. Stephen Weber
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  2. Hubert Hilbi
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Correspondence to Hubert Hilbi .

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Editors and Affiliations

  1. U1047, UFR Médecine, INSERM, Université Montpellier 1, Nîmes, France

    Annette C. Vergunst

  2. U1047, UFR Médecine, INSERM, Université Montpellier 1, Nîmes, France

    David O'Callaghan

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Weber, S., Hilbi, H. (2014). Live Cell Imaging of Phosphoinositide Dynamics During Legionella Infection. In: Vergunst, A., O'Callaghan, D. (eds) Host-Bacteria Interactions. Methods in Molecular Biology, vol 1197. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1261-2_9

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  • DOI: https://doi.org/10.1007/978-1-4939-1261-2_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1260-5

  • Online ISBN: 978-1-4939-1261-2

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