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Subcellular Localization of Legionella Dot/Icm Effectors

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Book cover Legionella

Part of the book series: Methods in Molecular Biology ((MIMB,volume 954))

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Abstract

The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.

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Abbreviations

AYE:

ACES Yeast Extract

BCYE:

Buffered Charcoal Yeast Extract

BSA:

Bovine Serum Albumin

DAPI:

4′,6-Diamidino-2-phenylindole

DMEM:

Dulbecco’s Modified Eagle Medium

EGFP:

Enhanced green fluorescent protein

HA:

Hemagglutinin

IPTG:

Isopropyl-β-d-thio-galactoside

LB:

Luria–Bertani

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

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Acknowledgments

This work was supported by the Australian National Health and Medical Research Council and the Australian Research Council (ARC). ELH is an ARC Future Fellow. AV is supported by a University of Melbourne MDHS Faculty Research (Trust) Scholarship. Aurelie Mousnier is supported by the Wellcome Trust.

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Melbourne, Melbourne, VIC, Australia

    Adam J. Vogrin & Elizabeth L. Hartland

  2. Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, Imperial College of London, London, UK

    Aurelie Mousnier

  3. Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, Imperial College London, London, UK

    Gad Frankel

Authors
  1. Adam J. Vogrin
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  2. Aurelie Mousnier
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  3. Gad Frankel
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  4. Elizabeth L. Hartland
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Corresponding author

Correspondence to Elizabeth L. Hartland .

Editor information

Editors and Affiliations

  1. , Biology of Intracellular Bacteria, Institut Pasteur, Rue du Dr. Roux 28, Paris, 75724, France

    Carmen Buchrieser

  2. Max von Pettenkofer Institute, Ludwig-Maximilians-University, Pettenkoferstrasse 9a, Munich, 80336, Germany

    Hubert Hilbi

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Vogrin, A.J., Mousnier, A., Frankel, G., Hartland, E.L. (2013). Subcellular Localization of Legionella Dot/Icm Effectors. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 954. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-161-5_20

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  • DOI: https://doi.org/10.1007/978-1-62703-161-5_20

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-160-8

  • Online ISBN: 978-1-62703-161-5

  • eBook Packages: Springer Protocols

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