Legionella pp 333-344 | Cite as

Subcellular Localization of Legionella Dot/Icm Effectors

  • Adam J. Vogrin
  • Aurelie Mousnier
  • Gad Frankel
  • Elizabeth L. Hartland
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 954)

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.

Key words

Legionella pneumophila HEK293T cells Epitope tagging Laser scanning confocal microscopy Transfection Infection Type IV secretion 

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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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Adam J. Vogrin
    • 1
  • Aurelie Mousnier
    • 2
  • Gad Frankel
    • 3
  • Elizabeth L. Hartland
    • 1
  1. 1.Department of Microbiology and ImmunologyUniversity of MelbourneMelbourneAustralia
  2. 2.Division of Cell and Molecular BiologyCentre for Molecular Microbiology and Infection, Imperial College of LondonLondonUK
  3. 3.Division of Cell and Molecular BiologyCentre for Molecular Microbiology and Infection, Imperial College LondonLondonUK

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