Unit

UNIT 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation

  1. Simon Urwyler,
  2. Ivo Finsel,
  3. Curdin Ragaz,
  4. Hubert Hilbi

Published Online: 1 MAR 2010

DOI: 10.1002/0471143030.cb0334s46

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Urwyler, S., Finsel, I., Ragaz, C. and Hilbi, H. 2010. Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation. Current Protocols in Cell Biology. 46:3.34:3.34.1–3.34.14.

Author Information

  1. Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland

Publication History

  1. Published Online: 1 MAR 2010
  2. Published Print: MAR 2010

Abstract

The environmental bacterium Legionella pneumophila naturally parasitizes free-living amoebae. L. pneumophila is an opportunistic human pathogen that grows in macrophages, thus causing a life-threatening pneumonia termed Legionnaires' disease. The bacteria replicate intracellularly in environmental and immune phagocytes within a unique compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is a complex and robust process involving >150 secreted bacterial effector proteins, which are believed to subvert host cell signaling and vesicle trafficking pathways. This unit describes a simple approach to purify intact LCVs from Dictyostelium discoideum amoebae. The method comprises a two-step purification protocol that includes immuno-magnetic separation by means of an antibody against an effector protein specifically binding to LCVs, followed by density gradient centrifugation. The use of D. discoideum producing a fluorescent LCV marker and fluorescently labeled L. pneumophila allow tracking the enrichment of LCVs by light microscopy. Curr. Protoc. Cell Biol. 46:3.34.1-3.34.14. © 2010 by John Wiley & Sons, Inc.

Keywords:

  • bacterial pathogenesis;
  • Dictyostelium;
  • GTPase;
  • macrophage;
  • pathogen vacuole;
  • phagosome;
  • phosphoinositide;
  • vesicle trafficking