Isolation of Legionella‐Containing Vacuoles by Immuno‐Magnetic Separation

Simon Urwyler1, Ivo Finsel1, Curdin Ragaz1, Hubert Hilbi1

1 Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.34
DOI:  10.1002/0471143030.cb0334s46
Online Posting Date:  March, 2010
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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

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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Legionella‐Containing Vacuoles
  • Support Protocol 1: Growth of Dictyostelium discoideum
  • Support Protocol 2: Growth of Legionella pneumophila
  • Support Protocol 3: Analysis of LCV Purification by Fluorescence Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Isolation of Legionella‐Containing Vacuoles

  • Dictyostelium discoideum (see protocol 2)
  • HL5 medium (see recipe)
  • AYE medium (see recipe)
  • Legionella pneumophila (see protocol 3)
  • SorC buffer (see recipe), ice cold
  • HS buffer (see recipe), cold
  • Blocking reagent (e.g., NHS: normal human serum; Blutspendezentrum Zürich)
  • Primary antibody against a bacterial LCV marker (e.g., affinity‐purified polyclonal rabbit anti‐SidC serum; NeoMPS SA)
  • Secondary antibody coupled to MACS micro‐beads (e.g., MACS goat anti‐rabbit IgG micro‐beads; Miltenyi Biotec)
  • Histodenz (Sigma‐Aldrich) solution in PBS (see recipe)
  • 75‐cm2 tissue culture flasks
  • Incubators (23°C, 25°C, 37°C) with and without wheel or shakers
  • 15‐ml test tubes
  • Spectrophotometer
  • Tissue culture centrifuge with swing‐out rotors for plates, flasks, and 15‐ml tubes
  • Plastic cell scraper
  • 3‐ml plastic Luer‐lok syringes
  • Stainless‐steel ball homogenizer (8‐µm clearance, 0.5‐ml chamber; Isobiotec), cold
  • 15‐ml centrifuge tubes with screw caps
  • Overhead spinning wheel at 4°C
  • MACS‐MS separation columns (Miltenyi Biotec, cat. no. 130‐042‐201)
  • MACS separator (e.g., MACS Multistand; Miltenyi Biotec)
  • 1.5‐ml microcentrifuge tubes
  • Glass Pasteur pipets (230‐mm, plugged)

Support Protocol 1: Growth of Dictyostelium discoideum

  • HL5 medium (see recipe)
  • G418 (geneticin; Invitrogen)
  • D. discoideum wild‐type strain Ax3 bearing a plasmid for expression of calnexin‐GFP (Müller‐Taubenberger et al., )
  • SM/5 agar plates (see recipe)
  • Klebsiella pneumoniae (grown in LB medium; appendix 2A)
  • 75‐cm2 tissue culture flasks
  • 23°C incubator

Support Protocol 2: Growth of Legionella pneumophila

  • Frozen glycerol stocks of Legionella pneumophila bearing Ds‐Red Express on plasmid pSW001
  • Charcoal yeast extract (CYE) agar plates (see recipe)
  • Chloramphenicol (Cm)
  • ACES yeast extract (AYE) broth (see recipe)
  • 37°C incubator with and without shaker
  • 15‐ml test tubes
  • Spectrophotometer

Support Protocol 3: Analysis of LCV Purification by Fluorescence Microscopy

  • 0.1% (w/v) poly‐L‐lysine sterile solution (Sigma)
  • D. discoideum Infected with L. pneumophila (see protocol 1)
  • HS medium (see recipe), ice cold
  • 4% (w/v) paraformaldehyde in PBS (see recipe)
  • SorC buffer (see recipe)
  • Mounting medium (e.g., Vectashield; Vector Laboratories)
  • Round microscope coverslips, sterile
  • 24‐well flat‐bottomed tissue culture plate
  • Microscope slides
  • Epifluorescence microscope equipped with filters for GFP and Ds‐Red
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Literature Cited

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