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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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

     
 
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Table of Contents

  • Introduction
  • Basic Protocol: 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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Materials

Basic Protocol: Isolation of Legionella-Containing Vacuoles

 Materials
  • Dictyostelium discoideum (see Support Protocol 1)
  • HL5 medium (see recipe)
  • AYE medium (see recipe)
  • Legionella pneumophila (see Support Protocol 2)
  • 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

 Materials
  • 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., 2001)
  • 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

 Materials
  • 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

 Materials
  • 0.1% (w/v) poly-l-lysine sterile solution (Sigma)
  • D. discoideum Infected with L. pneumophila (see Basic Protocol)
  • 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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Figures

  •  FigureFigure 3.34.1 D. discoideum amoebae infected with L. pneumophila. D. discoideum producing the ER/LCV marker calnexin-GFP (green) was infected with DsRed-Express labeled L. pneumophila Philadelphia-1 wild-type strain JR32 (red) at an MOI of 100 and incubated 1 hr at 25°C. The nucleus of D. discoideum is stained with DAPI. Image kindly provided by Stefan S. Weber.
    View Image
  •  FigureFigure 3.34.2 Immuno-magnetic separation of Legionella-containing vacuoles. (A) Flow-through and (B) eluate of a MACS (magnetic cell separation) column loaded with antibody-treated homogenate of L. pneumophila-infected D. discoideum. Homogenates of D. discoideum producing calnexin-GFP (green) infected with DsRed-labeled L. pneumophila (red) were treated with a primary antibody (polyclonal serum) against the bacterial LCV marker SidC and a secondary antibody coupled to magnetic micro beads. Intact LCVs on a MACS column were selectively retained by a magnet and eluted upon removal of the magnet. The insets show magnifications of the marked areas.
    View Image
  •  FigureFigure 3.34.3 Density centrifugation of Legionella-containing vacuoles. The eluate from a MACS (magnetic cell separation) column was centrifuged through a linear (10% to 35%) Histodenz density gradient and collected in 8 fractions of 1.5 ml. Intact LCVs accumulate in the fractions 4 and 5 (image shown). The inset shows a magnification of the marked area.
    View Image

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Literature Cited

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