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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Isolation of Legionella‐Containing Vacuoles

  Materials
  • 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

  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., )
  • 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 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
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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.
  •   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.
  •   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.

Videos

Literature Cited

Literature Cited
   Feeley, J.C., Gibson, R.J., Gorman, G.W., Langford, N.C., Rasheed, J.K., Mackel, D.C., and Baine, W.B. 1979. Charcoal‐yeast extract agar: Primary isolation medium for Legionella pneumophila. J. Clin. Microbiol. 10:437‐441.
   Fernandez‐Mora, E., Polidori, M., Lührmann, A., Schaible, U.E., and Haas, A. 2005. Maturation of Rhodococcus equi‐containing vacuoles is arrested after completion of the early endosome stage. Traffic 6:635‐653.
   Fields, B.S., Benson, R.E., and Besser, R.E. 2002. Legionella and Legionnaires' disease: 25 years of investigation. Clin. Microbiol. Rev. 15:506‐526.
   Garin, J., Diez, R., Kieffer, S., Dermine, J.F., Duclos, S., Gagnon, E., Sadoul, R., Rondeau, C., and Desjardins, M. 2001. The phagosome proteome: Insight into phagosome functions. J. Cell Biol. 152:165‐180.
   Gotthardt, D., Warnatz, H.J., Henschel, O., Bruckert, F., Schleicher, M., and Soldati, T. 2002. High‐resolution dissection of phagosome maturation reveals distinct membrane trafficking phases. Mol. Biol. Cell 13:3508‐3520.
   Gotthardt, D., Blancheteau, V., Bosserhoff, A., Ruppert, T., Delorenzi, M., and Soldati, T. 2006. Proteomics fingerprinting of phagosome maturation and evidence for the role of a Gα during uptake. Mol. Cell Proteomics 5:2228‐2243.
   Hägele, S., Kohler, R., Merkert, H., Schleicher, M., Hacker, J., and Steinert, M. 2000. Dictyostelium discoideum: A new host model system for intracellular pathogens of the genus Legionella. Cell Microbiol. 2:165‐171.
   Hilbi, H., Weber, S.S., Ragaz, C., Nyfeler, Y., and Urwyler, S. 2007. Environmental predators as models for bacterial pathogenesis. Environ. Microbiol. 9:563‐575.
   Isberg, R.R., O'Connor, T.J., and Heidtman, M. 2009. The Legionella pneumophila replication vacuole: Making a cosy niche inside host cells. Nat. Rev. Microbiol. 7:13‐24.
   Kima, P.E. and Dunn, W. 2005. Exploiting calnexin expression on phagosomes to isolate Leishmania parasitophorous vacuoles. Microb. Pathog. 38:139‐145.
   Lu, H. and Clarke, M. 2005. Dynamic properties of Legionella‐containing phagosomes in Dictyostelium amoebae. Cell Microbiol. 7:995‐1007.
   Lührmann, A. and Haas, A. 2000. A method to purify bacteria‐containing phagosomes from infected macrophages. Methods Cell Sci. 22:329‐341.
   Malchow, D., Nagele, B., Schwarz, H., and Gerisch, G. 1972. Membrane‐bound cyclic AMP phosphodiesterase in chemotactically responding cells of Dictyostelium discoideum. Eur. J. Biochem. 28:136‐142.
   Mampel, J., Spirig, T., Weber, S.S., Haagensen, J.A.J., Molin, S., and Hilbi, H. 2006. Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions. Appl. Environ. Microbiol. 72:2885‐2895.
   Mills, S.D. and Finlay, B.B. 1998. Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis‐containing phagosomes from infected mouse macrophages: Y. pseudotuberculosis traffics to terminal lysosomes where they are degraded. Eur. J. Cell Biol. 77:35‐47.
   Müller‐Taubenberger, A., Lupas, A.N., Li, H., Ecke, M., Simmeth, E., and Gerisch, G. 2001. Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis. EMBO J. 20:6772‐6782.
   Ragaz, C., Pietsch, H., Urwyler, S., Tiaden, A., Weber, S.S., and Hilbi, H. 2008. The Legionella pneumophila phosphatidylinositol‐4 phosphate‐binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication‐permissive vacuole. Cell Microbiol. 10:2416‐2433.
   Segal, G., Feldman, M., and Zusman, T. 2005. The Icm/Dot type‐IV secretion systems of Legionella pneumophila and Coxiella burnetii. FEMS Microbiol. Rev. 29:65‐81.
   Shevchuk, O., Batzilla, C., Hägele, S., Kusch, H., Engelmann, S., Hecker, M., Haas, A., Heuner, K., Glockner, G., and Steinert, M. 2009. Proteomic analysis of Legionella‐containing phagosomes isolated from Dictyostelium. Int. J. Med. Microbiol. 299:489‐508.
   Solomon, J.M., Rupper, A., Cardelli, J.A., and Isberg, R.R. 2000. Intracellular growth of Legionella pneumophila in Dictyostelium discoideum, a system for genetic analysis of host‐pathogen interactions. Infect. Immun. 68:2939‐2947.
   Sturgill‐Koszycki, S., Schlesinger, P., Chakraborty, P., Haddix, P., Collins, H., Fok, A., Allen, R., Gluck, S., Heuser, J., and Russell, D. 1994. Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton‐ATPase. Science 263:637‐639.
   Sturgill‐Koszycki, S., Haddix, P.L., and Russell, D.G. 1997. The interaction between Mycobacterium and the macrophage analyzed by two‐dimensional polyacrylamide gel electrophoresis. Electrophoresis 18:2558‐2565.
   Sussman, M., 1987. Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. Methods Cell Biol. 28:9‐29.
   Urwyler, S., Brombacher, E., and Hilbi, H. 2009a. Endosomal and secretory marker of the Legionella‐containing vacuole. Commun. Integr. Biol. 2:107‐109.
   Urwyler, S., Nyfeler, Y., Ragaz, C., Lee, H., Mueller, L.N., Aebersold, R., and Hilbi, H. 2009b. Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases. Traffic 10:76‐87.
   Watts, D.J. and Ashworth, J.M. 1970. Growth of myxameobae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochem. J. 119:171‐174.
   Weber, S.S., Ragaz, C., Reus, K., Nyfeler, Y., and Hilbi, H. 2006. Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole. PLoS Pathog. 2:E46.
   Weber, S.S., Ragaz, C., and Hilbi, H. 2009. The inositol polyphosphate 5‐phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole and binds to the bacterial effector LpnE. Cell Microbiol. 11:442‐460.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library