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Analysis of Legionella Metabolism by Pathogen Vacuole Proteomics

  • Christian Manske
  • Ivo Finsel
  • Christine Hoffmann
  • Hubert Hilbi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1841)

Abstract

The causative agent of Legionnaires’ disease, Legionella pneumophila, replicates in free-living amoebae as well as in macrophages of the innate immune system within a distinct membrane-bound compartment, the “Legionella-containing-vacuole” (LCV). LCV formation is a complex process and requires the bacterial Icm/Dot type IV secretion system, which translocates approximately 300 different “effector” proteins. Intact LCVs from infected Dictyostelium discoideum amoebae or RAW 264.7 murine macrophages can be purified using a straightforward protocol. In the first step, the LCVs in cell homogenates are tagged with an antibody directed against an L. pneumophila effector protein specifically localizing to the pathogen vacuole membrane and isolated by immunomagnetic separation using a secondary antibody coupled to magnetic beads. In the second step, the LCVs are further enriched by density gradient centrifugation through a Histodenz cushion. LCVs thus purified are analyzed by mass spectrometry-based proteomics and characterized by biochemical and cell biological approaches.

Key words

Amoeba Density gradient centrifugation Dictyostelium discoideum Effector protein Immunomagnetic separation Legionella pneumophila Macrophage Metabolism Pathogen vacuole Type IV secretion 

Abbreviations

ACES

N-(2-acetamido)-2-aminoethanesulfonic acid

GFP

Green fluorescent protein

HEPES

N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid

Icm/Dot

Intracellular multiplication/defective organelle trafficking

LCV

Legionella-containing vacuole

T2SS

Type II secretion system

T4SS

Type IV secretion system

Notes

Acknowledgments

This work received financial support from the Swiss National Science Foundation (SNF; 31003A_153200), the German Research Foundation (DFG; SPP1316, SPP1617) and the “Bundesministerium für Bildung und Forschung” (BMBF; “Medical Infection Genomics” project 0315834C, and “ERA-NET Infect-ERA” EUGENPATH project 031A401A).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Christian Manske
    • 1
  • Ivo Finsel
    • 1
  • Christine Hoffmann
    • 1
  • Hubert Hilbi
    • 2
  1. 1.Max von Pettenkofer InstituteLudwig-Maximilians UniversityMunichGermany
  2. 2.Institute of Medical MicrobiologyUniversity of ZürichZürichSwitzerland

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