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Analysis of Legionella Metabolism by Pathogen Vacuole Proteomics

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Microbial Proteomics

Part of the book series: Methods in Molecular Biology ((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.

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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

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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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Authors and Affiliations

  1. Max von Pettenkofer Institute, Ludwig-Maximilians University, Munich, Germany

    Christian Manske, Ivo Finsel & Christine Hoffmann

  2. Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland

    Hubert Hilbi

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  1. Christian Manske
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  2. Ivo Finsel
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  3. Christine Hoffmann
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  4. Hubert Hilbi
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Correspondence to Hubert Hilbi .

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  1. Department of Microbial Proteomics, Institute for Microbiology, University Greifswald, Greifswald, Germany

    Dörte Becher

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Manske, C., Finsel, I., Hoffmann, C., Hilbi, H. (2018). Analysis of Legionella Metabolism by Pathogen Vacuole Proteomics. In: Becher, D. (eds) Microbial Proteomics. Methods in Molecular Biology, vol 1841. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8695-8_6

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  • DOI: https://doi.org/10.1007/978-1-4939-8695-8_6

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