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Immunomagnetic Purification of Fluorescent Legionella-Containing Vacuoles

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Dictyostelium discoideum Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 983))

Abstract

Protozoa are natural reservoirs of the environmental bacterium Legionella pneumophila. Upon inhalation of Legionella-laden aerosols, the amoeba-resistant bacteria replicate within human alveolar macrophages causing the severe pneumonia “Legionnaires’ disease.” Within host cells, including Dictyostelium discoideum, L. pneumophila establishes a custom-tailored compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system and involves a plethora of “effector” proteins, some of which specifically decorate the LCV membrane. This unique feature of LCVs is exploited to isolate the pathogen vacuole by immunomagnetic separation using an antibody against the effector protein SidC. LCV purity is further increased by a subsequent density gradient centrifugation step. The use of red fluorescent L. pneumophila and D. discoideum producing the LCV marker calnexin-GFP allows following the purification by fluorescence microscopy.

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Acknowledgments

This work was supported by the Max von Pettenkofer Institute, Ludwig-Maximilians University Munich, the “Bundesministerium für Bildung und Forschung” (BMBF “Medical Infection Genomics”), and the German Research Foundation (DFG, HI 1511/3-1).

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Authors and Affiliations

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

    Ivo Finsel, Christine Hoffmann & Hubert Hilbi

Authors
  1. Ivo Finsel
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  2. Christine Hoffmann
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  3. Hubert Hilbi
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Corresponding author

Correspondence to Hubert Hilbi .

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Editors and Affiliations

  1. , Centre for Biochemistry, University of Cologne, Joseph-Stelzmann-Str. 52, Köln, 50931, Nordrhein-Westfalen, Germany

    Ludwig Eichinger

  2. , Centre for Biomedical Research, University of Hull, Hull, HU6 7RX, Montserrat

    Francisco Rivero

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Finsel, I., Hoffmann, C., Hilbi, H. (2013). Immunomagnetic Purification of Fluorescent Legionella-Containing Vacuoles. In: Eichinger, L., Rivero, F. (eds) Dictyostelium discoideum Protocols. Methods in Molecular Biology, vol 983. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-302-2_24

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  • DOI: https://doi.org/10.1007/978-1-62703-302-2_24

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-301-5

  • Online ISBN: 978-1-62703-302-2

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