Abstract
Legionella pneumophila replicates intracellularly in environmental and immune phagocytes within a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is strictly dependent on the Icm/Dot type IV secretion system and the translocation of “effector” proteins into the cell. Some effector proteins decorate the LCV membrane and subvert host cell vesicle trafficking pathways. Here we describe a method to purify intact LCVs from Dictyostelium discoideum amoebae and RAW 264.7 murine macrophages. The method comprises a two-step protocol: first, LCVs are enriched by immuno-magnetic separation using an antibody against a bacterial effector protein specifically localizing to the LCV membrane, and second, the LCVs are further purified by density gradient centrifugation. The purified LCVs can be characterized by proteomics and other biochemical 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
- T4SS:
-
Type IV secretion system
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Acknowledgments
This work was supported by the Max von Pettenkofer Institute, Ludwig-Maximilians University Munich, and the German Research Foundation (BMBF “Medical Infection Genomics”, HI 1511/3-1).
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Hoffmann, C., Finsel, I., Hilbi, H. (2013). Pathogen Vacuole Purification from Legionella-Infected Amoeba and Macrophages. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 954. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-161-5_18
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DOI: https://doi.org/10.1007/978-1-62703-161-5_18
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