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Purification of Pathogen Vacuoles from Legionella-infected Phagocytes
Published on: June 19, 2012
Pathogen vacuole purification from legionella-infected amoeba and macrophages
Christine Hoffmann1, Ivo Finsel, Hubert Hilbi
1Max von Pettenkofer Institute, Ludwig-Maximilians University, Munich, Germany.
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.
Insights
Researchers developed a new method to purify intact Legionella-containing vacuoles (LCVs) from host cells. This technique enables detailed biochemical analysis of LCVs and their associated bacterial effector proteins.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Legionella pneumophila is an intracellular pathogen that resides within a unique vacuole called the Legionella-containing vacuole (LCV).
- LCV formation relies on the Icm/Dot type IV secretion system, which delivers bacterial effector proteins into the host cell.
- These effectors can alter host cell processes, including vesicle trafficking, to promote bacterial survival.
Purpose of the Study:
- To establish a robust method for purifying intact Legionella-containing vacuoles (LCVs).
- To enable detailed biochemical and proteomic analysis of LCVs and their associated components.
Main Methods:
- A two-step purification protocol was developed for LCVs.
- Step 1: Immuno-magnetic separation using antibodies targeting LCV-specific bacterial effector proteins.
- Step 2: Further purification of enriched LCVs via density gradient centrifugation.
Main Results:
- The described method successfully purifies intact LCVs from both Dictyostelium discoideum and RAW 264.7 murine macrophages.
- Purified LCVs are suitable for subsequent proteomic and biochemical characterization.
Conclusions:
- This purification method provides a valuable tool for studying the molecular mechanisms of Legionella intracellular replication.
- Understanding LCV composition and function can lead to new strategies for combating Legionella infections.
