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Purification of Pathogen Vacuoles from Legionella-infected Phagocytes
Published on: June 19, 2012
Isolation of Legionella-containing vacuoles by immuno-magnetic separation
Simon Urwyler1, Ivo Finsel, Curdin Ragaz
1Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland.
Abstract:
The environmental bacterium Legionella pneumophila naturally parasitizes free-living amoebae. L. pneumophila is an opportunistic human pathogen that grows in macrophages, thus causing a life-threatening pneumonia termed Legionnaires' disease. The bacteria replicate intracellularly in environmental and immune phagocytes within a unique compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is a complex and robust process involving >150 secreted bacterial effector proteins, which are believed to subvert host cell signaling and vesicle trafficking pathways. This unit describes a simple approach to purify intact LCVs from Dictyostelium discoideum amoebae. The method comprises a two-step purification protocol that includes immuno-magnetic separation by means of an antibody against an effector protein specifically binding to LCVs, followed by density gradient centrifugation. The use of D. discoideum producing a fluorescent LCV marker and fluorescently labeled L. pneumophila allow tracking the enrichment of LCVs by light microscopy.
Insights
Researchers developed a simple method to purify intact Legionella-containing vacuoles (LCVs) from amoebae. This technique aids in studying how Legionella pneumophila, a pathogen causing Legionnaires
Area of Science:
- Microbiology
- Cell Biology
- Pathogen Research
Background:
- Legionella pneumophila is an environmental bacterium and opportunistic pathogen causing Legionnaires' disease by parasitizing amoebae and human macrophages.
- L. pneumophila replicates within a unique compartment, the Legionella-containing vacuole (LCV), which is formed via complex subversion of host cell pathways by bacterial effector proteins.
Purpose of the Study:
- To describe a straightforward method for purifying intact LCVs from Dictyostelium discoideum.
- To facilitate the study of LCV formation and the role of effector proteins in host-pathogen interactions.
Main Methods:
- A two-step purification protocol involving immuno-magnetic separation using an anti-effector protein antibody.
- Density gradient centrifugation to further purify the LCVs.
- Utilized fluorescent markers in D. discoideum and L. pneumophila for tracking LCV enrichment via light microscopy.
Main Results:
- Successfully purified intact LCVs from D. discoideum using the described method.
- Demonstrated the efficacy of immuno-magnetic separation and density gradient centrifugation for LCV isolation.
- Visual tracking confirmed the enrichment of LCVs throughout the purification process.
Conclusions:
- The developed method provides a simple and effective means to isolate intact LCVs.
- This purification technique will be valuable for future research into L. pneumophila pathogenesis and LCV biology.
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