Isolation of Legionella-containing vacuoles by immuno-magnetic separation.
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.
Urwyler
Simon
S
Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland.
Finsel
Ivo
I
Ragaz
Curdin
C
Hilbi
Hubert
H
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D004023:Dictyostelium / Q000166:cytology / Q000254:growth & development / Q000382:microbiology; D018189:Immunomagnetic Separation / Q000379:methods*; D007875:Legionella / Q000166:cytology / Q000254:growth & development / Q000302:isolation & purification* / Q000502:physiology; D008856:Microscopy, Fluorescence; D014617:Vacuoles / Q000382:microbiology*