Immunomagnetic Purification of Fluorescent Legionella-Containing Vacuoles

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.