title: Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages.
abstract: Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.
authors.affiliation: Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg.
authors.forename: Anna Lena
authors.initials: AL
authors.lastname: Jung
authors.affiliation: Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Hoffmann
authors.affiliation: Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg.
authors.forename: Christina E
authors.initials: CE
authors.lastname: Herkt
authors.affiliation: Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg.
authors.forename: Christine
authors.initials: C
authors.lastname: Schulz
authors.affiliation: Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Bertrams
authors.affiliation: Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg; German Center for Lung Research; Department of Medicine, Pulmonary and Critical Care Medicine, University Medical Center Giessen and Marburg; bernd.schmeck@uni-marburg.de.
authors.forename: Bernd
authors.initials: B
authors.lastname: Schmeck
mesh_terms: D002460:Cell Line; D002462:Cell Membrane; D004797:Enzyme-Linked Immunosorbent Assay; D000067128:Extracellular Vesicles / Q000378:metabolism; D016952:Legionella pneumophila / Q000302:isolation & purification* / Q000378:metabolism; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism* / Q000382:microbiology; D014617:Vacuoles / Q000378:metabolism / Q000382:microbiology