<em>Legionella pneumophila</em> 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.

Proinflammatory Microenvironment During Kingella kingae Infection Modulates Osteoclastogenesis

“…It is in contrast with the previous report, in which K. kingae-derived OMV can promote a proinflammatory microenvironment by releasing IL-6 and granulocyte-M-CSF secretion from fibroblast, synoviocytes, and osteoblasts (4). The plausible dual OMV role in opposite outcome has been extensively reported for other Gram-negative bacteria (41)(42)(43)(44)(45)(46). However, differences in K. kingae strain and/or in the preparation of OMV could also be implicated (4).…”

Heterogeneous Populations of Outer Membrane Vesicles Released from Helicobacter Pylori SS1 with Distinct Biological Properties

“…Macrophages treated with OMVs from P. gingivalis were found to produce high levels of IFNβ, TNFα, IL-10, IL-12p70, and IL-6 [75] . Moreover, OMVs isolated from Legionella pneumophila (L.pneumophila) have elected a pro-inflammatory response in macrophages [76] . Similar observations were found in the current work as the results showed the ability of ExHp-HBS, ExHp-FBS, ExHp-CD, and ExHp-Chlos to produce IL-4 and IL-10 that represents the election of antiinflammatory response.…”

Small RNAs in Outer Membrane Vesicles and Their Function in Host-Microbe Interactions

“…OMVs can be typically separated from cell-free culture supernatants, liquid cultures, or body fluids (Jung et al, 2017) by differential centrifugation steps, filtration (through 0.22-0.45 μm porosity filters), and ultracentrifugation (Moon et al, 2012;Dean et al, 2019) although this isolation method does not completely remove small contaminants such as protein aggregates or membrane debris (Klimentova and Stulik, 2015). Gel filtration and density gradient ultracentrifugation are other two widely employed procedures to isolate and purify Gram-negative OMVs (Nevot et al, 2006;Chutkan et al, 2013;van de Waterbeemd et al, 2013;Antenucci et al, 2017;Kohl et al, 2018).…”