Jung Anna Lena, Hoffmann Kerstin, Herkt Christina E, Schulz Christine, Bertrams Wilhelm, Schmeck Bernd
Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg.
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;
J Vis Exp. 2017 Feb 22(120):55146. doi: 10.3791/55146.
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.
细菌能够通过各种分泌系统分泌多种分子。除了将分子分泌到细胞外空间或直接分泌到另一个细胞中，革兰氏阴性菌还能形成外膜囊泡（OMV）。这些膜囊泡能够将其携带的物质远距离运输，并且携带的物质可免受蛋白酶和核酸酶的降解。嗜肺军团菌是一种细胞内革兰氏阴性病原体，可引起严重形式的肺炎。在人类中，它感染肺泡巨噬细胞，在其中阻断溶酶体降解并形成特殊的复制液泡。此外，嗜肺军团菌在各种生长条件下都会产生OMV。为了了解OMV在人类巨噬细胞感染过程中的作用，我们建立了一个从液体培养物中纯化细菌膜囊泡的方案。该方法基于差速超速离心。随后对富集的OMV进行蛋白质和脂多糖（LPS）含量分析，然后用于处理人类单核细胞系或小鼠骨髓来源的巨噬细胞。通过酶联免疫吸附测定分析这些细胞的促炎反应。此外，分析后续感染中的变化。为此，通过菌落形成单位测定研究嗜肺军团菌在巨噬细胞中的细菌复制。在此，我们描述了一种通过超速离心从液体培养物中纯化嗜肺军团菌OMV及其对巨噬细胞促炎潜力的下游分析的详细方案。
