Einfluss von Legionella pneumophila outer membrane vesicles auf die bakterielle Replikation in Makrophagen

Gramnegative Bakterien treten über die Sekretion verschiedenster Moleküle mit ihrer Umwelt in Kontakt. Die Freisetzung von Proteinen und Nukleinsäuren kann aber nicht nur über die bakteriellen Sekretionssysteme vermittelt werden, sondern auch über outer membrane vesicles (OMVs) erfolgen. Diese klein...

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Bibliographic Details
Main Author: Jung, Anna Lena
Contributors: Schmeck, Bernd (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2016
Online Access:PDF Full Text
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Gram-negative bacteria secrete various molecules to communicate with their environment. The secretion of proteins and nucleic acids cannot only be mediated via secretion systems, but also via outer membrane vesicles (OMVs). These small, spheroid membrane vesicles are produced by all Gram-negative bacteria and can deliver their cargo via long distances, as the transported proteins are protected from proteolytic degradation. OMVs from pathogenic bacteria can activate the innate immune system and can be immuno-modulatory. Legionella pneumophila (L. pneumophila) is a causative agent of severe, atypical pneumonia. These Gram-negative, rod-shaped bacteria occur ubiquitously and replicate in amoeba. Inhalation of L. pneumophila in human lungs leads to the infection of alveolar macrophages. In this work, the influence of L. pneumophila OMVs on human and murine macrophages was analyzed. Differentiated THP-1 cells were incubated with increasing OMV doses, which led to a TLR2-dependent pro-inflammatory activation of the cells. The cytokine secretion was induced through TLR2 activation and subsequent signal transduction via NF-κB. Furthermore, the impact of OMVs on a following infection with L. pneumophila was examined. In an early phase of infection, OMV pre-treatment of macrophages resulted in reduced bacterial replication, whereas at a later time point, replication was increased. Macrophages were more permissive for an infection and had more Legionella-containing vacuoles per cell. The increased bacterial replication was independent of the transported proteins and nucleic acids, but could be associated with TLR2 activation. Additionally, it could be shown that the intracellular signal transduction was mediated via IRAK-1 and the nuclear translocation of p65. Macrophages responded with a reduced expression of pro-inflammatory genes to an infection with L. pneumophila when they were pre-incubated with OMVs. The induction of the anti-inflammatory microRNA-146a (miR-146a) after OMV stimulation was TLR2- and NF-κB-mediated and resulted in a degradation-dependent absence of IRAK-1 in macrophages. Transfection experiments could demonstrate that the overexpression of miR-146a as well as the siRNA-mediated knockdown of IRAK-1 could induce L. pneumophila replication. In summary, the experiments demonstrated that L. pneumophila OMVs are potent pro-inflammatory stimulators for human und murine macrophages and that they activate them via TLR2. The subsequent infection with L. pneumophila results in an increased bacterial replication which depends on miR-146a and IRAK-1. OMVs can hereby promote the bacterial replication and possibly lead to the spreading of the infection in human lungs.