miRNAs und bioreaktive Substanzen mit Einfluss auf Legionella pneumophila verursachte Infektionen

Legionella pneumophila ist ein gramnegatives Bakterium, das sich als intrazelluläres Pathogen, neben Amöben als natürlichen Wirt, auch in alveolaren Makrophagen vermehren kann. Dort löst es das Pontiac-Fieber, oder in schwerwiegenderen Fällen die Legionärskrankheit aus, welche durch eine schwere Pne...

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Bibliographic Details
Main Author: Ringshandl, Stephan
Contributors: Becker, Anke (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2022
Online Access:PDF Full Text
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Legionella pneumophila is a gram-negative bacterium which acts as an intracellular pathogen. Its natural host are amoeba but it can also replicate in human alveolar macrophages. It causes the so called pontiac fever, or in severe cases the so called Legionnaires' disease. This disease is a form atypical pneumonia. The aim of this work was to develop and establish a high-throughput method to facilitate the testing of a large number of biological and chemical samples within the scope of Legionella infection. With the analysis of these samples, it was the goal to increase the knowledge about the interaction between the Legionella bacteria and macrophages. Samples originated from a miRNA library as well as a self-created set of bioreactive substances. Dependent on the treatment the output of the high-throughput method was the ratio between infected and non-infected cells as well as the intensity of infection. As an experimental model, differentiated THP-1 cells were used to mimic alveolar macrophages that were subsequently infected with the Legionella pneumophila strain Corby. Even though the analysis of the miRNA library displayed technical difficulties, miRNA-101-3p, miRNA-106a-5p and miRNA-107 could be identified and validated to have a stimulating effect on legionella infection. Mass spectrometry was performed to shed light on which proteins are regulated by these miRNAs and how this results in the increase of infection. Several classes of proteins and pathways could be identified to be regulated upon miRNA transfection and could be involved in the observed stimulating effect on infection. The analysis of bioreactive substances revealed five novel substances that were previously not known in this infection context. These were 25-hydroxy cholesterol, H-89, fluoxetine, forskoline and W-7. As amitriptylin and fluoxetin act as inhibitors of acid sphingomyelinase two other inhibitors of this protein were subsequently tested. Benztropine as well as perhexiline showed a similar strong downregulation of infection, which highlights the potential relevance of this protein during infection. This is further reinforced as macrophages as well as bacteria seem to regulate the acid sphingomyelinase. In summary, a high-throughput method was established that, with some limitations, facilitates testing of an increased number of samples on their effect on the Legionella pneumophila – macrophage interaction. Three miRNAs and five bioreactive substances were found to have an effect in this setting and regulated protein classes and pathways will be a substantial basis for further scientific work.