Biogenesis and function of outer membrane vesicles shed by Legionella pneumophila

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Legionella pneumophila, the causative agent of Legionnaires‘ disease, sheds outer membrane vesicles (OMVs) during all phases of bacterial growth. We have performed a proteome analysis of OMVs from L. pneumophila and collected evidence that OMVs promote bacterial dissemination within infected tissues by proteolytic and lipolytic enzyme activities. In the envisioned project our aims are to unravel the biogenesis of OMVs and to functionally understand OMV interactions with host structures. For the first aim a transposon mutant library will be screened to characterize mutants with exceptionally high or low OMV production. In addition we will compare the lipid and LPS composition of OMVs and the outer membrane fraction of L. pneumophila. To understand how OMVs contribute to the process of bacterial dissemination within the lung, we will perform histopathologic analyses of human lung tissue sections and a transcriptome analysis of the tissue after OMV exposure. Additionally, we will use an extracellular matrix degradation assay and a transwell-based lung epithelium barrier model to specify the contribution of OMV constituents to tissue destruction and to L. pneumophila dissemination. On the cellular level we will analyze uptake of OMVs, intercellular transport and effects of OMV constitutients after host cell contact by fluorescence microscopy and FRET spectroscopy measurements.