Biogenesis and function of outer membrane vesicles shed by Legionella pneumophila

嗜肺军团菌是军团病的病原体，在细菌生长的所有阶段都会脱落外膜小泡(OMV)。我们对嗜肺乳杆菌的OMV进行了蛋白质组学分析，并收集了OMV通过蛋白水解酶和脂解酶活性促进细菌在感染组织内传播的证据。在设想的项目中，我们的目标是揭开OMV的生物发生并从功能上了解OMV与宿主结构的相互作用。对于第一个目标，将筛选一个转座子突变体文库，以鉴定具有异常高或低OMV产量的突变体。此外，我们还将比较OMVS和嗜肺乳杆菌外膜部分的脂类和内毒素组成。为了了解OMV如何促进细菌在肺内的传播过程，我们将对接触OMV后的人肺组织切片进行组织病理学分析和转录组分析。此外，我们将使用细胞外基质降解试验和基于Transwell的肺上皮屏障模型来明确OMV成分对组织破坏和嗜肺乳杆菌传播的贡献。在细胞水平上，我们将通过荧光显微镜和FRET光谱测量来分析OMV的摄取、细胞间转运以及OMV组分在宿主细胞接触后的影响。

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.