Transregional Collaborative Research Center SFB-TR 84 - “Innate Immunity of the Lung: Mechanisms of Pathogen Attack and Host Defence in Pneumonia“

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C1-Subproject

MicroRNA in the pathogenesis of Legionella pneumonia
(Schmeck / Vingron)

 

Legionella pneumophila (L. pneumophila) is an important intracellular pathogen causing pneumonia. It is highly adapted to intracellular replication and manipulates important host cell functions like vesicle trafficking and gene expression with specific virulence factors. Small non-coding RNAs from mammals (microRNA) and bacteria (sRNA) have been found to modulate infection processes.

In the first funding period, we performed deep sequencing of different RNA fractions from L. pneumophila infected human macrophages. From these data we identified and validated (1) changes in host cell microRNA (miRNA) expression, and (2) putative L. pneumophila sRNAs with a newly developed preliminary bioinformatics approach for small RNA gene finding. In a next step, we predicted in silico miRNAs that could influence bacterial replication as well as inflammatory host cell response and validated them experimentally. To elucidate the underlying regulatory mechanisms, we characterized so far unknown miRNA promoters by combining a newly developed computational approach for miRNA promoter recognition with the evidence from miRNA sequencing and chromatin immunoprecipitation sequencing (ChIP-seq) data. Bioinformatic analysis of ChIP-seq data lead to the recognition of TNFAIP2 as a host cell factor involved in intracellular replication of L. pneumophila.

In addition, preliminary data and in silico analysis showed (a) a complex regulatory crosstalk between human macrophages and alveolar epithelial cells in L. pneumophila infection, (b) an exchange of miRNA-containing exosomes between these cells, and (c) epithelial cell activation by L. pneumophila outer membrane vesicles. Therefore, we developed the hypothesis that the innate immune response against L. pneumophila infection in the alveolar compartment is regulated in part by microvesicle-packed small RNAs from host or bacterial origin. To test this hypothesis, we will develop a dual-RNA-seq approach to profile miRNA, sRNA and mRNA expression of host cells, bacteria, and microvesicles during infection. Furthermore, we will complement and validate the transcriptomic data by an unbiased host cell proteomics technique and further functional assays. Based on these data, we aim to achieve an even more comprehensive picture by identifying new L. pneumophila factors directly influencing miRNA expression. By high-end bioinformatics we aim at constructing an intra- and inter-cellular regulatory network of the alveolar compartment, and validate our findings both in human lung tissue culture and murine infection models.