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Summary:
Legionella pneumophila (L.p.) is a gram-negative, intracellular pathogen and a common cause of severe community-acquired pneumonia. In humans, L.p. replicates primarily within alveolar macrophages. It manipulates vital host cell functions such as vesicle trafficking and gene expression by the secretion of over 300 effector proteins into the host cell cytosol. Thus, L.p. modifies its host cell to promote its own replication. An unbiased and global analysis of the molecular changes and biological processes that are associated with bacterial infections of human cells can provide new insights into host-pathogen interactions. Therefore, one goal of this study was to characterize expression changes of different RNA species in response to infection with L.p. in human primary blood-derived macrophages (BDMs) or differentiated THP-1 cells. This work is structured into two parts: (1) a functional study on how miRNA manipulations can alter L.p. replication in macrophages and (2) an in depth analysis of transcriptomic events in host and pathogen during infection. (1) In the last few decades, miRNAs have been established as critical modulators of immune function. Therefore, one aim of this study was to identify the miRNA profile of L.p.-infected macrophages and to determine the functional impact of a miRNA manipulation on L.p. replication. BDMs of healthy donors were infected with L.p. strain Corby. Small RNA sequencing revealed the miRNA profile in BDMs following L.p. infection. An upregulation of miR-146a and miR-155, as well as downregulation of miR-221 and miR-125b was validated by qPCR in macrophages. miRNA regulation in response to infection seems to be due to transcriptional regulation of miRNA promoters, since the histone acetylation levels at the promoter and the pri-miR expression correlated with the miRNA expression upon L.p.- infection. Overexpression and knock down experiments of miR-125b, miR-221 and miR-579 in combination were performed for functional characterization and showed an influence of all three miRNAs on bacterial replication. A SILAC approach revealed the protein MX1 as downregulated following simultaneous overexpression of all three miRNAs. MX1 is an interferon-induced GTP-binding protein important for antiviral defence. As shown by validation experiments, MX1 knockdown in macrophages led to an increased replication of L.p., as seen following overexpression of the miRNAs. Since in silico analysis predicted no binding sites for either miRNA in the 3’UTR of MX1, Ingenuity pathway analysis was performed to find the linking molecules. DDX58 (RIG-I), a sensor for cytosolic RNA, was validated as a target for miR-221, while the tumour suppressor TP53 was shown to be targeted by miR-125b via luciferase reporter assays. An siRNA-mediated knockdown of both, TP53 and DDX58, respectively, led to an enhanced replication of L.p. in macrophages. Thus, DDX58 and TP53 were validated as linking molecules between the three miRNAs and MX1. Additionally, the aforementioned SILAC approach revealed a downregulation of LGALS8 which was later validated as a target of miR-579. LGALS8 is a cytosolic lectin which binds carbohydrates and localizes to damaged vesicles. Knockdown of LGALS8 enhanced intracellular replication in macrophages. Thus, MX1 and LGALS8 were identified as targets of the three miRNAs (miR-125b, miR-221, miR-579) and to be responsible for the restriction of L.p. replication within human macrophages. (2) The transcriptional profile of L.p. during the course of infection in human macrophages was next to be established. Dual RNA-Sequencing was performed to determine the regulation of coding and non-coding RNA species during the course of infection of both, host and pathogen, simultaneously. After adaptation and optimization of existing protocols, macrophages were infected using a GFP-expressing L.p. strain Corby. To separate infected cells (gfp+) from the non-invaded bystander cells (gfp-), flow cytometry sorting was performed. Furthermore, Pam3CSK4 was used to generate TLR2-activated cells. RNA from all different samples, and also RNA from cultivated Legionella, was sequenced. Differential gene expression analysis was performed using DESeq2 resulting in 4,144 differentially expressed human genes (across multiple conditions) and 2,707 differentially expressed Legionella genes (across two time points). The DESeq analysis of the separated RNA fractions from host cells revealed differentially expressed mRNAs (3,504), lncRNAs (495), and miRNAs (145). 1,128 differentially expressed genes were exclusively significantly regulated in invaded cells (gfp+ at 8 and 16 h). Some of these were validated via qPCR including BCL10, SOD1, IRS1, CYR61, ATG5, RND3 and JUN. In addition, the simultaneous upregulation of the genes ZFAND2A and HSPA1 in the bystander and in Legionella-invaded cells was validated. The analysis of the bacterial mRNAs revealed a switch of gene usage, i.e. inverse regulation at 8 and 16 h post infection. This switch included genes which are involved in iron metabolism, stress response, glycolysis and lipid biosynthesis. Hence, differentially expressed genes within different growth phases of the infection cycle were identified. This dataset is the first of its kind to cover a respiratory pathogen. The dual RNA-Sequencing performed in this study provides data to encapsulate the RNA landscape of coding and non-coding RNAs in pathogen and host. In summary, the results have deepened our insight into the infection process and the molecular interaction of L.p. and its host cells and will help to understand the complex interplay between host and pathogen by allowing for the in silico re-construction of an RNA interaction network. Furthermore, the present study will help to establish potential new candidates for diagnosis and therapy.

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