Differentially expressed miRNAs after Legionella pneumophila infection of human macrophages

Abstract

Legionella pneumophila ( L.p. ) is a common cause of severe community-acquired pneumonia. This pathogen replicates within alveolar macrophages and manipulates the host by interfering with intracellular signaling pathways. MicroRNAs (miRNAs) have emerged as critical regulators of mRNAs on posttranscriptional level. They are involved in the innate immunity and could have an important role during L.p. infections. The aim of this work was to identify deregulated miRNAs following infection by means of small RNA sequencing experiments and advanced bioinformatics analysis. Primary blood-derived human macrophages of healthy donors were infected for 24 and 48 hrs with a multiplicity of infection of 0.25. miRNA libraries were prepared for Illumina small RNA sequencing. The analysis of deep-sequencing data consisted of four main steps: mapping of reads against the human genome for detection of known and novel miRNAs according to a biogenesis model; normalization and differential expression analysis between different libraries; identification of miRNA isoforms, mutations and RNA editing products and selection and filtering of miRNA-associated targets. Our analysis revealed infection-specific and statistically significant changes of miRNA expression in human macrophages. miRNA deregulation seems to be due to transcriptional regulation of miRNA promoters. Overexpression or knock down experiments of miRNAs were performed for functional characterization and showed an influence of selected miRNAs on bacterial replication. In summary, the results have deepened our insight in the molecular interaction of L.p. and its host cells and might help to establish potential new gene candidates for diagnosis and therapy.