Abstract
BackgroundLegionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. It is highly adapted to intracellular replication and manipulates host cell functions like vesicle trafficking and mRNA translation to its own advantage. However, it is still unknown to what extent microRNAs (miRNAs) are involved in the Legionella-host cell interaction.MethodsWT and MyD88-/- murine bone marrow-derived macrophages (BMM) were infected with L. pneumophila, the transcriptome was analyzed by high throughput qPCR array (microRNAs) and conventional qPCR (mRNAs), and mRNA-miRNA interaction was validated by luciferase assays with 3´-UTR mutations and western blot.ResultsL. pneumophila infection caused a pro-inflammatory reaction and significant miRNA changes in murine macrophages. In MyD88-/- cells, induction of inflammatory markers, such as Ccxl1/Kc, Il6 and miR-146a-5p was reduced. Induction of miR-125a-3p was completely abrogated in MyD88-/- cells. Target prediction analyses revealed N-terminal asparagine amidase 1 (NTAN1), a factor from the n-end rule pathway, to be a putative target of miR-125a-3p. This interaction could be confirmed by luciferase assay and western blot.ConclusionTaken together, we characterized the miRNA regulation in L. pneumophila infection with regard to MyD88 signaling and identified NTAN1 as a target of miR-125a-3p. This finding unravels a yet unknown feature of Legionella-host cell interaction, potentially relevant for new treatment options.
Highlights
Legionella pneumophila (L. pneumophila), a gram-negative bacterium, is described as a causative pathogen of lung inflammation and life-threatening pneumonia [1]
WT and MyD88-/- murine bone marrow-derived macrophages (BMM) were infected with L. pneumophila, the transcriptome was analyzed by high throughput qPCR array and conventional qPCR, and mRNA-miRNA interaction was validated by luciferase assays with 3 ́-UTR mutations and western blot
We characterized the miRNA regulation in L. pneumophila infection with regard to MyD88 signaling and identified NTAN1 as a target of miR-125a-3p
Summary
Legionella pneumophila (L. pneumophila), a gram-negative bacterium, is described as a causative pathogen of lung inflammation and life-threatening pneumonia [1]. L. pneumophila form their replication niche inside macrophages, the Legionella-containing vacuole (LCV), where they replicate until host cell lysis. Infection of macrophages with L. pneumophila leads to a broad activation of signaling pathways, triggered by both extracellular and intracellular receptor molecules, such as Toll-like receptors (TLRs) that recognize pathogen associated molecular patterns (PAMPs). It is still unknown to what extent miRNAs are involved in L. pneumophila infection, either as part of the host response, or as part of host cell rewiring by the pathogen. We characterized miRNA expression in murine macrophages upon L. pneumophila infection in the context of a MyD88 knockout to shed more light on this particular aspect of host-pathogen interaction. It is still unknown to what extent microRNAs (miRNAs) are involved in the Legionella-host cell interaction
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