microRNA-125a-3p is regulated by MyD88 in Legionella pneumophila infection and targets NTAN1

PLoS One. 2017 Apr 26;12(4):e0176204. doi: 10.1371/journal.pone.0176204. eCollection 2017.

Abstract

Background: Legionella 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.

Methods: WT 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.

Results: L. 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.

Conclusion: Taken 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.

MeSH terms

  • 3' Untranslated Regions
  • Amidohydrolases / antagonists & inhibitors
  • Amidohydrolases / genetics
  • Amidohydrolases / metabolism*
  • Animals
  • Base Sequence
  • Chemokine CXCL1 / analysis
  • Genotype
  • Interleukin-6 / analysis
  • Interleukin-6 / genetics
  • Interleukin-6 / metabolism
  • Legionella pneumophila / physiology*
  • Legionnaires' Disease / genetics
  • Legionnaires' Disease / pathology
  • Macrophages / cytology
  • Macrophages / metabolism
  • Macrophages / microbiology
  • Mice
  • Mice, Knockout
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Myeloid Differentiation Factor 88 / deficiency
  • Myeloid Differentiation Factor 88 / genetics*
  • RAW 264.7 Cells
  • Sequence Alignment
  • Signal Transduction
  • Transcriptome

Substances

  • 3' Untranslated Regions
  • Chemokine CXCL1
  • Interleukin-6
  • MicroRNAs
  • Myd88 protein, mouse
  • Myeloid Differentiation Factor 88
  • Amidohydrolases
  • N-terminal asparagine amidohydrolase

Grants and funding

The work was in part supported by grants from the Deutsche Forschungsgesellschaft, (TR84), Bundesministerium für Bildung und Forschung (e:bio 0316175A, e:Med 01X1304E, Progress FKZ 01KI1010K) and Hessisches Ministerium für Wissenschaft und Kunst (LOEWE UGMLC and LOEWE Medical RNomics) to BS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.