A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1 Christina E. Herkt, a Brian E. Caffrey, b Kristin Surmann, c Sascha Blankenburg, c Manuela Gesell Salazar, c Anna L. Jung, a Stefanie M. Herbel, a Kerstin Hoffmann, a Leon N. Schulte, d Wei Chen, e Alexandra Sittka-Stark, a Uwe Völker, c Martin Vingron, b Annalisa Marsico, b,f Wilhelm Bertrams, a Bernd Schmeck a,g,h,i a Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps University Marburg, Marburg, Germany b Computational Molecular Biology, Max Planck Institute for Molecular Genetics, Berlin, Germany c Department of Functional Genomics, University Medicine Greifswald, Greifswald, Germany d Institute for Lung Research/iLung, Research Group RNA-Biology of Inflammation and Infection, Philipps University, Marburg, Germany e Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong, China f Institute of Computational Biology, Helmholtz Zentrum Muenchen, Neuherberg, Germany g Department of Medicine, Pulmonary and Critical Care Medicine, University Medical Center Giessen and Marburg, Philipps University, Marburg, Germany h Center for Synthetic Microbiology (SYNMIKRO), Philipps University Marburg, Marburg, Germany i German Center for Infection Research (DZIF), partner site Giessen-Marburg-Langen, Marburg, Germany Annalisa Marsico, Wilhelm Bertrams, and Bernd Schmeck contributed equally to this article. ABSTRACT Legionella pneumophila is an important cause of pneumonia. It invades alveolar macrophages and manipulates the immune response by interfering with signaling pathways and gene transcription to support its own replication. MicroRNAs (miRNAs) are critical posttranscriptional regulators of gene expression and are in- volved in defense against bacterial infections. Several pathogens have been shown to exploit the host miRNA machinery to their advantage. We therefore hypothesize that macrophage miRNAs exert positive or negative control over Legionella intracel- lular replication. We found significant regulation of 85 miRNAs in human macro- phages upon L. pneumophila infection. Chromatin immunoprecipitation and se- quencing revealed concordant changes of histone acetylation at the putative promoters. Interestingly, a trio of miRNAs (miR-125b, miR-221, and miR-579) was found to significantly affect intracellular L. pneumophila replication in a cooperative manner. Using proteome-analysis, we pinpointed this effect to a concerted down- regulation of galectin-8 (LGALS8), DExD/H-box helicase 58 (DDX58), tumor protein P53 (TP53), and then MX dynamin-like GTPase 1 (MX1) by the three miRNAs. In sum- mary, our results demonstrate a new miRNA-controlled immune network restricting Legionella replication in human macrophages. IMPORTANCE Cases of Legionella pneumophila pneumonia occur worldwide, with potentially fatal outcome. When causing human disease, Legionella injects a plethora of virulence factors to reprogram macrophages to circumvent immune defense and create a replication niche. By analyzing Legionella-induced changes in miRNA expres- sion and genomewide chromatin modifications in primary human macrophages, we identified a cell-autonomous immune network restricting Legionella growth. This net- work comprises three miRNAs governing expression of the cytosolic RNA receptor DDX58/RIG-I, the tumor suppressor TP53, the antibacterial effector LGALS8, and MX1, which has been described as an antiviral factor. Our findings for the first time link TP53, LGALS8, DDX58, and MX1 in one miRNA-regulated network and integrate them into a functional node in the defense against L. pneumophila. KEYWORDS miRNA, infection, macrophage, MX1, bacteria, galectin-8, Legionella Citation Herkt CE, Caffrey BE, Surmann K, Blankenburg S, Gesell Salazar M, Jung AL, Herbel SM, Hoffmann K, Schulte LN, Chen W, Sittka-Stark A, Völker U, Vingron M, Marsico A, Bertrams W, Schmeck B. 2020. A microRNA network controls Legionella pneumophila replication in human macrophages via LGALS8 and MX1. mBio 11:e03155-19. https://doi.org/ 10.1128/mBio.03155-19. Editor Michele S. Swanson, University of Michigan—Ann Arbor Copyright © 2020 Herkt et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Bernd Schmeck, bernd.schmeck@uni-marburg.de. Received 20 December 2019 Accepted 28 February 2020 Published RESEARCH ARTICLE Host-Microbe Biology crossm March/April 2020 Volume 11 Issue 2 e03155-19 ® mbio.asm.org 1 24 March 2020 on July 23, 2020 by guest http://mbio.asm.org/ Downloaded from