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. 2020 Mar 24;11(2):e03155-19.
doi: 10.1128/mBio.03155-19.

A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1

Affiliations
Free PMC article

A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1

Christina E Herkt et al. mBio. .
Free PMC 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 involved 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 intracellular replication. We found significant regulation of 85 miRNAs in human macrophages upon L. pneumophila infection. Chromatin immunoprecipitation and sequencing 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 downregulation 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 summary, 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 expression and genomewide chromatin modifications in primary human macrophages, we identified a cell-autonomous immune network restricting Legionella growth. This network 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: DDX58; Legionella pneumophila; MX1; RIG-I; TP53; bacteria; galectin-8; infection; infectious disease; inflammation; macrophage; macrophages; miRNA; microRNA.

Figures

FIG 1
FIG 1
Differentially expressed miRNAs in blood-derived macrophages after L. pneumophila infection. BDMs from three different donors were infected with L. pneumophila at an MOI of 0.25 or left untreated as a control (ctr). Samples were taken 24 and 48 h postinfection. RNA was isolated and used for library preparation and enriched for small RNAs using a TruSeq small RNA kit. Sequencing was performed in a multiplexed run of 1 × 51 cycle +7 (index). (A) Heatmap of the miRNA log2 fold change over the respective controls. The expression of distinct upregulated (B and C) and downregulated (D and E) miRNAs in BDMs and THP-1 cells after L. pneumophila infection was validated by qPCR using TaqMan assays and are displayed as log2 fold changes. Boxes show the upper and lower quartiles with medians and whiskers indicating minimal and maximal values of five to nine independent biological replicates for dysregulated miRNAs. Paired t tests were performed. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ .001; ****, P ≤ 0.0001 (compared to the corresponding control).
FIG 2
FIG 2
Infection-related chromatin changes on miRNA promoters. The alterations of the acetylation pattern at the miRNA promoter regions of miR-155, miR-146a, miR-221, and miR-125b after L. pneumophila infection in BDMs were investigated. BDMs were infected with L. pneumophila for 1 h, and ChIP was performed using a pan-ac-H4-antibody (based on data published by Du Bois et al. [32]). (A) After sequencing, the enrichment of H4 acetylation at the miR promoters was analyzed. As confirmation for active sites in the promoter region, IP data for H3K4me3 and H3K27Ac provided by the Encode UCSC browser are shown. Horizontal dark green lines indicate the genomic locations of the promoter regions (2,000 bp) for the four analyzed miRNAs corresponding to given coordinates. Circles highlight the mature transcript of each miRNA. The expression of the pri-miRs (B, C, D, and E) in BDMs after L. pneumophila infection (MOI of 0.25 or 0.5) was examined by qPCR and is displayed as log2 fold changes. Boxes show the upper and lower quartiles, with medians (when n = 3 independent biological replicates) and whiskers indicating minimal and maximal values (when n = 4 independent biological replicates). Paired t tests were performed. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (compared to the corresponding control).
FIG 3
FIG 3
Influence of miRNAs on bacterial replication in macrophages through downregulation of MX1 protein. THP-1 cells were transfected with miR-125b/-221/-579 mimics or inhibitors plus scrambled control (scr) at a final concentration of 30 nM. At 24 h posttransfection, THP-1 cells were activated with PMA for another 24 h and then infected with L. pneumophila at an MOI of 0.5. (A) Bacterial replication was determined by a CFU assay at the indicated time points. The bacterial yield is depicted as the percent relative to CFU count of scr-transfected cells at every time point, and values are shown as means ± the standard errors of the mean (SEM) of three to four independent biological replicates. (B) SILAC-supported quantitative proteomics was used to analyze the proteome of THP-1 cells treated as in panel A. (C) The numbers of proteins that were downregulated after simultaneous precursor transfection of all three miRNAs compared to scramble transfection are shown. (D) The relative numbers of MX1-positive cells upon miRNA transfection were analyzed by flow cytometry. Boxes show the upper and lower quartiles with median of three independent biological replicates. Significance was assessed by two-way ANOVA with Tukey’s (for CFU assay) or Sidak’s (for MX1 staining) correction. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (compared to the corresponding control). ##, P ≤ 0.01 (for global treatment effect).
FIG 4
FIG 4
Downregulation of MX1 enhances L. pneumophila replication in macrophages. THP-1 cells or BDMs were transfected with an siRNA-pool targeting MX1 (siMX1) or with scrambled siRNA as a control (scr). At 24 h posttransfection, THP-1 cells were stimulated with PMA (20 nM) for another 24 h. Differentiated THP-1 cells or BDMs were infected as shown. Knockdown of MX1 protein in THP-1 cells (A) and BDMs (B) was verified and quantified by Western blotting. CFU were determined 24, 48, and 72 h postinfection after MX1 knockdown in THP-1 cells (C) or BDMs (D). Mean values ± the SEM of three to seven independent biological experiments are depicted. Significance was assessed by two-way ANOVA with Sidaks correction. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (compared to the corresponding control). #, P ≤ 0.05, ##, P ≤ 0.01 (for global treatment effects).
FIG 5
FIG 5
MX1 is not directly targeted by the miRNA-pool. IPA was performed to investigate the connection between the miRNA pool (miR-125b, miR-221, and miR-579) and the MX1 protein. Filters were set to only include experimentally observed or high-confidence-predicted miRNA-mRNA interaction partners. Output was limited to 13 total candidates. The newly found miRNA targets were interconnected using all available data sources in IPA with restriction to experimentally validated or high-confidence-predicted interactions. Direct or indirect relationships between molecules are indicated by solid or dashed lines, respectively. Blue lines depict positive regulations, while red lines indicate negative regulations. Molecule classes (cytokine, enzyme, ion channel, ligand-dependent nuclease, phosphatase, transcription factor, other, or mature miRNAs) are indicated by distinct symbols.
FIG 6
FIG 6
DDX58 is targeted by miR-221 and influences bacterial replication. THP-1 cells were transfected either with miR-221 mimic, miR-579 mimic, or the miRNA mimic pool (miR-125b, miR-221, and miR-579) at a final concentration of 30 nM. As a control, a scrambled precursor (scr) was transfected. At 24 h posttransfection, THP-1 cells were activated with PMA for another 24 h and then infected with L. pneumophila at an MOI of 0.5. (A) DDX58 expression was determined via qPCR and is displayed as the log2 fold change. Luciferase reporter assay analyses were carried out in HEK-293T cells. The plasmid harbored either the wild-type (DDX58WT) or the mutated (DDX58mut) version of the 3′ UTR of DDX58. (B) Ratios of Renilla and firefly luciferase luminescence were normalized to the vector without insert. (C) Intracellular MX1 upon miRNA pool transfection was quantified by flow cytometric analysis, and the relative numbers of MX1-positive cells were calculated. (D) BDMs were transfected with a small interfering RNA-pool targeting DDX58 (siDDX58) or with a scrambled siRNA as control (scr) and infected at an MOI of 0.5 or left untreated. The downregulation of DDX58 expression with siRNA was verified by qPCR and is displayed as the log2 fold change. (E) The CFU of Legionella were determined 24, 48, and 72 h postinfection after a knockdown of DDX58. Boxes show the upper and lower quartiles, with medians (when n = 3 independent biological replicates) and whiskers indicating minimal and maximal values (when n = 4 to 6 independent biological replicates). A two-way ANOVA with Sidak’s correction was performed. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (compared to scramble). #, P ≤ 0.05 compared to the wild type (B); ##, P ≤ 0.01 (for global treatment effects) (E).
FIG 7
FIG 7
TP53 is targeted by miR-125b and affects L. pneumophila replication. THP-1 cells were transfected with either miRNA-125b mimic or an miRNA mimic pool (miR-125b, miR-221, and miR-579) at a final concentration of 30 nM. As a control, a scrambled precursor (scr) was transfected. At 24 h posttransfection, THP-1 cells were activated with PMA for another 24 h and then infected with L. pneumophila at an MOI of 0.5. (A) TP53 expression was examined via qPCR and is displayed as the log2 fold change. Luciferase reporter assay analyses were performed in HEK-293T cells. The plasmid contained either the wild-type (TP53WT) or the mutated (TP53mut) version of the 3′ UTR of TP53. Ratios of Renilla and firefly luciferase luminescence were normalized to the vector without insert. BDMs were transfected with an siRNA pool targeting TP53 (siTP53) or with a scrambled siRNA as a control (scr) and infected at an MOI of 0.5 or left untreated. (C) Downregulation of TP53 expression with siRNA was verified by qPCR and is displayed as the log2-fold change. (D) The CFU of Legionella were determined 24, 48, and 72 h postinfection after a knockdown of TP53. Boxes show the upper and lower quartiles with median (when n = 3 independent biological replicates) and whiskers indicate minimal and maximal values (when n = 4 independent biological replicates). A two-way ANOVA with Sidak’s correction was performed. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 (compared to scramble). ####, P ≤ 0.0001 (compared to the wild type) (B). #, P ≤ 0.05 (for global treatment effects) (D).
FIG 8
FIG 8
LGALS8 is targeted by miR-579 and influences L. pneumophila replication. THP-1 cells were transfected with either the miR-579 mimic or the miRNA mimic pool (miR-125b, miR-221, and miR-579) at a final concentration of 30 nM. As a control, a scrambled precursor (scr) was transfected. At 24 h posttransfection, THP-1 cells were activated with PMA for another 24 h and then infected with L. pneumophila at an MOI of 0.5. (A) LGALS8 expression was examined with qPCR and is displayed as the log2 fold change. Luciferase reporter assay analyses were performed in HEK-293T cells. The plasmid contained either the wild-type (LGALS8WT) or the mutated (LAGLS8mut) version of the 3′ UTR of LGALS8. (B) Ratios of Renilla and firefly luciferase luminescence were normalized to the vector without insert. BDMs were transfected with an siRNA pool targeting LGALS8 (siLGALS8) or with a scrambled siRNA as a control (scr) and infected at an MOI of 0.5 or left untreated as a control. (C) Downregulation of LGALS8 expression with siRNA was verified by qPCR and is displayed as the log2-fold change. (D) CFU of Legionella were determined 24, 48, and 72 h postinfection after a knockdown of LGALS8. Boxes show the upper and lower quartiles, with medians (when n = 3 independent biological replicates) and whiskers indicating minimal and maximal values (when n = 4 or 5 independent biological replicates). Two-way ANOVA with Sidak’s correction was performed. *, P ≤ 0.05; **, P ≤ .01; ***, P ≤ 0.001; ****, P ≤ 0.0001 (compared to the corresponding control). #, P ≤ 0.05; ##, P ≤ 0.01; ###P ≤ 0.001 (compared to wild type) (B). ##, P ≤ 0.01 (for global treatment effects) (D).
FIG 9
FIG 9
Scheme of MX1 and LGALS8 regulation by miRNA precursor pool transfection to influence L. pneumophila replication in macrophages. Precursor transfection of miR-125b reduces the expression of TP53, while DDX58 (RIG-I) is targeted by miR-221. Both targets further regulate the protein expression of MX1, which has an antibacterial effect on Legionella. Furthermore, miR-579 acts on LGALS8 to reduce its expression. Thus, reduction of MX1 and LGALS8 expression leads to an increased replication of Legionella in macrophages. In conclusion, these miRNAs are influencing the expression of their targets and have an impact on Legionella replication.

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