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Cited by 10RNA and protein synthesis mechanismsBacterial Genetics and BiotechnologyImmune Response and Inflammation

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Coordination of transcriptional and translational regulations in human epithelial cells infected by Listeria monocytogenes

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Besic, Vinko, Habibolahi, Fatemeh, Noël, Benoît, Rupp, Sebastian, Genovesio, Auguste, Lebreton, Alice·2020·RNA Biology
DOI 10.1080/15476286.2020.1777380PMC PMC7549700

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Abstract

Abstract The invasion of mammalian cells by intracellular bacterial pathogens reshuffles their gene expression and functions; however, we lack dynamic insight into the distinct control levels that shape the host response. Here, we have addressed the respective contribution of transcriptional and translational regulations during a timecourse of infection of human intestinal epithelial cells by an epidemic strain of Listeria monocytogenes , using transcriptome analysis paralleled with ribosome profiling. Upregulations were dominated by early transcriptional activation of pro-inflammatory genes, whereas translation inhibition appeared as the major driver of downregulations. Instead of a widespread but transient shutoff, translation inhibition affected specifically and durably transcripts encoding components of the translation machinery harbouring a 5’-terminal oligopyrimidine motif. Pre-silencing the most repressed target gene ( PABPC1 ) slowed down the intracellular multiplication of Listeria monocytogenes , suggesting that the infected host cell can benefit from the repression of genes involved in protein synthesis and thereby better control infection.

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Figure 1. Lm infection has a low impact on total translation activity in LoVo epithelial cells. (A) Principle of the metabolic labelling of newly synthesized proteins with homopropargylglycine (HPG) over an infection time course. LoVo cells, infected or not for 2, 5 or 10 h with Lm LL195 constitutively expressing eGFP, were treated with HPG for 1 h prior to recovery. Cell infection was monitored by immunofluorescence staining on coverslips. DAPI staining of cell nuclei is displayed in white, F-actin staining by fluorescently labelled phalloidin is in red, and eGFP-expressing bacteria are in green. After cell lysis, HPG residues that had been incorporated into newly-synthesized proteins were conjugated with sulpho-Cy5-azide by copper-catalysed alkyne-azide cycloaddition. (B) In-gel fluorescence detection of HPG incorporation into newly synthesized proteins. Following cycloaddition, protein samples were separated by SDS-PAGE, and Cy5 fluorescence was recorded (top panel) before the gel was stained with colloidal Coomassie as a loading control (bottom panel). (C) Quantification of HPG incorporation. The integrated density of Cy5 fluorescence was measured for each lane and normalized to the corresponding integrated density of Coomassie staining. NI, non-infected. Data are average and standard deviation from independent experiments; n = 3 for CHX, 2 and 5 h p.i.; n = 2 for 10 h p.i. p-values were calculated by two-tailed t-tests.
Figure 1. Lm infection has a low impact on total translation activity in LoVo epithelial cells. (A) Principle of the metabolic labelling of newly synthesized proteins with homopropargylglycine (HPG) over an infection time course. LoVo cells, infected or not for 2, 5 or 10 h with Lm LL195 constitutively expressing eGFP, were treated with HPG for 1 h prior to recovery. Cell infection was monitored by immunofluorescence staining on coverslips. DAPI staining of cell nuclei is displayed in white, F-actin staining by fluorescently labelled phalloidin is in red, and eGFP-expressing bacteria are in green. After cell lysis, HPG residues that had been incorporated into newly-synthesized proteins were conjugated with sulpho-Cy5-azide by copper-catalysed alkyne-azide cycloaddition. (B) In-gel fluorescence detection of HPG incorporation into newly synthesized proteins. Following cycloaddition, protein samples were separated by SDS-PAGE, and Cy5 fluorescence was recorded (top panel) before the gel was stained with colloidal Coomassie as a loading control (bottom panel). (C) Quantification of HPG incorporation. The integrated density of Cy5 fluorescence was measured for each lane and normalized to the corresponding integrated density of Coomassie staining. NI, non-infected. Data are average and standard deviation from independent experiments; n = 3 for CHX, 2 and 5 h p.i.; n = 2 for 10 h p.i. p-values were calculated by two-tailed t-tests.CC BY-NC-ND · PubMed Central
Figure 2. Transcriptional up- & translational down-regulations dominate gene expression response to Lm in the first hours of infection. LoVo intestinal epithelial cells were infected for 2 to 10 h. Cell lysates were processed for total cytoplasmic RNA-seq and ribosome profiling (Ribo-seq). (A-C) Scatter plots of changes in normalized RFP (Y-axis) vs RNA (X-axis) levels along the course of infection, when comparing (A) 2 h vs non-infected, (B) 5 vs 2 h or (C) 10 vs 5 h. (D-E) Volcano plots highlighting genes being significantly up- (right) or down- (left) regulated in (D) RNA-seq, (E) Ribo-seq, or (F) translation efficiency (TE) at 5 h p.i. compared to 2 h p.i. Data points coloured in blue, green or red represent genes with padj below 0.05 (above dashed grey horizontal line; -log10padj = 1.3) and an FC below or above 1.5 (vertical dashed grey lines; log2 FC = ± 0.58). Genes for which padj < 0.5 in TE are highlighted in red in D and E. (A-E) Data from three independent replicates (except for RFPs at 10 h). FC, foldchange; padj, adjusted p-value [DESeq false discovery rate (FDR)].
Figure 2. Transcriptional up- & translational down-regulations dominate gene expression response to Lm in the first hours of infection. LoVo intestinal epithelial cells were infected for 2 to 10 h. Cell lysates were processed for total cytoplasmic RNA-seq and ribosome profiling (Ribo-seq). (A-C) Scatter plots of changes in normalized RFP (Y-axis) vs RNA (X-axis) levels along the course of infection, when comparing (A) 2 h vs non-infected, (B) 5 vs 2 h or (C) 10 vs 5 h. (D-E) Volcano plots highlighting genes being significantly up- (right) or down- (left) regulated in (D) RNA-seq, (E) Ribo-seq, or (F) translation efficiency (TE) at 5 h p.i. compared to 2 h p.i. Data points coloured in blue, green or red represent genes with padj below 0.05 (above dashed grey horizontal line; -log10padj = 1.3) and an FC below or above 1.5 (vertical dashed grey lines; log2 FC = ± 0.58). Genes for which padj < 0.5 in TE are highlighted in red in D and E. (A-E) Data from three independent replicates (except for RFPs at 10 h). FC, foldchange; padj, adjusted p-value [DESeq false discovery rate (FDR)].CC BY-NC-ND · PubMed Central
Figure 3. Early transcriptional regulation of inflammatory response precedes a translational repression of the translational equipment. (A) Over-representation analysis (ORA) of GO Biological Process terms for up- or down-regulated genes in RNA-seq, Ribo-seq or TE over all time-points. For each time-point, DRGs were selected by comparison to the non-infected condition. (B-F) Induction of inflammation and of the integrated stress response by Lm infection of LoVo cells. The variation of RNA (B, D) and RFP (C, E) levels was quantified for selected NF-κB transcriptional target genes related to inflammation (B, C) or genes involved in the integrated stress response (D, E) during infection. Data represent DESeq normalized read counts from three independent experiments and error bars indicate standard deviation. (F) The phosphorylation status of the mTOR substrate S6K1, and of the target of ISR eIF2 kinases eiF2α were assessed by immunoblotting in cells infected for 2, 5 or 10 h by wild-type Lm or by an Lm strain where the hlyA gene encoding LLO had been deleted (∆hlyA). Treatments by tunicamycin and rapamycin were used, respectively, as an inducer of the ISR through endoplasmic reticulum stress, and as a repressor of mTOR activity. (G) Transcripts sharing similar TE profiles over time were clustered by fuzzy clustering. For each cluster, only genes having more than 70% membership are displayed. (H) Functional categories were assigned to each cluster by ORA of GO Biological Process terms.
Figure 3. Early transcriptional regulation of inflammatory response precedes a translational repression of the translational equipment. (A) Over-representation analysis (ORA) of GO Biological Process terms for up- or down-regulated genes in RNA-seq, Ribo-seq or TE over all time-points. For each time-point, DRGs were selected by comparison to the non-infected condition. (B-F) Induction of inflammation and of the integrated stress response by Lm infection of LoVo cells. The variation of RNA (B, D) and RFP (C, E) levels was quantified for selected NF-κB transcriptional target genes related to inflammation (B, C) or genes involved in the integrated stress response (D, E) during infection. Data represent DESeq normalized read counts from three independent experiments and error bars indicate standard deviation. (F) The phosphorylation status of the mTOR substrate S6K1, and of the target of ISR eIF2 kinases eiF2α were assessed by immunoblotting in cells infected for 2, 5 or 10 h by wild-type Lm or by an Lm strain where the hlyA gene encoding LLO had been deleted (∆hlyA). Treatments by tunicamycin and rapamycin were used, respectively, as an inducer of the ISR through endoplasmic reticulum stress, and as a repressor of mTOR activity. (G) Transcripts sharing similar TE profiles over time were clustered by fuzzy clustering. For each cluster, only genes having more than 70% membership are displayed. (H) Functional categories were assigned to each cluster by ORA of GO Biological Process terms.CC BY-NC-ND · PubMed Central
Figure 4. Translational repression of 5ʹ-terminal oligopyrimidine-containing transcripts, including PABPC1, during Lm infection. (A-D) Violin plots representing fold changes in TE over time for transcripts that have been experimentally verified to contain (A) functional TOP, (B) uORF, or (C) IRES motifs, or (D) predicted to contain a TISU in their 5ʹ-UTR regions. (E-H) Translation efficiency profiles of individual transcripts containing either (E) TOP, (F) uORF, (G) IRES or (H) TISU motifs. Transcripts for which the adjusted p-value from Riborex analysis was below 0.5 are displayed in black line (except PABPC1 and PABPC4, in dotted green and blue lines, respectively), while transcripts for which TE changes were not significant are displayed in grey. (I) Profiles of RNA-seq (top) and Ribo-seq (bottom) reads aligned at the PABPC1 locus. Average values of read counts per genomic position from three independent experiments, normalized for library size, are represented for each time-point. (J-K) Quantification of the variation of PABPC1 and PABPC4 RNA (J) and RFP (K) levels during infection. Data represent DESeq normalized counts from three independent experiments and error bars indicate standard deviation. (L) Silencing of PABPC1 reduces Lm intracellular replication rate. LoVo cells were transfected with siRNA against PABPC1 mRNA or a scrambled siRNA (siCtrl) for 48-h before infection. Two independent experiments were carried out using distinct siRNA against PABPC1 (siPABPC1 (1) and (2)). Bacterial entry and replication were assessed by gentamicin protection assay followed by serial dilution plating of infected cell lysates on agar plates. In order to focus on intracellular multiplication rather than differences in the entry, the log2 ratio of colony-forming units (CFU) counts per cell at each time-points relative to time 2 h post-infection were plotted. Values are averages and standard deviation from three infected wells per condition, each counted thrice.
Figure 4. Translational repression of 5ʹ-terminal oligopyrimidine-containing transcripts, including PABPC1, during Lm infection. (A-D) Violin plots representing fold changes in TE over time for transcripts that have been experimentally verified to contain (A) functional TOP, (B) uORF, or (C) IRES motifs, or (D) predicted to contain a TISU in their 5ʹ-UTR regions. (E-H) Translation efficiency profiles of individual transcripts containing either (E) TOP, (F) uORF, (G) IRES or (H) TISU motifs. Transcripts for which the adjusted p-value from Riborex analysis was below 0.5 are displayed in black line (except PABPC1 and PABPC4, in dotted green and blue lines, respectively), while transcripts for which TE changes were not significant are displayed in grey. (I) Profiles of RNA-seq (top) and Ribo-seq (bottom) reads aligned at the PABPC1 locus. Average values of read counts per genomic position from three independent experiments, normalized for library size, are represented for each time-point. (J-K) Quantification of the variation of PABPC1 and PABPC4 RNA (J) and RFP (K) levels during infection. Data represent DESeq normalized counts from three independent experiments and error bars indicate standard deviation. (L) Silencing of PABPC1 reduces Lm intracellular replication rate. LoVo cells were transfected with siRNA against PABPC1 mRNA or a scrambled siRNA (siCtrl) for 48-h before infection. Two independent experiments were carried out using distinct siRNA against PABPC1 (siPABPC1 (1) and (2)). Bacterial entry and replication were assessed by gentamicin protection assay followed by serial dilution plating of infected cell lysates on agar plates. In order to focus on intracellular multiplication rather than differences in the entry, the log2 ratio of colony-forming units (CFU) counts per cell at each time-points relative to time 2 h post-infection were plotted. Values are averages and standard deviation from three infected wells per condition, each counted thrice.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Besic, V., Habibolahi, F., Noël, B., Rupp, S., Genovesio, A., & Lebreton, A.
Affiliations
Centre National de la Recherche Scientifique (FR)
Corresponding author
Lebreton, Alice
ORCID
Venue
RNA Biology
Publisher
Informa UK Limited
Published
2020-06-25
Volume
17
Issue
10
Pages
1492-1507
ISSN
  • 1547-6286 (ISSN-L)
  • 1555-8584
DOI
10.1080/15476286.2020.1777380
PMC
PMC7549700
OpenAlex
W3037073818
Cited by
10
References
4
Citations by year
2026: 1 · 2025: 1 · 2024: 1 · 2023: 2 · 2022: 2 · 2021: 3
FWCI
0.57
Type
journal article
Language
en
Open access
Licence
Locations
  • RNA Biology (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by-nc-nd)
Funders
  • Investissements d’Avenir (ANR-10-LABX-54)
  • Fondation pour la Recherche Médicale (FRM-AJE20131128944)
  • Inserm ATIP-Avenir and Mairie de Paris
  • SNF Early Postdoc Mobility (P2BEP3_168721)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
Keywords
Listeria monocytogenes, host-pathogen interaction, intestinal epithelial cells, transcriptional regulation, translational regulation, protein synthesis inhibition, ribosome profiling, transcriptome, PABPC1, pro-inflammatory gene expression, host response to infection
MeSH terms
Cells, Cultured, Epithelial Cells, Humans, Listeriosis, Listeria monocytogenes, RNA, Messenger, Time Factors, Transcription, Genetic, Protein Biosynthesis, Host-Pathogen Interactions

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