, The structure of trp RNA-binding attenuation protein, Nature, vol.374, pp.693-700, 1995.
, Translation elongation factor EF-Tu is a target for Stp, 2005.
, New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria, Appl. Environ. Microbiol, vol.70, pp.6887-6891, 2004.
,
, Mice lacking the type I interferon receptor are resistant to Listeria monocytogenes, J. Exp. Med, vol.200, pp.527-533, 2004.
, TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a toroid-shaped molecule that binds transcripts containing GAG or UAG repeats separated by two nucleotides, Proc. Natl. Acad. Sci. USA, vol.92, pp.7916-7920, 1995.
, , 2010.
, The human SWI/SNF subunit Brm is a regulator of alternative splicing, Nat. Struct. Mol. Biol, vol.13, pp.22-29, 2006.
, Listeriomics: an Interactive Web Platform for Systems Biology of Listeria. mSystems, vol.2, pp.186-202, 2017.
, Type I interferon sensitizes lymphocytes to apoptosis and reduces resistance to Listeria infection, 2004.
, J. Exp. Med, vol.200, pp.535-540
, RIG-I Recognizes the 5 0 Region of Dengue and Zika Virus Genomes, Cell Rep, vol.24, pp.320-328, 2018.
, Probing mRNA structure and sRNA-mRNA interactions in bacteria using enzymes and lead(II), Methods Mol. Biol, vol.540, pp.215-232, 2009.
URL : https://hal.archives-ouvertes.fr/hal-00477293
, RIG-I and Other RNA Sensors in Antiviral Immunity, Annu. Rev. Immunol, vol.36, pp.667-694, 2018.
, Identification of small Hfq-binding RNAs in Listeria monocytogenes, RNA, vol.12, pp.1383-1396, 2006.
, AlienTrimmer: a tool to quickly and accurately trim off multiple short contaminant sequences from high-throughput sequencing reads, Genomics, vol.102, pp.500-506, 2013.
,
, Comparative genome analysis of Listeria bacteriophages reveals extensive mosaicism, programmed translational frameshifting, and a novel prophage insertion site, J. Bacteriol, vol.191, pp.7206-7215, 2009.
, The bacterial pathogen Listeria monocytogenes and the interferon family: type I, type II and type III interferons, Front. Cell. Infect. Microbiol, vol.4, pp.3047-3048, 2014.
URL : https://hal.archives-ouvertes.fr/pasteur-01145465
, , 2001.
, Comparative genomics of Listeria species, Science, vol.294, pp.849-852
, Characterization of a Listeria monocytogenes-specific protein capable of inducing delayed hypersensitivity in Listeria-immune mice, Mol. Microbiol, vol.4, pp.1091-1099, 1990.
, Listeria marthii sp. nov., isolated from the natural environment, Finger Lakes National Forest, Int. J. Syst. Evol. Microbiol, vol.60, pp.1280-1288, 2010.
, RIG-I detects triphosphorylated RNA of Listeria monocytogenes during infection in non-immune cells, PLoS One, vol.8, p.62872, 2013.
, RNA-binding proteins in bacteria, Nat. Rev. Microbiol, vol.16, pp.601-615, 2018.
, Fast gapped-read alignment with Bowtie 2, Nat. Methods, vol.9, pp.357-359, 2012.
, Crystal structure of human nucleophosmin-core reveals plasticity of the pentamer-pentamer interface, Proteins, vol.69, pp.672-678, 2007.
, F-Type Bacteriocins of Listeria monocytogenes: a New Class of Phage Tail-Like Structures Reveals Broad Parallel Coevolution between Tailed Bacteriophages and High-Molecular-Weight Bacteriocins, J. Bacteriol, vol.198, pp.2784-2793, 2016.
, The Sequence Alignment/Map format and SAMtools, Genome Project Data Processing Subgroup, vol.25, pp.2078-2079, 1000.
, featureCounts: an efficient general purpose program for assigning sequence reads to genomic features, Bioinformatics, vol.30, pp.923-930, 2014.
, Nuclear-resident RIG-I senses viral replication inducing antiviral immunity, Nat. Commun, vol.9, p.3199, 2018.
, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, Genome Biol, vol.15, p.550, 2014.
, Inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity, PLoS Pathog, vol.9, 2013.
URL : https://hal.archives-ouvertes.fr/pasteur-01113535
, A Secreted RNA Binding Protein Forms RNA-Stabilizing Granules in the Honeybee Royal Jelly, Mol. Cell, vol.74, pp.598-608, 2019.
, A riboswitch-regulated antisense RNA in Listeria monocytogenes, Proc. Natl. Acad. Sci. USA, vol.110, pp.13132-13137, 2013.
, Type I interferon production enhances susceptibility to Listeria monocytogenes infection, J. Exp. Med, vol.200, pp.437-445, 2004.
, Infection Reveals a Modification of SIRT2 Critical for Chromatin Association, Cell Rep, vol.23, pp.1124-1137, 2018.
URL : https://hal.archives-ouvertes.fr/pasteur-01857244
, OrfX, a Nucleomodulin Required for Listeria monocytogenes Virulence, MBio, vol.8, pp.1550-1567, 2017.
URL : https://hal.archives-ouvertes.fr/pasteur-01740237
, deepTools2: a next generation web server for deep-sequencing data analysis, Nucleic Acids Res, vol.44, issue.W1, pp.160-165, 2016.
, , 2010.
, Subcellular Localizations of RIG-I, TRIM25, and MAVS Complexes, J. Virol, vol.91, pp.1155-1171, 2017.
, Comparative analysis of viral RNA signatures on different RIG-I-like receptors, p.11275, 2016.
URL : https://hal.archives-ouvertes.fr/hal-01483406
,
, Characterization of the interferon-producing cell in mice infected with Listeria monocytogenes, 2009.
, PLoS Pathog, vol.5, 1000355.
, A systematic analysis of the RNA-targeting potential of secreted bacterial effector proteins, Sci. Rep, vol.7, p.9328, 2017.
, Running in reverse: the structural basis for translocation polarity in hexameric helicases, Cell, vol.139, pp.523-534, 2009.
, Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria, Gene, vol.102, pp.99-104, 1991.
, SARTools: A DESeq2-and EdgeR-Based R Pipeline for Comprehensive Differential Analysis of RNA-Seq Data, PLoS One, vol.11, p.157022, 2016.
URL : https://hal.archives-ouvertes.fr/hal-01344179
, MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins, Nat. Cell Biol, vol.13, pp.423-433, 2011.
, Cells were then incubated for 1 h with primary antibody, washed in PBS, incubated for 45 min with secondary antibody/DAPI, washed again as above and mounted in Vectashield
, 70% (m/v) saccharose). Samples were centrifuged for 500 3 g, 4 C). The supernatant was isolated as the cytosolic fraction and recentrifuged as before to eliminate cell nuclear debris. The pellet was washed in buffer B (10 mM HEPES pH 8, 0.1 mM EDTA, 100 mM NaCl, 25% (v/v) glycerol) and centrifuged 5 min at 500 3 g, 4 C. Buffer A, B and SR were supplemented with 0.15 mM spermidine, 0.15 mM spermine, 1 mM DTT and protease inhibitor. The washed pellet was resuspended in sucrose buffer, Cell were resuspended in buffer A (20 mM HEPES pH 7, 0.15 mM EDTA, 0.15 mM EGTA, 10 mM KCl). 1% NP40 was added, followed by SR buffer (50 mM HEPES pH 7, 0.25 mM EDTA, 10 mM KCl, vol.10, 2018.
, Concentrated medium was then supplemented with 0,05% of Triton X-100, centrifuged again (18407 3 g, 20 min, 4 C) and left on ice while preparing bacterial cytosolic extracts. The bacterial pellet was washed thrice in ice-cold PBS and lysed by mechanical shaking in a FastPrep apparatus (described above) in 1 mL of lysis buffer (25mM Tris pH 7.4, 150mM KCl, 1mM DTT, 0.05% Triton X-100) supplemented with protease inhibitors mixture. Bacterial cytosol was recovered by centrifugation (two serial centrifugations at 18407 3 g, 20 min, 4 C) and protein concentration determine by Bradford assay. One-milliliter of bacterial cytosol and concentrated culture medium (corresponding to 50 mL and 10 mL of the bacterial cytosol and culture medium, respectively) were individually loaded on a Superose 6 10/300 GL column pre-equilibrated with lysis buffer without Triton X-100. About 161 fractions of 220 mL were collected, and one out of every seven fractions was concentrated by acetone precipitation and the presence of Zea and EF-Tu analyzed by immunoblotting after wet transfer onto a nitrocellulose membrane. Fractions containing the complexes (A or B) were then pooled and processed for immunoprecipitation assays. Briefly, each sample was incubated overnight at 4 C with shacking, with a mix of 30 mg of anti-Zea antibodies (10 mg of each antibody) or 30 mg of normal rabbit IgG (CellSignaling) . Then, 50 mL of Protein A, Live/Dead Bacterial Staining L. monocytogenes was grown in 20 mL of BHI until stationary phase. Bacteria were pelleted (2862 3 g, 20 min, room temperature) and then stained with LIVE/DEAD BacLight (Molecular Probes), following the manufacturer's recommendation. Bacterial suspension (20 mL) was then deposited on a glass coverslip and immediately imaged by using a Zeiss AxioObserver
, Turbo DNase buffer. Samples were vortexed for 30 s after the addition of 200 mL of acid phenol (Ambion). Then, 50 mL chloroform
, Samples were centrifuged (30 min, 10 000 3 g, 4 C), and RNA was washed once with 70% ethanol before being resuspended in 25 mL of nuclease-free water, RNA was analyzed with the Bioanalyser RNA pico kit
, purified by ethanol precipitation and quality-controlled with the Bioanalyser, as described above. Directional RNA-seq libraries were prepared with 30 ng of purified RNA for each sample by using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) according to the manufacturer's instructions. When required, the RNA samples were spiked-in with a synthetic in vitro-transcribed AdML splicing reporter (Allemand et al., 2016) in order to have 30 ng of total RNA, Purified RNA was fragmented with the ''RNA fragmentation reagents'' (Thermofisher)
, A parallel culture (same conditions) was set-up to check the OD and arrest the bacterial growth when the strains reached the same OD. The culture medium was then recovered by centrifugation (2862 3 g, 20 min, 4 C) and filtered (0.22 mm). The bacterial pellet was stored at À80 C for subsequent RNA extraction. 10 mL of the filtered culture medium were desalted and the RNA was extracted as described above. The quality of the RNA was checked by using the Bioanalyser RNA nano kit. The amount of recovered RNA was similar in all the samples. Total secreted RNA (5 mg) was ribodepleted by using the Ribo Zero rRNA removal kit (Illumina) following the manufacturer's instructions, RIP-Seq Data Analysis The L. monocytogenes EGD-e genome (NC_003210) and a list of 3160 transcripts (genes, small-RNAs, tRNAs, and rRNAs) were downloaded from the Listeriomics database, vol.1, 2009.
, and saved to BAM files after indexation. Read Per Million coverage files were saved in BigWig format using bamCoverage package from deepTools 3.1.3 (Ramírez et al., 2016). The quality of the sequencing and mapping was assessed using FastQC 0.10.1 and MultiQC 0, Sequencing of Secreted L. monocytogenes RNA The RNA-seq datasets were first trimmed to keep only reads longer than, p.45, 2009.
, 50 mL of bacterial culture were processed as follows. Bacteria were pelleted at 2862 3 g, 20 min, 4 C and culture supernatant was filtered and processed (5 mL) for total RNA purification (input, 10%), by performing two sequential phenol/chloroform extractions followed by ethanol/sodium acetate precipitation. The RNA pellet was washed once with ethanol 70% and resuspended in 20 mL nuclease-free water, RIP-qPCR L. monocytogenes bacterial cultures (Dzea+zea + strain) were processed essentially as described for the RIP-seq experiment unless otherwise stated. In summary, L. monocytogenes was grown until the stationary phase (OD 600nm = 3.5) and, for every sample
, Beads were washed four times with lysis buffer, treated with Turbo DNase and processed for RNA extraction. For qPCR analysis, 100 ng of purified RNA were subjected to reverse transcription in 20 mL final volume using the Reverse Transcription Kit (QIAGEN) according to the manufacturer's instructions. Reactions were then diluted by adding 180 mL of nuclease-free water. qPCR was assayed in 10 mL reactions with Brillant III Ultra Fast SYBR-Green qPCR Master Mix (Agilent). Reactions were carried out in a Stratagene MX3005p system with the following thermal profile: 5 min at 95 C, 37 cycles of 10 s at 95 C and 12 s at 60 C, 2006.
, Quantitative Real-Time PCRs For qPCR of L. monocytogenes secreted RNA (phage, lma-monocin and rli143 RNAs), bacterial strains were grown in MM until exponential phase (OD 600nm = 0.4). L. monocytogenes wt, Dzea and Dzea+zea + strains were used for the phage and lma-monocin quantification
, Dzea, zea + and lmo2595 + L. monocytogenes strains were used for the quantification of rli143
, Gene expression levels were normalized to the OVA mRNA, and the fold change was calculated using the DDCT method. For qPCR of Listeria genes from total (intracellular) L. monocytogenes RNA, the RNA was extracted, as described in the RNA extraction section and treated, as described above, except that the OVA mRNA was not included in the reverse transcription reaction. Gene expression levels were normalized to the rpob gene, and the fold change was calculated using the DDCT method. For qPCR of Hfq-associated RNAs, RNA was extracted from immunoprecipitated Hfq, by using the protocol described for the RIPseq of Zea. DNase-treated RNA (120 ng) was subjected to reverse transcription. Gene expression levels were normalized to the input fractions, and the fold change was calculated using the DDCT method. For qPCR of IFNb, IFNg and IL-8, mammalian RNA was extracted, as described in the RNA extraction section, zea-pAD and lmo2595-pAD strains were employed for the quantification of rli143. The bacterial OD was measured, and the cultures were recovered when OD was equal for all the strains. MM was collected by centrifugation, filtered and processed for RNA extraction (as described above)
, a special mapping software allowing variability in reads sequence. Mapping files were filtered to keep uniquely mapped reads using SAMtools 0.1.19 (samtools view -b -q 1 parameters, Data Analysis of RLR-Associated RNAs Due to the high number of eukaryotic RNAs in the datasets and presence of insertions and deletions, the reads were trimmed, vol.1, 2009.
, 2013)) were used as positive controls. Cells were lysed 24 h post-transfection with 200 mL Passive Lysis buffer (Promega). The Firefly luciferase activity was measured using the Bright-Glo Luciferase Assay System (Promega) following the manufacturer's recommendation, Transfection of Zea-Interacting RNAs The ISRE reporter cells (STING-37 cell line, vol.26, p.10, 2000.
, An RNA-Binding Protein Secreted by a Bacterial Pathogen Modulates RIG-I Signaling, Cell Host & Microbe, 2019.
URL : https://hal.archives-ouvertes.fr/pasteur-02390001