Caractérisation de l'histone H3 lysine désacétylation au cours de l'infection par Listeria monocytogenes

Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.De nombreuses bacteries pathogènes sont capables d'affecter les programmes transcriptionnels de la cellule hôte pendant l'infection. Cependant, les mécanismes contrôlant ce processus restent largement méconnus. En investigant les effets de la Listerai monocytogenes sur les modifications des histones de l'hôte, nous avons mis en évidence un nouveau mecanisme de régulation de transcription nécessaire pour la répression de certains gènes, pendant l'infection. Lors de l'infection par L. monocytogenes, le facteur de virulence sécrété, InlB, se lie au récepteur c-Met et active la signalisation par les intermédiaires PI3K et Akt. cette plateforme de signalisation est nécessaire pour la relocalisation de la deacetylase d'histone, SIRT2, au noyau et l'association à la chromatine.En caractérisant me mécanisme gouvernant la relocalisation nucléaire de SIRT2 lors de l'infection, nous avons démontrés que SIRT2 subit une modification post-traductionnelle. SIRT2 est déphosphorylée à un nouveau site de phosphorylation localisé à la partie terminale de la protéine. SIRT2 est recrutée au site de démarrage de la transcription des gènes réprimés lors de l'infection menant à la deacetylation de H3K18 et la répression transcriptionnelle. Nous avons mis en évidence que SIRT2 est détournée par L. monocytogenes et provoque une croissance des bactéries intracellulaires. Ces résultats démontrent un clef de SIRT2 en provoquant la deacetylation de H3K18 mors de l'infection et dévoilent un nouveau mécanisme imposée par les bactéries pathogènes dans le but de reprogrammer la cellule hôte

AFM Experimental Data

Here included are data sets of AFM images, corresponding cell height profiles for all time points, time stamp, and text describing experimental conditions

Data from: Mechanical morphotype switching as an adaptive response in Mycobacteria

<p><span>Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report a new virulence mechanism by which changes to the mechanical stiffness of the mycobacterial cell surface confers refraction to killing during infection. Long-Term Time-Lapse Atomic Force Microscopy was used to reveal a process of "mechanical morphotype switching" in mycobacteria exposed to host intracellular stress. A "soft" mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Both pharmacologic treatment, with bedaquiline, and a genetic mutant lacking <em>uvrA</em> modified the basal mechanical state of mycobacteria into a "soft" mechanical morphotype, enhancing survival in macrophages. Our study proposes microbial cell mechanical adaptation as a critical axis for surviving host-mediated stressors.</span></p><p>Funding provided by: European Molecular Biology Organization<br>Crossref Funder Registry ID: https://ror.org/04wfr2810<br>Award Number: 191-2014</p><p>Funding provided by: European Molecular Biology Organization<br>Crossref Funder Registry ID: https://ror.org/04wfr2810<br>Award Number: 750-2016</p><p>Funding provided by: Cystic Fibrosis Foundation<br>Crossref Funder Registry ID: https://ror.org/00ax59295<br>Award Number: 002510I221</p><h2><span>AFM image datasets of Height, DMT modulus (stiffness), and Peak Force Error channels for M. smegmatis imaged in axenic conditions of growth and stress</span></h2> <h3><span>AFM imaging</span></h3> <p><span>Coverslips were prepared as previously described </span><span>(3)</span><span>. Polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning) at a ratio of 15:1 (elastomer:curing agent) and cut 1:10 with Hexane to reduce 10-fold the spin-coated layer, while equally increasing the hydrophobicity of the surface. Aliquots of mycobacteria isolated from axenic culture or from infection of macrophages were filtered through a 0.5 µm pore size PVDF filter (Millipore) to remove cell clumps and enrich single cells. Aliquots were deposited on the hydrophobic surface of a PDMS-coated coverslip. 7H9 growth medium was supplied. Where indicated, antibiotic was added to the growth medium. The medium was maintained at 37°C using a custom-made heating element within the sample space and a TC2-80-150 temperature controller (Bioscience tools). Bacteria were imaged by peak force tapping using a Nanoscope 5 controller (Veeco Metrology) at a scan rate of 0.25 – 0.5 Hz and a maximum Z-range of 5 µm. A ScanAsyst fluid cantilever (Bruker) was used. Continuous scanning provided snapshots at 2–30 min intervals. Height, peak force error, adhesion, dissipation, deformation modulus and log modulus were recorded for all scanned images. Peak force error yields a fine representation of the height on the order of 10 nm in the Z-axis; this is computed as the difference between the peak force setpoint and the actual value. Images were processed using Gwyddion (Department of Nanometrology, Czech Metrology Institute). ImageJ was used for extracting bacterial cell surface height and modulus values and generating dynamic and quantitative mean values of the mechanical properties of individual cells.</span></p> <h3><span>Correlated fluorescence and AFM</span></h3> <p><span>Correlated fluorescence and AFM images were acquired as described previously</span><span>. Briefly, fluorescence images were acquired with an electron-multiplying-charge-coupled device (EMCCD) iXon Ultra 897 camera (Andor) mounted on an IX71 inverted optical microscope (Olympus) equipped with a UAPON100XOTIRF x100 oil immersion objective (Olympus) with the x2 magnifier in place. Illumination was provided by a monolithic laser combiner (MLC) (Agilent) using the 488 or 561 nm laser output coupled to an optical fibre with appropriate filter sets: F36-526 for Calcein-AM and F71-866 for mCherry-Wag31 or cytosolic RFP. The AFM was mounted on top of the inverted microscope and images were acquired with a customised Icon scan head (Bruker) using ScanAsyst fluid cantilevers (Bruker) with a nominal spring constant of 0.7 N m<sup>-1</sup> in peak force tapping mode at a setpoint <2 nN and typical scan rates of 0.5 Hz. The samples were maintained at 37°C in 7H9 growth medium heated by a custom-made coverslip heating holder controlled by a TC2-80-150 temperature controller (Bioscience tools).</span></p> <p> </p> <h2>Analysis of TnSeq experimentation to identify conditionally essential Tn Insertion Mutants isolated from Low and High Buoyancy Fractions</h2> <div> <h3><strong>Transposon library construction, genomic DNA Extraction, and sequencing.</strong></h3> <p>Transposon library after buoyancy centrifugation was collected and resuspended in 400ul 10mM Tris (pH=9). After beads-beating, genomic DNA was extracted by Phenol-Chloroform method. DNA concentration was measured and quantified by Nanodrop and Qubit. For building transposon sequencing library, approximately 5ug genomic DNA was resuspended in 150ul TE buffer and transferred to a Covaris tube, Genomic DNA was disrupted to 200-500bp size range by sonication with the following parameters: duty cycle (10%), intensity (4), cycles/burst (200), time (80s). The fragmented genomic DNA was size-selected and purified by AMPure XP beads. The fragmented genomic DNA was further subjected to end repair and dA tailing. Annealed adapter was ligated to the dA tailed fragmented genomic DNA and the linked ligated DNA fragment was used as template of 1<sup>st</sup> round nested PCR to amplify fragments containing adaptor and transposon junction. Indexed barcoded sequencing and illumina sequencing adaptor was added by 2<sup>nd</sup> nested PCR. All sequence libraries were examined by Agilent 2100 Bioanalyzer and subjected to next generation sequencing. </p> <p> </p> <h3><strong>Transposon mutagenesis.</strong></h3> <p><em>Mycobacterium smegmatis</em> mc<sup>2</sup>155 strain was grown to stationary phase (OD>6) in 50ml of 7H9 growth medium. Bacterial cultures were washed and resuspended in 5ml MP buffer (50mM Tris, 150mM NaCl, 10mM MgSO4, 2mM CaCl2). To transduce bacteria with MycoMarT7 phage, approximately 10<sup>11</sup> plaque forming units of phage (PFU) was added to the bacterial suspension in MP buffer and incubated at 37°C for 4h. Immediately after transduction, ~300-400ul of the transduction mixture was plated on 15-cm LB agar plates, containing 20ug/ml kanamycin and 0.1% Tween80. After 3 days, library size was determined, and bacteria was scrapped and stored in 7h9 medium plus 15% glycerol as library stock. The transposon library was made in triplicate. The transposon library was cultured to an OD<sub>600nm</sub> of 0.8 and 1ml of sample was loaded onto 10ml of stock isotonic percoll medium, with buoyant density beads as fiducial markers. Buoyancy centrifugation was conducted at 18°C, and spinning at 20k rpm (~50,000 g), for 1h20m. Three buoyancy fractions were isolated: "high" (>1.02 g cm<sup>-3</sup>, <1.064 g cm<sup>-3</sup>), "middle" (>1.064 g cm<sup>-3</sup>, <1.102 g cm<sup>-3</sup>), and "low" (>1.102 g cm<sup>-3</sup>). Three biological replicates of the buoyancy centrifugation were conducted for the transposon library made in triplicate each of the three transposon libraries: 3 (libraries) x 3 (buoyancy centrifugation experiments) x 3 (buoyancy fractions) = 27 individual samples.</p> <p> </p> <h3><strong>Transposon mapping and analysis.</strong></h3> <p>Reads processing and TA loci mapping were performed through software TRANSIT (<em>31</em>). Loci that were differentially disrupted by transposon were analysed using resampling test in TRANSIT with default parameter. Different buoyancy fractions were compared with input libraries and genes that are over-represented <u>(log2FC < -1, adjusted p-value < 0.05)</u> and under-represented <u>(Log2FC >1, p-value < 0.05)</u> were plotted.</p> </div&gt

Caractérisation de l'histone H3 lysine désacétylation au cours de l'infection par Listeria monocytogenes

De nombreuses bacteries pathogènes sont capables d'affecter les programmes transcriptionnels de la cellule hôte pendant l'infection. Cependant, les mécanismes contrôlant ce processus restent largement méconnus. En investigant les effets de la Listerai monocytogenes sur les modifications des histones de l'hôte, nous avons mis en évidence un nouveau mecanisme de régulation de transcription nécessaire pour la répression de certains gènes, pendant l'infection. Lors de l'infection par L. monocytogenes, le facteur de virulence sécrété, InlB, se lie au récepteur c-Met et active la signalisation par les intermédiaires PI3K et Akt. cette plateforme de signalisation est nécessaire pour la relocalisation de la deacetylase d'histone, SIRT2, au noyau et l'association à la chromatine.En caractérisant me mécanisme gouvernant la relocalisation nucléaire de SIRT2 lors de l'infection, nous avons démontrés que SIRT2 subit une modification post-traductionnelle. SIRT2 est déphosphorylée à un nouveau site de phosphorylation localisé à la partie terminale de la protéine. SIRT2 est recrutée au site de démarrage de la transcription des gènes réprimés lors de l'infection menant à la deacetylation de H3K18 et la répression transcriptionnelle. Nous avons mis en évidence que SIRT2 est détournée par L. monocytogenes et provoque une croissance des bactéries intracellulaires. Ces résultats démontrent un clef de SIRT2 en provoquant la deacetylation de H3K18 mors de l'infection et dévoilent un nouveau mécanisme imposée par les bactéries pathogènes dans le but de reprogrammer la cellule hôte.Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

Evaluation of SARS-CoV-2 serology assays reveals a range of test performance.

Appropriate use and interpretation of serological tests for assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, infection and potential immunity require accurate data on assay performance. We conducted a head-to-head evaluation of ten point-of-care-style lateral flow assays (LFAs) and two laboratory-based enzyme-linked immunosorbent assays to detect anti-SARS-CoV-2 IgM and IgG antibodies in 5-d time intervals from symptom onset and studied the specificity of each assay in pre-coronavirus disease 2019 specimens. The percent of seropositive individuals increased with time, peaking in the latest time interval tested (>20 d after symptom onset). Test specificity ranged from 84.3% to 100.0% and was predominantly affected by variability in IgM results. LFA specificity could be increased by considering weak bands as negative, but this decreased detection of antibodies (sensitivity) in a subset of SARS-CoV-2 real-time PCR-positive cases. Our results underline the importance of seropositivity threshold determination and reader training for reliable LFA deployment. Although there was no standout serological assay, four tests achieved more than 80% positivity at later time points tested and more than 95% specificity

Test performance evaluation of SARS-CoV-2 serological assays.

Background:Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. Method:We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. Results:Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed. Conclusion:Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies

Test performance evaluation of SARS-CoV-2 serological assays.

Background:Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. Method:We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. Results:Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed. Conclusion:Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies