TFEB induces mitochondrial itaconate synthesis to suppress bacterial growth in macrophages

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Antibodies and reagents

The following primary antibodies and dyes were used for immunofluorescence staining (IF) or Western Blot (WB): anti-IRG1 (IF 1:100; Abcam, ab222411), anti-HSP60 (IF 1:1,000; CST, MA5-15836), anti-TFEB (WB: 1:3,000; IF: 1:1,000; Bethyl Laboratories, A303-673A), anti-Salmonella Typhimurium control serum (IF 1:10,000; TS1624, Sifin), anti-Rab32 (WB 1:2,000, LS-C204248, LSBio), anti-actin (WB 1:10,000; SantaCruz, sc-47778). The following secondary antibodies were used: antirabbit HRP-linked (WB 1:10,000; CST, 7074), antigoat HRP-linked (WB 1:10,000; ThermoFisher, 31402), antirabbit Alexa Fluor 647-conjugated (IF 1:500; ThermoFisher, A-21244) and antirabbit Cy3-conjugated (IF 1:1,000; Jackson Immuno Research Laboratories, 111-165-144). LysoTracker Red DND-99 (L7528) was purchased from ThermoFisher. The following stimuli were used: IFNγ (50 ng ml−1; PeproTech, AF-315-05), LPS (20 or 100 ng ml−1; InvivoGen, tlrl-pb5lps), macrophage colony stimulating factor (20 ng ml−1; PeproTech, 315-02), heat-killed M. tuberculosis (hk Mt; 10 µg ml−1; InvivoGen, tlrl-hkmt), heat-killed S. aureus (HKSA; 106 particles per ml; InvivoGen, tlrl-hksa), heat-killed Salmonella Typhimurium (HKST; five particles per BMDM; InvivoGen, tlrl-hkst2). The following chemicals were purchased from Sigma Aldrich: TFEBa (2-hydroxypropyl-β-cyclodextrin 5 mM; H-107); 2-deoxy-d-glucose (2-DG; 10 mM, D6134), 3-methyladenine (3-MA; 10 mM; M9281), l-arabinose (10839), Bafilomycin A1 (Baf, 100 nM, B1793), mouse serum (M5905) and UK5099 (4 µM, PZ0160).

Mice

Mice were maintained in specific pathogen-free conditions under protocols approved by the animal care committee of the Regierungspräsidium Freiburg, Germany, in compliance with all relevant ethical regulations. Mice were housed under controlled conditions, namely 20–21 °C, 55–65% relative humidity and 12/12 h light/dark cycle. Food was available ad libitum for all animals. Eight- to 22-week-old animals were euthanized by carbon dioxide asphyxiation followed by cervical dislocation, and bone marrow or spleens were harvested postmortem.

The following mice were used: C57BL/6J, Tfebfl/fl Vav-iCre or Tfebfl/fl or Lyz2-Cre mice. Tfebfl/fl mice17 were kindly provided by A. Ballabio (Faculty of Medicine, Frederico II University of Naples, Italy). Irg1−/− mice (C57BL/6NJ-Acod1em1(IMPC)J/J), Irf1−/− mice (B6.129S2-Irf1tm1Mak/J) and Hps4−/− mice (B6.C3-Pde6brd1 Hps4le/J) were purchased from the Jackson Laboratories. Ifnar1−/− mice (B6.129s2-Ifnartm(Neo)Agt) and Souris−/− mice (C57BL/6J-Lystbg-Btlr/Mmucd) were kindly provided by P. Stäheli (Institute of Virology, University Clinics Freiburg, Germany) and P. Aichele (Institute for Microbiology and Hygiene, University Clinics Freiburg, Germany), respectively. K. Simons provided bones from Atg7fl/fl Vav-iCre mice (Kennedy Institute of Rheumatology, University of Oxford). For most mice, sex- and age-matched control littermates were used. Sex-matched Cre-negative Tfebfl/fl littermates were used as control for experiments with TFEB-deficient cells, since tested biological responses were unaffected by the presence or absence of Cre.

In vivo infection studies of 12–25-week-old female and male mice (Fig. 4d,g and Extended Data Fig. 4c,d) were infected intra-peritoneally with 5 × 104 CFU per mouse of S. Typhimurium SL1344 with arabinose-induced pFCcGi-Cb. Animals were kept for 3 days and TFEBa (0017; Acacetin, Sigma Aldrich) dissolved in PBS (20 mg kg−1, sonicated for 5 min) or relevant solvent (PBS) was injected daily. For Tfeb-deletion studies, mice with macrophage-specific Tfeb knockout were used (Tfebfl/fl Lyz2-Cre), annotated as TfebΔmac. Mice were euthanized by CO2 and cervical dislocation and spleens were harvested. Cell suspensions were obtained by homogenizing the spleens using 70-μm cell strainers. Erythrocyte lysis (ACK lysing buffer, Gibco A1049201) was performed and unspecific binding was blocked with anti-CD16/32 for 15 min before cells were stained for F4/80 (1:200, BM8, BioLegend, 123137), Cd11b (1:500, M1/70, eBioscience, 50-0112-82) and live/dead (1:200, eBioscience, 65-0866-14) in cold PBS (Gibco) for 1 h. Cells were fixed for 15 min using the Foxp3 transcription factor staining buffer set (eBioscience, 00-5523-00). Data was acquired on a LSR Fortessa (BD) and analysed with FlowJo software (BD, v.10). During analysis, doublets were excluded. For the gating strategy, please see Supplementary Fig. 1.

BMDM culture

Bone marrow was isolated from femur, tibia and pelvic bone of 8–12-week-old male and female mice. BMDMs were differentiated in BMDM medium (RPMI 1640, 10% FCS, 100 U ml−1 penicillin and 0.1 mg ml−1 streptomycin) containing 20 ng ml−1 macrophage colony stimulating factor at 37 °C and 5% CO2. Cells were grown for 6 days and then gathered with 0.25% trypsin. For most experiments, BMDMs were plated in BMDM medium. For Salmonella infection assays, BMDMs were plated in BMDM infection medium (DMEM, 10% FCS, 10% L929 supernatant, 1 mM sodium pyruvate, 4 mM glutamine).

Retrovirus

pBMN-TFEB-GFP and ΔNLS-TFEB-GFP plasmids were kind gifts from R. Youle and S. Ferguson, respectively54,55. The pBMN-Irg1-BFP plasmid was generated by replacing the Tfeb gene in pBMN-TFEB-GFP plasmid with the Irg1 sequence from the pCMV6-Entry-Irg1-Myc-DDK-tagged plasmid purchased from Origene and the GFP was replaced by the blue fluorescent protein (BFP) sequence. New plasmids were generated using the CloneAmp HiFi PCR Premix and In-Fusion HD Cloning Kit (Takara) according to the manufacturer’s instructions. For production of viral particles, 2.5 × 106 PlatE cells were plated and the following day transfected with pBMN-plasmid DNA using Lipofectamine 3000 according to the manufacturer’s instructions. Viral supernatant was collected every 24 h for 4 days.

BMDM transduction and sorting

For BMDM transduction, viral particles (diluted 1:3 in BMDM medium) were added to the bone marrow culture on day 2 of BMDM differentiation. After 18 h, transduction medium was removed and cells were cultured for three additional days. Where necessary, virus-targeted BMDMs were sorted using the BD FACSAria III cell sorter FACSDiva (BD, v.8.0.1).

RNA-seq

RNA isolation was performed using the RNeasy MinElute Cleanup Kit according to the manufacturer’s instructions. Complementary DNA libraries were prepared by the Deep Sequencing facility at the Max Planck Institute of Immunobiology and Epigenetics using the TrueSeq stranded mRNA protocol (Illumina) and sequenced on a HiSeq 3000 (Illumina) platform to a depth of 16 million reads per sample. Sequencing data were analysed using the Galaxy platform provided by the Bioinformatics Core Facility of the Max Planck Institute of Immunobiology and Epigenetics and the University of Freiburg. The STAR aligner56 was used for trimming and mapping, GRCm38 as the reference genome. Quantification of the mapped reads was performed with featureCounts57 (https://doi.org/10.1093/bioinformatics/btt656) and differential gene expression determined using the DESeq2 algorithm58 (https://doi.org/10.1186/s13059-014-0550-8). Expression data were further processed and filtered using R (Lucent Technologies). For biological pathway enrichment analysis, significantly upregulated genes (adjusted P ≤ 0.01) in TFEB-GFP- versus GFP-expressing BMDMs were subjected to the PANTHER classification system v.13 (http://www.pantherdb.org) using the Gene List analysis (Statistical overrepresentation test, Annotation Data Set, PANTHER GO-Slim Biological Process; Reference List, Default Mus musculus genes) to define overrepresented biological processes.

ATAC-seq

BMDMs were collected with 0.25% trypsin and 50,000 BMDMs per sample were lysed in ice-cold lysis buffer (10 mM Tris-Cl, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) Igepal CA-630, pH 7.4), immediately followed by centrifugation at 500g, 4 °C. Pellets containing BMDM nuclei were subjected to transposition reaction using the Nextera DNA Flex Library Prep Kit (Illumina). DNA libraries were sequenced in paired-end mode (75 cycles) on a HiSeq 3000 (Illumina) by the Deep Sequencing facility at the Max Planck Institute of Immunobiology and Epigenetics with a reading depth of 50 million reads per sample in two biological replicates per condition. ATAC-seq was run in two replicates per condition. Adapter sequences were trimmed with Trimmomatic (v.0.36)59 and the Bowtie2 (ref. 60) algorithm (v.2.1.0) using the «–very-sensitive» parameter for aligning ATAC-seq reads to the mouse genome version GRCm38/mm10. Samtools61 (v.0.1.19) were used for data filtering and file format conversion. Duplicate reads and chr M were removed before peak calling. MACS2 (ref. 62) (v.2.1.0) algorithm was used for ATAC-seq peak identification with a P value cut-off of 1 × 103. Genomic regions that are common or different from a set of peak files were identified with BEDTools63 (v.v.2.25.0). All .bam files were converted to bedgraphs with genomeCoverageBed a subcommand of BEDTools63. Gene annotation (100 kb upstream and 50 kb downstream from the transcription start site) and genomic distribution of accessible regions identified by MACS2 (ref. 62) were performed with BEDTools63 and -closetBed and -intersectBed subcommands, respectively. Clustering of regions was generated with ComputeMatrix function of DeepTools64, using reference-point–referencePoint centre -b 3000 -a 3000 -R<bed files>-S<bigwig files> as parameters. The function plotHeatmap from the same package was used for displaying the average profiles heatmap. Differentially accessible chromatin regions were scanned for enriched short-sequence motifs using HOMER65 software with the ‘findMotifsGenome.pl’ command. To search for a set of sequences for occurrences of specific known motifs FIMO66 from the MEME suite67 was used. For the Irg1 gene a window of 1 kb upstream and downstream from the start and end of the two significant gained narrow peaks was analysed with BEDTools 5 subcommand -slopBed -b 1000 and motif occurrences with a P value of less than 0.0001 were chosen.

ChIP

ChIP was performed as previously described68. Briefly, for each ChIP experiment 8–10 million cells were cross-linked with 1% formaldehyde (Pierce) for 10 min at room temperature, nuclei were isolated and chromatin was sonicated at 4 °C using a Bioruptor (Diagenode) for 25 cycles (30 s ‘ON’ and 30 s ‘OFF’, power setting high). For each immunoprecipitation 18 μl of antibody against TFEB (Cell Signaling, no. 37785, D2O7D) were incubated with chromatin at 4 °C with rotation overnight. Chromatin was washed, crosslink was reversed at 65 °C overnight and DNA was isolated using Agencourt AMPure magnetic beads (Beckman Coulter). Subsequently, qPCR was performed (StepOne, Applied Biosystems) using ChIP and input DNA amplifying different regions around the transcriptional start site of Acod1 (Irg1). Enrichment of TFEB binding was calculated as ChIP–DNA relative to input-DNA PCR signal for each primer pair and normalized to a negative control region (non-accessible heterochromatin region).

Lysosomal mass measurements

BMDMs were incubated with 75 nM LysoTracker Red for 30 min at 37 °C in BMDM medium. Cells were washed three times with prewarmed BMDM culture medium, gathered with 20 mM EDTA and incubated for 15 min on ice with Live Dead Fixable Viability eFluor 780 (1:1,000) in PBS. Samples were measured on the BD LSR Fortessa cell analyser (BD Biosciences). Data were analysed and graphs generated in FlowJo v.10.

Real-time qPCR

BMDMs were gathered in 250 µl TriReagent per well and RNA was isolated by phenol-chloroform extraction. cDNA synthesis was performed with the QuantiTect Reverse Transcription Kit according to the manufacturer’s instructions. As template, 200 ng of RNA were used. The reverse transcription reaction was performed for 30 min at 42 °C. Measurement of Irg1, Tfeb, β-actin and β2-microglobulin mRNA expression was carried out in a 96-well plate using the Thermo Scientific ABsolute Blue QPCR SYBR Green Low ROX Mix according to the manufacturer’s instructions using 1 µl of cDNA and 22 ng of the respective primers. Samples were measured in the 7500 Fast Real-Time PCR System (Applied Bioscience) and analysed via StepOne Software (AB, v.2.0). To quantify relative Irg1 mRNA expression, Irg1 mRNA levels were normalized to the expression of the housekeeping genes β-actin and β2-microglobulin. Relative mRNA expression values were calculated using the 2(-ΔΔCT) method and normalized to unstimulated control samples. For Fig. 3g and Extended Fig. 3d (2-DG treatment), Irg1 expression levels were normalized to Tfeb expression levels per sample because the genotype of the cells or the treatment affected Tfeb expression levels.

Seahorse flux analysis

Extracellular acidification rate and oxygen consumption rate were determined with a Seahorse Flux Analyser XF96 (Agilent Technologies) from GFP- or TFEB-GFP-expressing BMDMs. Seahorse XF base medium was supplemented with 25 mM glucose, 2 mM glutamine, 1 mM sodium pyruvate and 1% FCS. Seahorse measurements were normalized to protein content, determined with the Pierce BCA Protein Assay Kit or cell numbers by using in situ Hoechst staining of nuclei. Nuclear stainings were acquired with the BioTek Cytation 1/5. Nuclei counting was performed with the Seahorse XF and Cell Counting Software and the Wave v.2.6 Software (Agilent Technologies). Oxygen consumption rate data were calculated as area under the curve and values were plotted using GraphPad Prism v.8.2.1.

Metabolic tracing

Metabolic tracing with 13C -glucose, 13C-glutamine and 13C-palmitate was performed with gas chromatography coupled to tandem mass spectrometry (GC–MS/MS). For glucose and glutamine tracing, BMDMs were incubated for 6 h in glucose- or glutamine-free BMDM medium supplemented with 11 mM 13C-glucose or 4 mM 13C -glutamine, respectively. For palmitate tracing, full BMDM medium containing 20 µM BSA-conjugated 13C-palmitic acid was used, as BMDMs were dying in lipid-deprived medium. To extract metabolites, BMDMs were washed once with ice-cold 0.9% NaCl in MilliQ-H2O, shock frozen in an ethanol-dry ice bath and collected with a cell lifter in ice-cold 80% methanol containing 1 µg ml−1 norvaline and 1 µg ml−1 adipic acid (internal standards). Cell debris was removed by centrifugation for 5 min at 20,000g and 4 °C. Methanol supernatants were collected and dried in a Genevac EZ-2 (SP Scientific). Metabolites were resuspended in 10 µl D27/methoxyamine mix (10 mg ml−1 methoxyamine hydrochloride, 0.2 µg ml−1 myristic-D27 acid in pyridine) for 1 h at 30 °C. Then 7.5 µl of the mix were derivatized with 15 µl of N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamid, with 1% tert-butyldimethyl-chlorosilane (375934 Sigma Aldrich) for 60 min at 80 °C. Isotopomer distributions were measured using a DB5-MS GC column in a 7890 GC system (Agilent Technologies) combined with a 5977 MS system (Agilent Technologies). Data from tracing experiments are presented as 13C-labelled metabolite fractions of total respective metabolite level.

Intracellular itaconate measurements

Polar metabolome quantifications were performed with LC–MS. BMDMs were stimulated as indicated. For metabolite extraction, cells were washed once with ice-cold 3% glycerol in MilliQ-H2O, followed by 5 min incubation on ice in prechilled 80% methanol. Methanol supernatants were collected and cell debris was removed by centrifugation for 5 min at 15,000g and 4 °C. Metabolite solutions were dried in a Genevac EZ-2 (SP Scientific) and subsequently resuspended in 15 µl of 90% acetonitrile containing 13C-yeast-standard (ISOtopic Solutions) as loading control. Suspensions were centrifuged for 10 min at 3,300g and 4 °C and 10 µl of each sample were transferred to a fresh container and used for mass spectrometry. Targeted metabolite quantification by LC–MS was carried out using an Agilent 1290 Infinity II UHPLC in line with an Agilent 6495 triple quadrupole–MS operating in MRM mode. MRM settings were optimized separately for all compounds using pure standards. LC separation was on a Phenomenex Luna propylamine column (50 × 2 mm, 3-μm particles), with, a solvent gradient of 100% buffer B (5 mM ammonium carbonate in 90% acetonitrile) to 90% buffer A (10 mM NH4 in water). Flow rate was from 1,000 to 750 µl min−1. Autosampler temperature was 5 °C and injection volume 2 µl. Values represent the area of the metabolite peaks from mass spectrometry as arbitrary units.

Western blotting

BMDMs were collected in ice-cold PBS with a cell lifter and pelleted by centrifugation for 5 min at 500g and 4 °C. Cell pellets were lysed for 15 min on ice with lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% Triton X-100, pH 7.4) with 1× Halt Protease Inhibitor Cocktail and 1× Phosphatase Inhibitor Cocktail and sheared with a 26 G insulin syringe. Cell debris was removed by centrifugation at 16,000g and 4 °C for 15 min. Then 25 to 35 µg of total protein was loaded on a 10 or 12% polyacrylamide gels. Protein transfer to a polyvinyl difluoride-membrane (Merck Millipore) was performed in a semidry blotting chamber for 90 min at 10 V. Membranes were blocked for 1 h in 5% milk in tris-buffered saline (TBS) with 0.1% Tween (TBST) at room temperature. Incubation with primary antibodies was performed overnight at 4 °C in buffers suggested for the specific antibody or in TBST containing 2% BSA. Incubation with secondary antibodies was performed for 1 h at room temperature in 5% milk in TBST. For signal detection, Amersham ECL Prime Western Blotting Detection Reagent was used and signals were acquired with the ChemiDoc Touch Gel Imaging System (Bio-Rad). Images were prepared for publication with the Image Lab v.5.2 TM Touch Software (Bio-Rad, v.1.0.0.15).

Immunofluorescence

BMDMs were plated in tissue culture treated 24-well plates containing fibronectin-coated 12 mm glass coverslips. To visualize TFEB, HSP60 or Salmonella, BMDMs were fixed for 15 min at room temperature in 4% paraformaldehyde, prewarmed to 37 °C, followed by permeabilization in 0.2% Triton X-100 in PBS. To visualize endogenous Irg1, BMDMs were fixed and permeabilized in ice-cold 100% methanol for 15 min at −20 °C. In both cases, unspecific binding sites were blocked afterwards for 1 h at room temperature in blocking buffer (0.1% Tween20, 5% FCS in PBS). Cells were incubated at 4 °C for 16 h with primary antibodies in blocking buffer. Secondary antibody stainings were performed in blocking buffer for 1 h at room temperature. BMDMs were mounted in Fluoromount-G supplemented with or without 4,6-diamidino-2-phenylindole (DAPI).

Confocal microscopy and image processing

Z-stacks were acquired with an inverted LSM880 or LSM780 Zeiss confocal microscope and ZEN software black edition (Carl Zeiss Microscopy, v.2.6). Brightness and contrast were adjusted and images prepared using Fiji ImageJ69,70. 3D-rendered images of mitochondria-pathogen interactions were generated using the surface tool of Imaris v.9.5.1 (Bitplane). For better visualization of differences in expression levels, Irg1 and Salmonella-mCherry fluorescent signals in Fig. 3b and Extended Fig. 4j were pseudocolored in ImageJ using the look-up table ‘red hot’.

Image analysis

To quantify nuclear TFEB-signals 3D nuclear masks generated from DAPI signal were generated, using the Imaris v.9.4.1 (Bitplane) surface tool (smoothing 0.51, signal intensities were set manually for each image). To quantify cellular TFEB levels in TFEB-GFP- or GFP-expressing BMDMs, cellular masks were generated on the basis of ectopically expressed GFP signals, as described for nuclear TFEB levels.

Mitochondria-pathogen interactions were determined from single slice images using the Pearson’s Coefficient ImageJ Jacob colocalization software tool (https://imagej.nih.gov/ij/plugins/track/jacop.html)69,70. Colocalized pixels were identified in individual slices using the ImageJ colocalization tool (channel cut-off 50)69,70.

To determine the bacterial load of individual macrophages by imaging (Extended Data Figure 5f,g), maximum intensity projections of images taken from Salmonella-mCherry infected BMDMs were transformed to binary images and signal per cell were measured using the ImageJ analysis tool69,70. Measured signals were presented as frequency distributions in Extended Data Fig. 5g. Data were normalized for bacterial signals in TFEBa relative to vehicle-treated BMDMs for each independent experiment. The proliferating Salmonella subpopulation was determined on the basis of signal representing >15 bacteria per cell. Signals containing the growing Salmonella subpopulation (from bins 10–14) were summarized for vehicle and TFEBa-treated BMDMs for each genotype and depicted as ratio TFEBa/vehicle-treated in Fig. 4k.

Salmonella infection of BMDMs

S. enterica serovar Typhimurium strain SL1344, harbouring the pFCcGi plasmid, was cultured for 16 h at 37 °C in a minimum MgMES medium (170 mM MES, 5 mM KCl, 7.5 mM (NH4)2SO, 0.5 mM K2SO4, 1 mM KH2PO4, 8 µM MgCl2, 38 M glycerol, 0.1% casamino acid, pH 5.8, 100 µg ml−1 ampicillin) supplemented with with 0.2% (w/v) l-arabinose and 100 µg ml−1 carbenicillin. Before infection of BMDMs, bacteria were opsonized for 20 min with 10% mouse serum in BMDM infection medium. BMDMs were pretreated or not with TFEBa for 3 h before opsonized living or heat-killed Salmonella were added. BMDMs were infected at a multiplicity of infection (MOI) of 5 for all experiments and incubated for 18.5 h postinfection. Host–bacteria interactions were synchronized by centrifugation for 10 min at 300g and room temperature. After 30 min, extracellular bacteria were killed by addition of gentamycin (100 µg ml−1) containing BMDM infection medium. After 30 min, gentamycin concentration was reduced to 10 µg ml−1, TFEBa or itaconate were added to BMDMs and cells were collected or incubated for further 18 h.

Analysis of Salmonella subpopulations by flow cytometry

To determine intracellular Salmonella subpopulations (growing, non-growing, host-killed) infected BMDMs were washed once with cold PBS and then collected on ice with a cell lifter in PBS. Per condition, three technical replicates were pooled. To assess Salmonella subpopulations on the basis of GFP and mCherry signals, samples were measured on a BD FACSAria III cell sorter with FACSDiva (BD, v.8.0.1). Control gates were set on the basis of there being uninfected or 30-min infected BMDMs (Extended Data Fig. 4d).

Plating assay to assess intra-macrophage bacterial survival rates

BMDMs infected for 18.5 h were washed once with cold PBS and immediately lysed in 1 ml of PBS with 0.1% Triton X-100. Serial dilutions (1:10, 1:100, 1:1,000) were plated on Luria-Bertani (LB) agar plates containing 100 µg ml−1 ampicillin and incubated at 37 °C for 16 h to allow Salmonella regrowth. CFUs were counted manually.

Luciferase assays

NanoLuc-ITA-Salmonella-infected BMDMs were lysed 18.5 h post-infection in Passive Lysis Buffer (E1941, Promega) and luciferase activity was determined using the Nano-Glo-Luciferase Assay System (N1110, Promega) according to the manufacturer’s instructions and a Centro LB 963 Microplate Luminometer (Berthold). In parallel, CFUs were determined and luminescence values were normalized to the ratio of CFUs between TFEBa-treated and control BMDMs. For Irg1-promotor luciferase measurements, mouse embryonic fibroblasts (ATCC CRL-2907) were cotransfected with the indicated Irg1-promoter-firefly luciferase constructs and GFP as control or the indicated TFEB-GFP constructs. Then 24 h after transfection, cells were treated with medium or hk SmT (105 particles per ml) for 3 h and luciferase expression was measured using the Glo-Luciferase Kit (Promega) according to the manufacturer’s instructions on a Centro LB 963 Microplate Luminometer (Berthold). Luciferase-expression levels were quantified as fold increase relative to Irg1-promoter-luciferase/GFP-coexpressing control cells.

Bacterial SPI-2 expression measurements

BMDMs were infected with Salmonella Typhimurium strain SL1344 carrying the PssaG::gfp plasmid71, encoding a GFP-reporter gene under the control of the bona fide SPI-2 promotor of the ssaG gene. Bacteria were grown overnight in LB medium containing 50 µg ml−1 chloramphenicol. Opsonization and BMDM infection were performed as described in the Salmonella infection assay. To assess SPI-2 GFP-reporter expression, BMDMs infected for 18.5 h were fixed at room temperature for 15 min in 4% paraformaldehyde before being measured on a BD LSR Fortessa cell analyser (BD Biosciences), with FACSDiva (BD, v.8.0.1). FACS data were analysed with FlowJo v.10 software.

In vitro Salmonella survival assay

Salmonella was grown in 50 ml of LB medium to an optical density (OD600) of 2. Subsequently, 2.5 ml of bacterial suspension were collected, spun down and bacteria were resuspended in 5 ml of either LB medium (100 µg ml−1 ampicillin) or minimal medium (170 mM MES, 5 mM KCl, 7.5 mM (NH4)2SO, 0.5 mM K2SO4, 1 mM KH2PO4, 8 µM MgCl2, 38 mM glycerol, 0.1% casamino acid, pH 5.8, 100 µg ml−1 ampicillin). Bacterial cultures were incubated with TFEBa (5 mM) and bacterial growth/survival were inferred from CFUs.

Quantification and statistical analysis

(Statistical analysis and data representation)

Graphs were generated and statistical analysis was carried out with GraphPad Prism v.8.2.1. To determine statistical significance, different tests were used as indicated in the figure legends. The number of experimental repeats is indicated in the figure legends. Proportional Venn diagrams for overlapping genes were generated with BioVenn72. Statistical significance of the overlap between the two groups of genes was calculated with a hypergeometric statistical test. Heatmaps were generated with Morpheus (Broad Institute) and schematics and figures with Adobe Illustrator CS5 (Adobe). Gene tracks of the Irg1 locus were generated with the Integrative Genomics Viewer (IGV)73.

Materials availability

No new, unique reagents, plasmids or mice were generated in this study. A Material Transfer Agreement exists for the use of Tfebflf/fl mice. These mice can only be shared via A.Ballabio.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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