Bacterial and viral strains
bacterial strain | mutant | resistance | overnight growth |
Salmonella enterica Typhimurium SL1344 | Wt, pGG2-dsRed | ampicillin | 37°C |
Shigella flexneri M90T | Wt, pGG2-dsRed | ampicillin | 37°C, +Kongo Red (agar plates) |
Citrobacter rodentium ICC169 | Wt, pGG2-dsRed | ampicillin | 37°C |
Vibrio cholerae | ClpV-mCherry | none | 37°C |
Yersinia enterocolitica MRS40 | Wt, pGG2-dsRed | ampicillin | 26°C |
Pathogenic Escherichia coli uti89 | Wt, RFP-expressing | kanamycin | 37°C |
Yersinia pseudotuberculosis IP2777 | Wt, pFPV-mCherry | ampicillin | 26°C |
Y. pseudotuberculosis IP2777 | pVY-, pGG2-dsRed | ampicillin | 26°C |
Y. pseudotuberculosis IP2777 | ΔyopE, pGG2-dsRed | ampicillin | 26°C |
Y. pseudotuberculosis IP2777 | ΔyopH, pGG2-dsRed | ampicillin | 26°C |
Y. pseudotuberculosis IP2777 | ΔyopE ΔyopH, pGG2-dsRed | ampicillin | 26°C |
Y. pseudotuberculosis IP2777 | ΔyopE ΔyopH ΔyopT, pGG2-dsRed | ampicillin | 26°C |
Y. pseudotuberculosis IP2777 | ΔyopJ, pFPV-mCherry | ampicillin | 26°C |
viral strain | genotype | titer | used MOI | co-infection delay |
Murine Adenovirus (mAdV) 1 | IX-FS2A-GFP | 1.3⋅107/ml | 2 | 6h |
Murine Adenovirus (mAdV) 2 | ΔE1A-GFP | 5.0⋅106/ml | 1 | 6h |
Murine Adenovirus (mAdV) 3 | IX-FS2A-GFP | 5.0⋅107/ml | 5 | 8h |
Murine Norovirus (MNV) 1 | wildtype | 1.3⋅107/ml | 1 | 6h |
Murine Norovirus (MNV) 2 | wildtype | 6.3⋅106/ml | 1 | 24h |
Murine Norovirus (MNV) 3 | wildtype | 3.0⋅107/ml | 3 | 24h |
Murine Norovirus (MNV) CR3 | wildtype | 2.0⋅106/ml | 0.25 | 20h |
Cell lines and culture
The following cell models were used throughout this study and, unless specified otherwise, grown at 37°C, 5% CO2 in the indicated media: RAW264.7 macrophages (ATCC, TIB-71) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco), containing 10% Fetal Calf Serum (FCS, Thermo Scientific). Bone-Marrow-derived macrophages (BMDMs, harvested from wildtype mice) in DMEM, supplemented with 10% FCS and 20% recombinant Macrophage Colony Stimulating Factor (MCSF, produced in the lab from L929 cells). Immortalized BMDMs (iBMDMs, previously produced in the lab) in DMEM containing 10% FCS and 10% MCSF.
RAW264.7 and iBMDMs were maintained by regular splitting into fresh media in tissue-culture treated flasks (TPP), after scraping and washing cells, and controlling for viability. BMDMs were thawed and seeded into non-tissue culture treated dishes (Falcon) for one day. For seeding, cells were detached by incubation at 4°C in pre-chilled Phosphate Buffer Saline (PBS, Gibco) for 15 minutes and counted in a Countess automated cell counter with Trypan Blue (Thermo Fisher Scientific) viability stain.
Screening of host-pathogen-pathogen interactions
The day prior to infection, 30000 RAW264.7 cells were seeded per well of a 96-well plate and primed for at least 6h with 10µg/ml m-IFNγ (Stemcell Technologies). Bacterial overnight cultures were started by inoculating 3ml LB-media containing appropriate antibiotics with a single colony from an agar plate, and grown at 30°C with agitation. For simultaneous co-infection, media was replaced with DMEM containing viral particles at the MOI indicated in the table of viral strains, which was determined prior to the screen to allow for dynamic range.
Cells were placed in a pre-heated centrifuge at 300G, while the bacterial infection was prepared. Bacterial overnight cultures were adjusted by OD600 and washed once in PBS. Assuming a host cell number of 50000 cells per well, bacteria were added at an MOI of 50 directly onto the viral infection media, as a control, cells were treated with 100µg/ml Lipo-Polysaccharide B5 (LPS, Invivogen). Cells were centrifuged for 5 minutes at 300G and placed at 37°C for 30 minutes. Subsequently, cells were washed once in pre-warmed PBS and the media was replaced with OptiMEM (Gibco) containing 20µg/ml gentamycin (Invitrogen), 12.5µg/ml propidium iodide (PI, Thermo Fisher Scientific) and 0.1% TritonX-100 (Tx100, ITW Reagents) where applicable, as indicated in the layout (conditions for the 12 columns indicated, rows: untreated, LPS control, each of the 6 bacterial pathogens)
virally uninfected | virus added |
+PI + Tx100 | media only | +PI | +PI | media only | +PI + Tx100 |
1 well | 2 wells | 3 wells | 3 wells | 2 wells | 1 well |
In the case of subsequent infections, the viral pre-infection was performed by replacing the priming media with viral suspension, and centrifugation for 1h at 37°C and 300G. The cells were maintained at 37°C as indicated in the strain table, which had been determined before conducting the screen, prior to bacterial infection, which was performed as described above.
For dynamic measurement, plates were placed in a plate reader (Biotek Cytation 5, serial number: 1602037, software version: 03/04/2017) and fluorescence intensity was recorded in two channels (1: excitation at 479/20nm, emission at 520/20nm; 2: excitation at 550/20nm, emission at 591/20nm) in 10-minute intervals for at least 16h at 37°C, 5% CO2.
Data cleanup and primary analysis
Cell death was calculated based on the fluorescence measurements obtained in channel 1. Firstly, the average fluorescence intensity obtained in wells containing DMEM + gentamycin was subtracted for each condition individually to account for background signal of the fluorophores expressed by the bacterial pathogen. Secondly, using the total lysis control (100%) and the uninfected control (0%) as reference points, cell death was quantified for each well. Thirdly, values were averaged across intervals of 30 minutes to reduce measurement artifacts. Finally, values below 0% or above 100% were set to 0% or 100% to maintain biological meaningfulness.
Similarly, pathogen growth was quantified using the intensity values obtained in wells containing DMEM + gentamycin (and hence no PI), using channel 1 for bacterial growth and channel 2 for viral growth, with respect to the uninfected controls. Smoothening was performed over intervals of 50 minutes and no capping was used.
Calculation of dynamic metrics and epistatic effects
The expected curve was calculated based on single infections, assuming Bliss independence: For bacterial and viral growth, the comparison occurred directly between singly and doubly infected condition, for cell death, the expected curve was calculated for each timepoint, as the product of the fraction of live cells quantified in the single infection controls.
Dynamic metrics were calculated and defined as follows: max. death: the 98th percentile of all values obtained for cell death; tonset: the time when cell death increases to more than 2% of max. death (with respect to the minimum cell death); t50: as tonset, but for 50% of max. death; gradient: difference between max. death and min. death, divided by the difference between tonset and tend (when 98% of max. death are reached); AUC score: The integral of the curve, based on the average Riemann sum.
In a next step, epistatic effects were calculated as the difference between the observed and the expected value for each metric. Z-transformation was applied to normalize distributions, by dividing the difference to the mean value with the standard deviation across all values for a given metric. Representation as heat maps was done in GraphPad Prism (version 10.2.0) and all raw data, cleaned values and calculated scores are available on Mendeley Data (DOI: 10.17632/thjzhzdpvc.1).
Quality control and replicate reproducibility
To evaluate data quality and replicate reproducibility, the two biological replicates were compared by Pearson correlation of each interaction pair, as well as assessing p-values of paired t-tests (see Mendeley Data DOI: 10.17632/thjzhzdpvc.1). Additionally, the dynamic range was taken into account to determine if a third biological replicate was necessary to ensure data reliability. This was only the case for mAdV3 subsequent infection, for which the average across all three replicates was used for further analysis.
Lactate dehydrogenase (LDH) assay
To validate detected interactions in cell death, cells were seeded, primed and infected as described above. After the initial bacterial infection, the media was replaced with OptiMEM containing 20µg/ml gentamycin in all wells and the plate was maintained at 37°C. At the timepoint that was to be validated, one uninfected and one virally infected well were chosen as total lysis control and Tx100 was added for a final concentration of 0.1% Tx100 for 15min. 30µl supernatant per well were added to 30µl LDH reagent (Sigma Aldrich, prepared as indicated by the manufacturer) for 20min. The reaction was stopped by adding 30µl 1M HCl and the plate was measured on a Biotek plate reader at 490nm.
Cell death for each well was calculated with respect to the uninfected (0% cell death) and the total lysis (100% cell death) controls. The total lysis control of the virally pre-infected sample was used as quality control to prevent artifacts from excessive cell death and reduction in host cell number prior to bacterial infection.
Quantification of Colony Forming units (CFUs)
Parallel to determining host cell death by LDH-release, CFUs were quantified to assess bacterial growth. To do so, all gentamycin-containing OptiMEM was removed and cells were washed once in pre-warmed PBS. 100µl 0.1% Tx100 were added to lyse host cells and serial 1:5 dilutions of the bacteria-containing lysate were prepared. 7µl of each dilution was spotted on an agar plate containing the appropriate selection antibiotic and grown at the appropriate condition for the respective pathogen. Colonies were counted in technical triplicates and CFUs/ml were quantified with respect to the dilution.
Calculation of validation rate
To assess the validation rate, the directionality (synergistic or antagonistic) of each tested interaction (combination of virus, bacterium, infection dynamic and readout) was assessed, and compared to the Bliss score obtained in the screen. For LDH-release, the expected value was calculated by subtracting the product of the fractions of alive cells in the single infections from 100%. For bacterial growth, the expected value was adjusted to the total host cell number, as approximated by the comparison of the total lysis controls for the virally infected and uninfected samples. The validation Bliss score was then calculated as the difference between the observed and the expected outcome, divided by the expected outcome.
Stable isotope labeling of amino acids in cell culture (SILAC) during viral infection
iBMDMs were seeded in tissue-culture treated 6-well plates (1 million cells per well) and primed for at least 6h with IFNγ. Infection with mAdV2 or mAdV3 was performed by adding 750µl viral suspension in iBMDM media containing R+ 10 (U-13C6 99%,15N2 99%, Cambridge Isotope Laboratories) and K+ 8 (U-13C6 99%; U-15N4 99%, Cambridge Isotope Laboratories) (SILAC heavy media), using iBMDM media containing R+ 6 (13C6, 99%, CIL) and K+ 4 (4,4,5,5-D4, 96–98%, CIL) (SILAC intermediate media) as uninfected control. 16hpi, media was removed, cells were washed twice in PBS and subsequently lysed in 500µl 100mM Tris pH7.5, containing 4% SDS (FASP-buffer), supplemented with 10mM DTT (Merck). Lysates were sonicated to shear DNA, boiled at 95°C for 5 minutes and cleared by centrifugation at full speed for 10min. Cleared lysates were further processed at the Protein Analysis Facility of the University of Lausanne.
After determination of protein concentration (tryptophan fluorescence method77), H and I samples were mixed at an equimolar ratio (total: 100µg) and digested (SP3 method78 using magnetic Sera-Mag Speedbeads (Cytiva 45152105050250, 50mg/ml). To alkylate, proteins were treated with 32mM iodoacetamide (final concentration) for 45min at RT in the dark. Precipitation was done on beads (10:1 (w:w) ratio beads:material) using ethanol (final concentration: 60%), and after 3 washes with 80% ethanol, beads were digested in 100mM ammonium bicarbonate containing 1µg trypsin (Promega #V5073), final volume 50µl, for 2h at 37°C. The same amount of trypsin was added for an additional 1h of digest. Supernatants were recovered and mixed with two sample volumes of isopropanol containing 1% TFA. Samples were desalted on a strong cation exchange (SCX) plate (Oasis MCX; Waters Corp., Milford, MA) by centrifugation, washed with isopropanol containing 1%TFA, eluted in 200µl 80% MeCN, 19% water, 1% (v/v) ammonia, and dried by centrifugal evaporation.
Fractionation and Liquid Chromatography / Mass Spectrometry (LC/MS)
Samples treated for fractionation and LC/MS as previously published by the Protein Analysis Facility at the University of Lausanne. In brief, samples were fractionated in 6 fractions using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific). The fractions collected were in 7.5, 10, 12.5, 15, 20 and 50% acetonitrile in 0.1% triethylamine (~ pH 10), redissolved in 2% acetonitrile with 0.5% TFA and used for LC-MS/MS analysis.
LC-MS/MS analysis was carried out on a Fusion Tribrid Orbitrap mass spectrometer (Thermo Fisher Scientific) connected through a nano-electrospray ion source to an Ultimate 3000 RSLCnano HPLC system (Dionex), via a FAIMS interface. Peptides were separated on a reversed-phase custom packed 45cm C18 column (75µm ID, 100Å, Reprosil Pur 1.9µm particles, Dr. Maisch, Germany, 4–90% acetonitrile gradient in 0.1% formic acid (total time 140min)). Cycling through three compensation voltages (-40, -50, -60V) was used to acquire full MS survey scans at 120'000 resolution. A data-dependent acquisition method in the Xcalibur software (Thermo Fisher Scientific) was set up, optimizing the number of precursors selected (“top speed”) of charge 2 + to 5 + from each survey scan, while maintaining a fixed scan cycle of 1s per FAIMS CV. Peptides were fragmented by higher energy collision dissociation (HCD) with a normalized energy of 32%. The precursor isolation window used was 1.6Th, and the MS2 scans were done in the ion trap. The m/z of fragmented precursors was then dynamically excluded from selection during 60s.
Proteomic data analysis and GO-term enrichment
As described in other recent publications79, analysis was performed with MaxQuant 2.1.4.080, using the Andromeda search engine81. The following modifications were selected: cysteine carbamidomethylation (fixed), methionine oxidation (variable), protein N-terminal acetylation (variable), SILAC heavy labeling (K+ 8 and R+ 10), SILAC intermediate labeling (K+ 4 and R+ 6). The mouse (Mus musculus) reference proteome based on the UniProt database (RefProt_Mus_musculus_20230301.fasta, from www.uniprot.org, version of January 2023, containing 55’309 sequences), and a “contaminant” database (most usual environmental contaminants, enzymes used for digestion) were used with a mass tolerance of 4.5ppm on precursors (after recalibration) and 20ppm on MS/MS fragments. All identifications were filtered at 1% FDR relative to hits against a decoy database (reversed protein sequences). Filtering and processing of MaxQuant outputs were performed using Perseus (version 1.6.15.0)82, contaminants were removed, and SILAC ratios were log2-transformed.
Heavy-to-intermediate-ratios were combined across replicates using the protein name to map across runs. Only proteins which had quantified ratios in at least two replicates were kept and p-values were calculated with respect to the null hypothesis that the logarithmic ratio between H and I was 0. For GO-term enrichment, selected proteins (logarithmic fold change larger 0.5, p-value smaller than 0.05) were queried for over-representation against the full list of GO terms for biological function (GO Ontology database DOI: 10.5281/zenodo.8436609 Released 2023-10-09) in the Panther database (PANTHER Overrepresentation Test (Released 20231017)), using the full mouse proteome as reference. Fisher’s exact test without correction was used for p-value analysis and only GO-terms (one per hierarchy group) with at least 3 proteins, a p-value smaller than 0.01 and a fold-enrichment larger than 3 were kept.
Sterile inflammasome stimulation with and without mAdV3 pre-infection
The day prior to the experiment, BMDMs were seeded in 96-well microscopy plates (Greiner) and stimulated the next morning with IFNγ, at least 6h prior to viral infection with mAdV3 (MOI of 5), which was performed as described above. Cells were left in the incubator overnight and subsequently treated with 100µg/ml LPS, by direct addition to the viral suspension / uninfected control. After 6h, media was removed and inflammasomes were stimulated by treatment with 5µg/ml nigericin (Sigma-Aldrich) or 12.5µM UCN-01 (Sigma Aldrich), transfection with 125ng/ml poly-dA-dT (using Lipofectamine LTX, Thermo Fisher Scientific) or infection with Salmonella which were subcultured for 3.5h (1.4 < OD600 < 1.8) at an MOI of 50. After 2h, plates were spun down, 100µl supernatant was harvested and used for LDH-release and ELISA for IL-1ß (R&D Systems).
The remaining supernatant was discarded, and the cells were washed twice in PBS. Cells were fixed with 4% paraformaldehyde (PFA, Electron Microscopy Sciences) for 10 minutes at RT and cell membranes were solubilized with 0.1% Tx100, 1% BSA (Thermo Scientific) in PBS. Antibody staining (anti-ASC, Adipogen) was performed at 1:1000 dilution overnight at 4°C in solubilization buffer and plates were subsequently washed and co-incubated in secondary antibody (Donkey-anti-Rabbit-Alexa-647, Invitrogen, 1:5000) in solubilization buffer at RT for 1h.
To stain nuclei, Hoechst 33342 (Fisher Scientific) was added for 30min at a 1:5000 dilution, and cells were washed three times prior to image acquisition. For microscopy, images (z-stack spanning the entire cell) were taken at 20x magnification on a Zeiss LSM800 confocal laser scanning microscope using Zeiss Zen Blue software (version 3.8) with the appropriate laser wavelengths and filters. At least 3 fields of view (z-stacks) were acquired for each biological replicate and ASC-speck-positive cells were quantified by automated counting of total cell number and cells with ASC-specks using ImageJ (version 1.53t), after summing all planes of each z-stack.
ELISA was performed according to the manufacturer’s protocol and sample input was diluted where required, according to LDH-release. Plates were read at 450nm and 570nm and the concentration of IL-1ß was calculated with respect to a standard curve using the absorption difference between the two channels, spanning 15.625 to 1000pg/ml (quadratic approximation).
Analysis of mAdV2-infected populations by FACS
To quantify bacterial uptake, BMDMs were seeded the day prior to viral infection in non-tissue culture treated 24-well plates (Eppendorff) and primed the next morning with IFNγ, at least 6h before mAdV2 infection at an MOI of 1, overnight. Bacteria were cultured overnight (at 37°C, Yersinia at 26°C) while shaking and Yersinia strains and mutants were subcultured (1:25 dilution) at 26°C for 1h and 37°C for 1h to induce the T3SS. Bacterial infection at MOI 5 were performed as described above, and the cells were harvested directly after invasion by incubating the infected cells for 15 minutes in chilled PBS at 4°C. Cells were detached and washed once in PBS, and subsequently stained with violet fixable violet live-dead dye (Thermo Scientific) as described by the manufacturer. Cells were washed once more and fixed in 4% PFA for 10 minutes. Subsequently, cells were transferred into 96-well U-bottom FACS plates (Brand) and flow cytometry was performed on a Cytoflex S (Beckman Coulter), where at least 20000 events were recorded for each condition in each replicate. Cells were gated by forward- and side-scatter (Cells), as well as side-scatter height and area (Single Cells) and by PB450-negativity (live cells). FITC-signal (virus) and ECD-signal (bacteria) were used for the quantification of uninfected, singly, and doubly infected cells, with respect to an uninfected control.
siRNA-mediated knockdown of Mprip
Pooled siRNAs for Mprip were ordered as SMARTpool from siGENOME which contains the following four siRNAs: GAUCAUCAGUGGGUGGUUA, GGAAAUGGCAGCGACGAUU, GGAUGGUGGUCGGAAAGUA, GCAAGUGUCAGAACUGCUU (M-058568-00-0005). Prior to the siRNA transfection, BMDMs were seeded in the appropriate format (50000 cells per well for 96-well plates (microscopy suited), 250000 cells per well for non-tissue culture treated 24-well plates). The transfection mix was prepared by mixing 100µl OptiMEM with 3µl XtremeGENE 9 transfection reagent (Sigma Aldrich) 0.25µM SMARTpool, and left incubating at RT for 15 minutes. 10µl were added to each well of a 96-well plate (50µl for 24-well plates) and cells were grown at 37°C for 48h. A pool of 4 non-targeting siRNAs was used as control and transfected using the same procedure.
Knockdown validation: Polyacrylamide Gel Electrophoresis (PAGE) and Western Blot
Cells were harvested through lysis in FASP-buffer, containing 5% ß-mercaptoethanol and sonicated to shear DNA. Samples were boiled for 5min at 95°C, subsequently loaded onto a precast Bis-Tris 4–12% gradient gel (mPAGE, Merck Millipore), and PAGE was performed at 120V for 1h. Semi-wet transfer onto Amersham Protran nitrocellulose membrane (Sigma Aldrich) was conducted in a Biorad Trans-Blot Turbo system using transfer buffer (25mM Tris, 192mM Glycine, pH8.3 containing 20% methanol): 10 minutes at 1.3A, 25V. Membranes were washed in TBS containing 0.1% Tween-20 (Sigma, TBS-T), and subsequently blocked for 1h in 5% milk in TBS-T while rocking at RT. Rabbit-anti-Mprip (Thermo Scientific) primary antibody was added at 1:1000 dilution and membranes were incubated overnight at 4°C while rocking. The next day, membranes were washed three times for 5 minutes in TBS-T and subsequently incubated for 1h in HRP-coupled secondary antibody (goat-anti-rabbit, 1:5000 in milk, SouthernBiotech) or in HRP-coupled anti-Tubulin antibody (Abcam, 1:5000 in milk). After three washes, membranes were incubated in ECL solution (BioRad) and images were acquired on an iBright Imaging System (Thermo Fisher Scientific), using an appropriate exposure time.
Microscopy image acquisition and quantification after mAdV2-co-infections
Infections, plate preparation and image acquisition were performed as described above. Additionally to Hoechst, cells were stained with phalloidin-Alexa647 (Abcam) for 1h at a concentration of 1:1000 in PBS. After image acquisition (4 fields of view, in a z-stack spanning at least 7 planes per condition in each replicate), the integrated intensities for DAPI, bacteria and phalloidin were quantified across planes in ImageJ. Subsequently, the phalloidin signal was normalized to DAPI (to quantify increase in actin intensity upon viral infection), and the bacterial signal was normalized to both phalloidin and DAPI (to assess changes in intracellular bacteria).
Statistical analysis
All analyses, significance testing and visualization was performed in Prism (version 10.2.0), taking into account the necessary prerequisites in test selection. Details of statistical analysis are indicated where required.