This readme file was generated on 2023-02-25 by Hannah Wong GENERAL INFORMATION Title of Dataset: Naturally-occurring serotype 3 Streptococcus pneumonia strains that lack functional pneumolysin and autolysis have attenuated virulence but induce localized protective immune responses Author/Principal Investigator Information Name: Hannah E Wong ORCID: 0000-0002-9530-7106 Institution: Department of Veterinary Medicine, University of Cambridge Address: Madingley Road, Cambridge, CB3 0ES Email: hew28@cam.ac.uk Author/Associate or Co-investigator Information Name: Clare E Bryant ORCID: 0000-0002-2924-0038 Institution: Department of Veterinary Medicine, University of Cambridge Address: Madingley Road, Cambridge, CB3 0ES Email: ceb27@cam.ac.uk Author/Associate or Co-investigator Information Name: Panagiotis Tourlomousis Institution: Department of Veterinary Medicine, University of Cambridge Address: Madingley Road, Cambridge, CB3 0ES Email: pt324@cam.ac.uk Author/Associate or Co-investigator Information Name: Gavin Paterson Institution: Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, Easter Bush Campus, Midlothian. Email: gavin.paterson@ed.ac.uk Date of data collection: 2012-10-01 to 2018-10-01 Geographic location of data collection: Cambridge, Cambridgeshire, UK. Information about funding sources that supported the collection of the data: This research was funded in whole by the Wellcome Trust [096413/Z/11/Z]. SHARING/ACCESS INFORMATION Licenses/restrictions placed on the data: For the purpose of open access, the author has applied a CC BY public copyright license to this data. Links to publications that cite or use the data: Naturally-occurring serotype 3 Streptococcus pneumoniae strains that lack functional pneumolysin and autolysis have attenuated virulence but induce localized protective immune responses. Wong HE, Tourlomousis P, Paterson GK, Webster S, Bryant CE. PlosOne Links to other publicly accessible locations of the data: see below Links/relationships to ancillary data sets: See below Was data derived from another source? This study uses genomic data that has been deposited in the public domain by Gavin Paterson et al. Strain A45 Accession HE983624.1 A small proportion of the data is reproduced here in the supplementary data for this publication by permission from Gavin Paterson. Recommended citation for this dataset: Naturally-occurring serotype 3 Streptococcus pneumonia strains that lack functional pneumolysin and autolysis have attenuated virulence but induce localized protective immune responses. Wong HE, Tourlomousis P, Bryant CE. 2023. DATA & FILE OVERVIEW File List: Wong_2023_data01. Single excel spreadsheet, with one tab containing data tables that relate to each figure in the paper. Equine pneumococcal strain genomic data.rtx. Accession numbers for complete genomic data from eight strains with ply-lytA region for seven strains in FASTA format. Relationship between files, if important: NA Additional related data collected that was not included in the current data package: None Are there multiple versions of the dataset? No If yes, name of file(s) that was updated: NA Why was the file updated? NA When was the file updated? NA METHODOLOGICAL INFORMATION Description of methods used for collection/generation of data: See publication Wong, Tourlomousis, Paterson, Webster & Bryant. Naturally-occurring serotype 3 Streptococcus pneumonia strains that lack functional pneumolysin and autolysis have attenuated virulence but induce localized protective immune responses. 2023 Brief methods: Sequencing done using Illumina hiSeq 20000 technology. S.pneumoniae prepared for in vitro or in vivo infection. Bacteria grown at 37c in static BHI broth until mid-log phase, then aliquoted and frozen. Frozen glycerol stocks of known titre used. Deoxycholate lysis. Bacteria grown at 37c in static BHI broth until mid-log phase. Bacterial counts and optical density measured before and after addition of sodium deoxycholate. Semiquantitative haemolysis assay. Bacterial lysates of known protein concentration were serially diluted in PBS. 5mM Dithiothreitol final concentration added to each well. Washed equine RBCs were added to all wells at a final concentration of 0.7% (v/v). The covered plate incubated at 37c for 30 mins, and then at 21c for 30 mins before visual assessment. Haemolytic titre is the reciprocal of the dilution factor. Immortalised murine bone marrow derived macrophages. iBMDMs culturerd in DMEM supplemented with 10% FCS, 2mM L-glutamine, 100U/ml penicillin and 100ug/ml streptomycin. iBMDM seeded in 96 well plates at 1 x 10*6 cells/ml unless otherwise stated. Animals. Mice were housed in a specific pathogen free facility and all animal work compiled with UK Home Office regulations. Primary bone marrow-derived macrophages were retrieved by flushing out femur bone marrow in to DMEM supplemented with 10% heat-inactivated FCS 5 mM L-glutamine and 20% L929 RPMI conditioned media. Primary BMDMs were seeded in 96 well plates at 1 x 10*6 cells/mL unless otherwise stated. Acute pneumonia model. Mice were lightly anaesthetised and received 50ul of innoculum. Euthanasia was with carbon dioxide and standard tissue processing procedures. Cell viability assays. Washed cells were lysed with 0.25% saponin at 37c of 20 min. LDH activity was measured using CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega) according to manufacturer's guidelines. Cytokine quantification. Standard ELISA kits used, and a custom flow cytometric multiplex bead array (ProcartaPLex). All cytokines were measured according to the manufacturer’s instructions. Methods for processing the data: GraphPad Prism 5 for Mac OS X (GraphPad) was used for the statistical analysis. Instrument- or software-specific information needed to interpret the data: GraphPad Prism 5 for Mac OS X (GraphPad) was used for the statistical analysis. Standards and calibration information, if appropriate: Any methods required standards or calibration were performed according to the manufacturers guidelines. Results outside the standard curve range were repeated. Environmental/experimental conditions: Ambient indoor laboratory conditions unless otherwise stated. Describe any quality-assurance procedures performed on the data: See publication Experiments involving cells were performed in triplicate. All experiments were repeated on different days. People involved with sample collection, processing, analysis and/or submission: Hannah Wong, Gavin Paterson, Panagiotis Tourlomousis, Clare Bryant DATA-SPECIFIC INFORMATION FOR: Equine pneumococcal strain genomic data.rtx Accession numbers for complete genomic data from eight strains. ply-lytA region for seven strains in FASTA format. DATA-SPECIFIC INFORMATION FOR: Wong_2023_data01 Fig 1. Outputs measured: TNFalpha pg/ml by ELISA, Cell viability % of uninfected by LDH cytotoxicity assay, IL-1beta pg/ml by ELISA Number of variables: 3 (uninfected, S. pneumoniae A66, S. pneumoniae 2721) Number of cases/rows: 2 (6 hr, 24 hr) Missing data codes: left blank Data analysed by One-way ANOVA with Tukey's multiple comparison test and adjusted P-values reported. Fig 2. Fig 2A. Outputs measured: TNFalpha pg/ml by ELISA Number of variables: 4 (wild type bone marrow derived macrophages (BMM), tlr4-/- BMM, tlr2-/- BMM, tlr2/4-/- BMM) Number of cases/rows: 2 (S. pneumoniae A66 MOI 1 6 hr post infection, S. pneumoniae 2721 Moi 1 6 hr post infection) Missing data codes: left blank Data analysed by One-way ANOVA with Dunnett's multiple comparison test and adjusted P-values reported. Fig 2B. Outputs measured: TNFalpha pg/ml by ELISA Number of variables: 3 (wild type immortalised bone marrow derived macrophages (iBMDM), myD88-/- iBMDM, tlr9-/- iBMDM) Number of cases/rows: 2 (S. pneumoniae A66 Moi 1 6 hr post infection, S. pneumoniae 2721 Moi 1 6 hr post infection) Missing data codes: left blank Data analysed by One-way ANOVA with Dunnett's multiple comparison test and adjusted P-values reported. Fig 2C. Outputs measured: Cell viability % of uninfected by LDH cytotoxicity assay Number of variables: 4 (wild type bone marrow derived macrophages (BMM), tlr4-/- BMM, tlr2-/- BMM, tlr2/4-/- BMM) Number of cases/rows: 2 (S. pneumoniae A66 moi 5 24hr post infection, S. pneumoniae 2721 moi 5 24 hr post infection) Missing data codes: left blank Data analysed by One-way ANOVA with Dunnett's multiple comparison test and adjusted P-values reported. Fig 2D. Outputs measured: Cell viability % of uninfected by LDH cytotoxicity assay Number of variables: 4 (wild type bone marrow derived macrophages (BMM), nlrp3-/- BMM, caspase1/11-/- BMM) Number of cases/rows: 2 (S. pneumoniae A66 moi 5 24hr post infection, S. pneumoniae 2721 moi 5 24 hr post infection) Missing data codes: left blank Data analysed by One-way ANOVA with Dunnett's multiple comparison test and adjusted P-values reported. Fig 2E. Outputs measured: Cell viability % of uninfected by LDH cytotoxicity assay Number of variables: 4 (wild type bone marrow derived macrophages (BMM), tlr4-/- BMM, tlr2-/- BMM, tlr2/4-/- BMM) Number of cases/rows: 1 (purified pneumolysin 10 ug/ml 24hr stimulation) Missing data codes: left blank Data analysed by One-way ANOVA with Dunnett's multiple comparison test and adjusted P-values reported. Fig 2F. Outputs measured: Cell viability % of uninfected by LDH cytotoxicity assay Number of variables: 4 (wild type bone marrow derived macrophages (BMM), nlrp3-/- BMM, caspase1/11-/- BMM) Number of cases/rows: 1 (purified pneumolysin 10 ug/ml 24hr stimulation) Missing data codes: left blank Data analysed by One-way ANOVA with Dunnett's multiple comparison test and adjusted P-values reported. Fig 3. Outputs measured: bacterial counts CFU/ml Number of variables: 2 (S. pneumoniae A66, S. pneumoniae 2721) Number of cases/rows: 3 (6 hr, 24 hr, 36 hr) Missing data codes: left blank Data analysed by Two-way ANOVA with Sidak's multiple comparison test and adjusted P-values reported. Fig 4. Outputs measured: cytokine production from lung tissue pg/ml. A.IFNg B.IL-6 C.TNFa D.RANTES E.IL-1a F.IL-1b Number of variables: 3 (uninfected control, S. pneumoniae A66, S. pneumoniae 2721) Number of cases/rows: 2 (24 hr, 36 hr) Missing data codes: left blank Data analysed by Two-tailed Mann Whitney U test. Fig 5. A,B & C Outputs measured: A,B,C histology score. Number of variables: 2 (S. pneumoniae A66, S. pneumoniae 2721) Number of cases/rows: 3 (6 hr, 24 hr, 36 hr) Missing data codes: left blank Data analysed by Two-way ANOVA with Sidak's multiple comparison test and adjusted P-values reported. Fig 5.G Outputs measured: semiquantitative bacterial score Number of variables: 2 (S. pneumoniae A66, S. pneumoniae 2721) Number of cases/rows: 3 (Interstitial, Airway/alveoli, pleura) Missing data codes: left blank Data analysed by Two-way ANOVA with Sidak's multiple comparison test and adjusted P-values reported. S2 Fig. Outputs measured: haemolytic titre Number of variables: 4 (S. pneumoniae A66, S. pneumoniae 06-3499, S. pneumoniae 2721, S. pneumoniae 7573) Number of cases/rows: 1 S3 Fig. Outputs measured: Optical density OD595. Number of variables: 4 (S. pneumoniae A66, S. pneumoniae 06-3499, S. pneumoniae 2721, S. pneumoniae 7573) Number of cases/rows: 3 choline %wt/vol (0, 0.02, 0.04) Missing data codes: left blank Data analysed by One-way ANOVA of 0.04% data with Dunnett's multiple comparison test and adjusted P-values reported. S4 Fig. S4.A Outputs measured: TNFalpha pg/ml by ELISA Number of variables: 10 (S. pneumoniae A66, S. pneumoniae 06-3499, S. pneumoniae 2721, S. pneumoniae A45, S. pneumoniae 409, S. pneumoniae 7573, S. pneumoniae A29, S. pneumoniae BLB1 no.5, S. pneumoniae BLB1 no.15, S. pneumoniae S/5528 S. pneumoniae 7573) Number of cases/rows: 2 (6hr, 24 hr) Missing data codes: left blank Data analysed by One-way ANOVA with Dunnett's multiple comparison test and adjusted P-values reported. S4.B Outputs measured: Cell viability % of uninfected by LDH cytotoxicity assay Number of variables: 10 (S. pneumoniae A66, S. pneumoniae 06-3499, S. pneumoniae 2721, S. pneumoniae A45, S. pneumoniae 409, S. pneumoniae 7573, S. pneumoniae A29, S. pneumoniae BLB1 no.5, S. pneumoniae BLB1 no.15, S. pneumoniae S/5528 S. pneumoniae 7573) Number of cases/rows: 2 (6hr, 24 hr) Missing data codes: left blank Data analysed by One-way ANOVA with Dunnett's multiple comparison test and adjusted P-values reported. S4.C Outputs measured: IL-1beta pg/ml by ELISA Number of variables: 10 (S. pneumoniae A66, S. pneumoniae 06-3499, S. pneumoniae 2721, S. pneumoniae A45, S. pneumoniae 409, S. pneumoniae 7573, S. pneumoniae A29, S. pneumoniae BLB1 no.5, S. pneumoniae BLB1 no.15, S. pneumoniae S/5528 S. pneumoniae 7573) Number of cases/rows: 1 (24 hr) Missing data codes: left blank Data analysed by One-way ANOVA with Dunnett's multiple comparison test and adjusted P-values reported. S5. Fig Outputs measured: cytokine production from blood serum pg/ml. A.IFNg B.IL-6 C.TNFa D.RANTES E.IL-1a F.IL-1b Number of variables: 3 (uninfected control, S. pneumoniae A66, S. pneumoniae 2721) Number of cases/rows: 1 (36 hr) Missing data codes: left blank Data analysed by Two-tailed Mann Whitney U test.