{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Richard Allen"],"organism":["mixed sample"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16250"],"description":["This experiment was conducted to uncover global transcription differences caused by Staphylococcal T7SS during early and mid-late macrophage infection."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic Acid Extraction - Total RNA was extracted from samples by bead beating with lysing matrix B beads of 0.1 mm (MP Biomedicals) in a FastPrep-24  5G bead beater (MP Biomedicals) at 6.5 m/s for 20 seconds and rest in ice for 3 minutes, carried out for 6 cycles. RNA was extracted by addition of TRIzol Reagent and chloroform following manufacturer's instructions. RNA was precipitated with isopropanol, centrifuged, and then washed with 75% ethanol and finally resuspended in DNase- and RNase-free water. DNA and RNA were quantified by Qubit Broad Range assays (Invitrogen). Contaminating DNA was removed using TURBO DNA-free Kit (Invitrogen) following instructions for rigorous treatment. Samples were further cleaned up with LiCl Precipitation Solution 7.5 M (Invitrogen). RNA integrity was assessed by Agilent 6000 RNA Pico bioanalyzer, and RNA purity and contaminants checked by NanoDrop One (ThermoFisher).","Sequencing - Sequencing was carried out by Genewiz Azenta. The sequencing libraries were multiplexed and loaded on the flow cell on the Illumina NovaSeq 6000 instrument according to manufacturer's instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration v1.5. Image analysis and base calling were conducted by NovaSeq Control Software v1.7 on the NovaSeq instrument. Raw sequence data (bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was allowed for index sequence identification.","Library Construction - Library construction was carried out by Genewiz Azenta. rRNA depletion was performed using NEBnext rRNA Depeletion Kit (Human/Mouse/Rat and/or bacteria). RNA sequencing library preparation was performed using NEBNext Ultra II RNA Library Prep Kit for Illumina by following manufacturer's instruction. First strand and second strand cDNA were subsequently synthesised. cDNA fragments were end-repaired and adenylated at 3'ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using NGS Kit","Sample Collection - THP-1 macrophages were infected with S. aureus JE2 WT or ΔessC strains in 12 well tissue culture treated plates for 1 hour before extracellular killing of bacteria. At both 2 and 6 hours post infection, wells were washed with PBS, before addition of LETs buffer (0.1M LiCl, 0.01 M Na2EDTA, 0.01 M Tris-Cl pH 7.4, 0.2% SDS) to the wells, and immediately frozen in dry ice."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Counts of gene expression were generated by LiBiNorm (without normalisation - HTSeq-compatible). Differentially expressed genes were generated by DeSEq2 R package (version 1.42.1), default normalisation using median-of-ratios size factor method. DEGs were considered significantly differentially expressed with adjusted p-values below 0.05 and log2 fold change values > 1 and <-1.","Sequence Alignment - FASTQ sequencing files were received from GeneWiz and were initially assessed by the FastQC Linux package. Adapter contamination of reads were trimmed by Trimmomatic tool (version 0.39) with the parameters: PE ILLUMINACLIP:adapters fa:2:30:10 SLIDINGWINDOW:4:20 MINLEN:32. Trimmed reads were aligned by HISAT2 v2.1.0 to either individual or concatenation of human genome GRCh38.p14 release 46 with comprehensive gene annotation for all regions (GENCODE) and S. aureus JE2 genome assembly ASM208552v1 (NCBI RefSeq assembly GCF_002085525.1). Mapping analysis of aligned genomes was done by Bowtie2."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["N/A","Illumina NovaSeq 6000","FastPrep-24 5G bead beater, Qubit Fluorometer","12 well TC-treated plate, LETs buffer, dry ice"],"pubmed_abstract":["The major human pathogen  Staphylococcus aureus is a facultatively intracellular bacterium that can replicate and survive within a range of host cells including macrophages. While macrophage-mediated immune modulation is pivotal in  S. aureus pathogenesis, bacterial manipulation of macrophage pathways remains poorly understood. The specialised type VII secretion system (T7SSb) is an important virulence-associated factor and immune modulator during staphylococcal infection, although, unlike its mycobacterial counterparts, its role in controlling staphylococcal-macrophage interactions remains unclear. Employing high-resolution time-lapse imaging, we demonstrated an induction of cell death corpses or pore-induced cellular traps (PITs) during  S. aureus infection of macrophages  in vitro , with a higher number of PITs forming in macrophages infected with  S. aureus lacking EssC (Δ  essC ), a central T7SS transporter. Interestingly, Δ  essC -infected macrophages displayed increased bacterial escape compared to wild type (WT)-infected cells, implicating a role for T7SS in delaying macrophage cell death. Δ  essC -infected macrophages were also efferocytosed less compared to WT  in vitro . We investigated the induction of cell death signalling markers in WT and Δ  essC -infected macrophages and demonstrated that the T7SS delayed the induction of key cell death pathways, necroptosis and pyroptosis. A dual RNAseq analysis of WT- and Δ  essC -infected macrophages further indicated a T7SS-dependent increase in ferroptosis later in infection, along with modulation of chemokines. We demonstrated that individual T7SS effectors, EsxA and EsxC, which were induced within macrophages, could individually delay macrophage cell death. Furthermore, in a murine skin infection model, we showed that effector and transporter mutants have distinct  S. aureus infection outcomes. Our findings suggest for the first time a key role for T7SSb proteins in controlling macrophage cell death, which impacts staphylococcal survival within the host during infection.  <h4>Importance</h4>  Staphylococcus aureus is a major hospital and community associated pathogen which causes an array of infections  . S. aureus can survive within immune cells, which may provide a niche for this pathogen to both persist and disseminate. Here we report a role for the staphylococcal type VII secretion system b, which secretes effectors that have been associated with bacterial virulence, in delaying macrophage cell death induced in response to this pathogen. T7SS functions through interfering with inflammatory cell death pathways, impacting the local cellular and immune environment. Our findings thus highlight a protective role for this system in macrophages, distinct to functions reported for the mycobacterial T7SS, and may implicate similar functions for T7SS proteins from related pathogens. Controlling macrophage cell death could be a critical mechanism by which  S. aureus modulates the local immune responses, enabling its survival within the host,"],"study_type":["RNA-seq of coding RNA"],"species":["mixed sample"],"pubmed_title":["The staphylococcal type VII secretion system delays macrophage cell death through modulating multiple cell death pathways"],"pubmed_authors":["Richard Allen","Richard D. Allen, Kate E. Watkins, Girihidar Chandrasekhan, Pooja Agarwal, Meera Unnikrishnan"],"additional_accession":[]},"is_claimable":false,"name":"Dual RNA-seq of THP-1 macrophages infected with JE2 WT and ΔessC at 2 and 6 hours post infection","description":"This experiment was conducted to uncover global transcription differences caused by Staphylococcal T7SS during early and mid-late macrophage infection.","dates":{"release":"2026-01-01T00:00:00Z","modification":"2026-05-28T10:35:24.155Z","creation":"2025-11-21T14:45:22.558Z"},"accession":"E-MTAB-16250","cross_references":{"ENA":["ERP185482"],"EFO":["EFO_0002944","EFO_0004170","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184"],"doi":["10.1101/2025.10.30.685513"]}}