Gene Expression
Nebulas
Gene Expression NebulasSummary: Non-typhoidal Salmonella (NTS) are among of the most important food-borne pathogens. Recently, a highly invasive multi-drug resistant S. Typhimurium of a distinct multilocus sequence type (MLST), ST313, has emerged across sub-Saharan Africa as a major cause of lethal bacteraemia in children and immunosuppressed adults. Encounters between dendritic cells (DCs) and invading bacteria determine the course of infection but whether or how ST313 might usurp DC mediated defence has not been reported. Here we utilised fluorescently labelled invasive and non-invasive strains of Salmonella combined with single-cell RNA sequencing to study the transcriptomes of individual infected and bystander DCs. The transcriptomes displayed a repertoire of cell instrinsic and extrinsic innate response states that differed between invasive and non-invasive strains. Gene expression heterogeneity was increased in DCs challenged with invasive Salmonella. DCs exposed but not harbouring invasive Salmonella exhibited a hyper-activated profile that likely facilitates trafficking of infected cells and dissemination of internalised intact bacteria. In contrast, invasive Salmonella containing DCs demonstrate reprogramming of trafficking genes required to avoid autophagic destruction. Furthermore, these cells displayed differential expression of tolerogenic IL10 and MARCH1 enabling CD83 mediated adaptive immune evasion. Altogether our data illustrate pathogen cell-to cell variability directed by a Salmonella invasive strain highlighting potential mechanisms of host adaption with implications for dissemination in vivo.
Overall Design: Single-cell RNA sequencing (SMARTSeq2) of 373 human monocyte derived dendritic cells infected with S. Typhimurium strain LT2 or D23580 or left uninfected
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| Growth Protocol: | Leukocyte Reduction System cones were obtained from the UK -tio-l Blood Centre with informed consent following local ethical guidelines. Blood was diluted in Phosphate-Buffered Saline (PBS) and layered on a standard density gradient (LymphoprepTM). Peripheral Blood Mononuclear Cells (PBMC) were collected from the interface and washed in PBS at 4 C. Monocytes were obtained by magnetic positive selection, using the human anti-CD14+ MicroBeads (Miltenyi Biotec, Germany) according to the manufacturer__ protocol. Freshly isolated monocytes were differentiated into Dendritic Cells (MoDCs) in the presence of 40 ng/mL of recombi-nt human (rh) Granulocyte Macrophage-Colony-Stimulating Factor (GM-CSF; Peprotech) and 40 ng/mL rh Interleukin-4 (IL-4; Peprotech). |
| Treatment Protocol: | DCs were infected with S. Tyhpimurium strain LT2 or D23580 at a MOI of 10 or left uninfected for 2h, 4h or 6h before sorting. |
| Extract Protocol: | Live single cells were sorted into 96-well plates filled with 4uL of lysis buffer (0.4% Triton, 2U/uL RNAse inhibitor (Clonotech, Takara), 7.5uM oligo dT (Biomers) and 10mM dNTP [supplemented with ERCC spike-in 1:10,000,000] (Invitrogen)) |
| Library Construction Protocol: | - |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 4000 |
| Strand-Specific: | Unspecific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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