Unknown,Transcriptomics,Genomics,Proteomics

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Host response to systemic bacterial challenge (sepsis) with E. coli strains CFT073 and F11 at 6 and 12 hours post inoculum

ABSTRACT: Host response to systemic bacterial challenge (sepsis) with E. coli strains CFT073 and F11 at 6 and 12 hours post inoculum 48 hpf embryos were manually dechorianated, briefly anesthetized with 0.77 mM ethyl 3-aminobenzoate methanesulfonate salt (tricaine) (Sigma-Aldrich), and embedded in 0.8% low melt agarose (MO BIO Laboratories) without tricaine. After the agarose solidified, embryos were immersed in E3 media lacking methylene blue. Prior to injection, 1 ml of bacterial culture was pelleted, washed with 1 ml sterile PBS, and re-suspended in PBS to obtain ~1X109 CFU/ml. PBS. One nl of this bacterial suspension containing ~1000 CFU was microinjected into the bloodstream via the circulation valley using an Olympus SZ61 or SZX10 stereomicroscope together with a YOU-1 micromanipulator (Narishige), a Narishige IM-200 microinjector, and a JUN-AIR model 3-compressor. For each experiment, the average CFU per injection was determined by adding 10 1-nl drops to 1 ml of 0.7% NaCl, which was then serial diluted and plated on Luria-Bertani (LB) agar plates. Mock-infected controls were inoculated with 1 nl sterile PBS. Following injection, embryos were removed from agar and placed individually into wells of a 48-well plate (Nunc) containing E3 medium and incubated at 28.5Â°C. Experiments were performed in biological quadruplicate.

ORGANISM(S): Danio rerio

SUBMITTER: Matthew Mulvey

PROVIDER: E-GEOD-79665 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos (Danio rerio) using transcriptomic profiling. Clobetasol propionate, a highly potent synthetic corticosteroid, which is known to have immunosuppressive effects on vertebrates including zebrafish, was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection, the embryos were injected with a mixture of different pathogen-associated molecular patterns (PAMPs) consisting of Pam3CSK4 (synthetic lipopeptide), poly(I:C) (synthetic viral dsRNA) and flagellin (isolated from Bacillus subtilis). Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test (OECD 236), 20 fertilized eggs were exposed to a sub-lethal concentration (250 nM) of clobetasol propionate for 48 hours under semi-static conditions. The test comprised of three treatments with clobetasol propionate and two negative control (NC) groups and was performed in triplicates. At 46 hours post fertilization (hpf), the eggs were manually dechorinated. At 48 hpf, the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection, 8 nL of the mixed PAMPs (0.33 µg/mL Pam3CSK4, 0.33 µg/mL poly(I:C) and 0.033 µg/mL flagellin) were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs, while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. After injection, the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

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Host response to systemic bacterial challenge (sepsis) with E. coli strains CFT073 and F11 at 6 and 12 hours post inoculum

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