Unknown,Transcriptomics,Genomics,Proteomics

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Deep sequencing of the innate immune transcriptomic response of zebrafish embryos to Salmonella infection

ABSTRACT: Salmonella enterica serovar Typhimurium (S. typhimurium) bacteria cause an inflammatory and lethal infection in zebrafish embryos. To characterize the embryonic innate host response at the transcriptome level, we have extended and validated previous microarray data by Illumina next-generation sequencing analysis. Comparison of tag-based sequencing (DGE or Tag-Seq) with full transcript sequencing (RNA-Seq) showed a strong correlation of sequence read counts per transcript and an overlap of 241 transcripts differentially expressed in response to infection. A slightly lower overlap of 165 transcripts was observed with previous microarray data. Based on the combined sequencing-based and microarray-based transcriptome data we compiled an annotated reference set of infection-responsive genes in zebrafish embryos, encoding transcription factors, signal transduction proteins, cytokines and chemokines, complement factors, proteins involved in apoptosis and proteolysis, proteins with anti-microbial activities, as well as many known or novel proteins not previously linked to the immune response. Furthermore, by comparison of the deep sequencing data of S. typhimurium infection in zebrafish embryos with previous deep sequencing data of Mycobacterium marinum infection in adult zebrafish we derived a common set of innate host defense genes that are expressed both in the absence and presence of a fully developed adaptive immune system and that provide a valuable reference for future studies of host-pathogen interactions using zebrafish infection models. Zebrafish embryos were infected with Salmonella typhimurium (strain SL1027) by microinjection of DsRED-labeled bacteria into the caudal vein close to the urogenital opening after the onset of blood circulation (27 hpf). An equal volume of PBS was injected in the control group. RNA samples were collected at 8 hours post infection (hpi) and samples from triplicate infection experiments were pooled. DGE libraries from the RNA pools (1 M-NM-

ORGANISM(S): Danio rerio

SUBMITTER: Oliver Stockhammer

PROVIDER: E-GEOD-22472 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Deep sequencing of the innate immune transcriptomic response of zebrafish embryos to Salmonella infection.

Ordas Anita A Hegedus Zoltan Z Henkel Christiaan V CV Stockhammer Oliver W OW Butler Derek D Jansen Hans J HJ Racz Peter P Mink Matyas M Spaink Herman P HP Meijer Annemarie H AH

Fish & shellfish immunology 20100908 5

Salmonella enterica serovar Typhimurium (S. typhimurium) bacteria cause an inflammatory and lethal infection in zebrafish embryos. To characterize the embryonic innate host response at the transcriptome level, we have extended and validated previous microarray data by Illumina next-generation sequencing analysis. We obtained 10 million sequence reads from control and Salmonella-infected zebrafish embryos using a tag-based sequencing method (DGE or Tag-Seq) and 15 million reads using whole transc ...[more]

PMID: 20816807

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Project description:Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks. In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait. In this work, we developed and tested a framework for analysis of tag-based, digital gene expression (DGE) profiles using Illumina’s high-throughput sequencing technology and the newly-assembled B73 maize reference genome. We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in determinacy of axillary meristems. The RA3 gene encodes a trehalose-6-phosphate-phosphatase and may act at the interface between developmental and metabolic processes. Deep sequencing of DGE libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20-21nt reads with frequencies spanning four orders of magnitude. Unique sequence tags were anchored to 3´-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing. We mapped 86% of non-redundant signature tags to the maize genome, which associated with 37,117 working gene models and un-annotated regions of expression. 66% of maize genes were detected at ? nine reads in immature maize ears. We used comparative genomics to leverage existing information from Arabidopsis and rice in functional analyses of differentially expressed maize genes. Results from this study provide a basis for analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene. 3' tag-based DGE libraries were generated using Illumina 1G technology. Libraries were prepared from 2mm ear samples and represent: 3 biological replicates of wild-type B73, 3 biological replicates of ramosa3 (ra3) mutants, and 1 technical replicate of a ra3 sample. Each sample was sequenced in a single lane of a flow cell. Each lane includes library preparations from both DpnII and NlaIII digests for that sample. Sequences from DpnII digests start with GATC and those from NlaIII digests start with ATG.

Project description:We use the zebrafish embryo model to study the similarities and differences in the innate immune response against three different bacterial pathogens. Therefore, we injected E. tarda, Salmonella, or M. marinum into the caudal vein of 28 hours post fertilization (hpf) zebrafish embryos and analysed their gene expression profile at 8 hours or 4 days after infection by microarrays. The results show that infections with the gram-negative bacteria E. tarda and S. typhimurium, which are lethal within 1-2 days, induce a strong early immune response at 8 hours after infection. In contrast, infection with M. marinum leads to a chronic infection that only induces a strong response at 4 days post infection. This microarray study was designed to determine the gene expression profile during infection with Salmonella typhimurium, Mycobacterium marinum, and Edwardsiella tarda. RNA was isolated from single embryos and each treatment group consisted of three embryos: (1) Wildtypes injected with PBS 8 hours post infection (hpi), (2) S. typhimurium-infected wildtypes 8hpi, (3) wildtypes injected with PBS/2%PVP 8hpi , (4) M. marinum-infected wildtypes 8hpi, (5) wildtypes injected with PBS/2%PVP 4 days post infection (dpi), (6) M. marinum-infected wildtypes 4dpi, (7) wildtypes injected with PBS 8hpi (E. tarda control), (8) E. tarda-infected wildtypes 8hpi. Embryos were grown at 28.5M-bM-^@M-^S30M-BM-0C in egg water and manually dechorionated at 24 hours post fertilization (hpf). Subsequently, embryos were infected at 28 hpf by micro-injecting 200 colony forming units (CFU) of S. typhimurium SL1027, E. tarda or Mycobacteria marinum M20 bacteria into the caudal vein, or were mock-injected with buffer as a control. After injections embryos were transferred into fresh egg water and incubated for 8 h or 4 days at 28M-BM-0C. After the incubation period, single embryos were snap-frozen in liquid nitrogen and RNA was isolated for microarray analysis. All treatment groups were analyzed using a common reference approach.

Dataset Information

Deep sequencing of the innate immune transcriptomic response of zebrafish embryos to Salmonella infection

Publications

Deep sequencing of the innate immune transcriptomic response of zebrafish embryos to Salmonella infection.

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