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Dual 3'Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells
Please always quote using this URN: urn:nbn:de:bvb:20-opus-143230
- Background: The interaction of eukaryotic host and prokaryotic pathogen cells is linked to specific changes in the cellular proteome, and consequently to infection-related gene expression patterns of the involved cells. To simultaneously assess the transcriptomes of both organisms during their interaction we developed dual 3'Seq, a tag-based sequencing protocol that allows for exact quantification of differentially expressed transcripts in interacting pro-and eukaryotic cells without prior fixation or physical disruption of the interaction.Background: The interaction of eukaryotic host and prokaryotic pathogen cells is linked to specific changes in the cellular proteome, and consequently to infection-related gene expression patterns of the involved cells. To simultaneously assess the transcriptomes of both organisms during their interaction we developed dual 3'Seq, a tag-based sequencing protocol that allows for exact quantification of differentially expressed transcripts in interacting pro-and eukaryotic cells without prior fixation or physical disruption of the interaction. Results: Human epithelial cells were infected with Salmonella enterica Typhimurium as a model system for invasion of the intestinal epithelium, and the transcriptional response of the infected host cells together with the differential expression of invading and intracellular pathogen cells was determined by dual 3'Seq coupled with the next-generation sequencing-based transcriptome profiling technique deepSuperSAGE (deep Serial Analysis of Gene Expression). Annotation to reference transcriptomes comprising the operon structure of the employed S. enterica Typhimurium strain allowed for in silico separation of the interacting cells including quantification of polycistronic RNAs. Eighty-nine percent of the known loci are found to be transcribed in prokaryotic cells prior or subsequent to infection of the host, while 75% of all protein-coding loci are represented in the polyadenylated transcriptomes of human host cells. Conclusions: Dual 3'Seq was alternatively coupled to MACE (Massive Analysis of cDNA ends) to assess the advantages and drawbacks of a library preparation procedure that allows for sequencing of longer fragments. Additionally, the identified expression patterns of both organisms were validated by qRT-PCR using three independent biological replicates, which confirmed that RELB along with NFKB1 and NFKB2 are involved in the initial immune response of epithelial cells after infection with S. enterica Typhimurium.…
Author: | Fabian Afonso-Grunz, Klaus Hoffmeier, Sören Müller, Alexander J. Westermann, Björn Rotter, Jörg Vogel, Peter Winter, Günter Kahl |
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URN: | urn:nbn:de:bvb:20-opus-143230 |
Document Type: | Journal article |
Faculties: | Medizinische Fakultät / Institut für Molekulare Infektionsbiologie |
Language: | English |
Parent Title (English): | BMC Genomics |
Year of Completion: | 2015 |
Volume: | 16 |
Issue: | 323 |
Source: | BMC Genomics (2015) 16:323. DOI: 10.1186/s12864-015-1489-1 |
DOI: | https://doi.org/10.1186/s12864-015-1489-1 |
Dewey Decimal Classification: | 6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit |
Tag: | Dual 3'seq; MACE; RNA-Seq; Salmonella enterica Typhimurium strain SL1344; SuperSAGE; complete genome sequence; deepSuperSAGE; epithelial cells; gene expression; gene expression profiling; genome wide; host pathogen interaction; human macrophages; infection; receptors; secretion systems; serovar Typhimurium; simultaneous; tag based; transcriptome |
Release Date: | 2017/11/03 |
Licence (German): | CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International |