Dual 3’Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells

Abstract

BackgroundThe 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.ResultsHuman 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.ConclusionsDual 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1489-1) contains supplementary material, which is available to authorized users.

Diminished nuclear RNA decay upon Salmonella infection upregulates antibacterial noncoding RNA s

“…Gene Ontology (GO) terms related to immune responses were significantly enriched for these genes (Table EV2). Moreover, our data confirmed the upregulation of several mRNAs previously reported to be transiently induced upon Salmonella infection, such as NFKB1 , NFKB2 , and RELB (Fig EV1B) (Afonso‐Grunz et al , ). Finally, of the 26,858 unstable nuclear ncRNA candidates detected in HeLa cells (Fig EV2B), 145 were upregulated with fold changes > 2 upon Salmonella infection with diverse induction kinetics (Fig A and B, Table EV3).…”

“…To define eRNAs expressing from enhancer regions, we selected the eRNAs that overlapped with enhancer regions identified by the co‐occurrence of H3K4me1, H3K4me3, and H3K27ac, obtained from the ENCODE project (ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeBroadHistone/wgEncodeBroadHistoneHelas3H3k04me1StdPk.broadPeak.gz, ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeBroadHistone/wgEncodeBroadHistoneHelas3H3k4me3StdRawDataRep1.fastq.gz, ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeBroadHistone/wgEncodeBroadHistoneHelas3H3k27acStdPk.broadPeak.gz). We also used the RNA‐seq data of Salmonella infection (DRA003692), MTR4 knockdown conditions (DRA003583), SAGE profiling (GSE61730) (Afonso‐Grunz et al , ), and Dual RNA‐seq (GSE60144) (Westermann et al , ).…”

Salmonella enterica serovar-specific transcriptional reprogramming of infected cells

“…The ability to stimulate transcriptional responses in infected cells is emerging as a central strategy in the pathogenesis of S . enterica serovars [ 17 – 21 ]. S .…”

“…S . Typhimurium, for example, stimulates transcriptional responses in intestinal epithelial cells that lead to the production of pro-inflammatory cytokines that initiate the inflammatory response that is central for its pathogenesis [ 17 – 21 ]. Furthermore, this transcriptional re-programming renders the infected cells more permissive for bacterial replication [ 19 ].…”

Targeting the ER-Mitochondrial Interface of Cell Death Sensitizes Leukemia Cells Towards Cytostatics

“…Jurkat cells visibly exhibited apoptotic phenotypes at concentrations >5 nM, induction of apoptosis was subsequently quantified applying higher concentrations which effectively escalated the percentage of apoptotic cells (Figure 34b 99 RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) and RNA amount determined using a NanoDrop Spectrophotometer ND-1000 (Peqlab, Erlangen, Germany). 4 µg of RNA was put in to prepare the cDNA library with the MACE Kit according to manufacturer's instructions (GenXPro, Frankfurt, Germany).…”