Dual 3'Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells.
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.
Institute for Molecular BioSciences, Goethe University Frankfurt am Main, Frankfurt am Main, Germany. FGrunz@rz.uni-frankfurt.de.|GenXPro GmbH, Frankfurt Biotechnology Innovation Center (FIZ), Frankfurt am Main, Germany. FGrunz@rz.uni-frankfurt.de.
Fabian
F
Afonso-Grunz
GenXPro GmbH, Frankfurt Biotechnology Innovation Center (FIZ), Frankfurt am Main, Germany. hoffmeier@genxpro.de.
Klaus
K
Hoffmeier
Institute for Molecular BioSciences, Goethe University Frankfurt am Main, Frankfurt am Main, Germany. s.mueller@bio.uni-frankfurt.de.|GenXPro GmbH, Frankfurt Biotechnology Innovation Center (FIZ), Frankfurt am Main, Germany. s.mueller@bio.uni-frankfurt.de.
Sören
S
Müller
Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany. alexander.westermann@uni-wuerzburg.de.
Alexander J
AJ
Westermann
GenXPro GmbH, Frankfurt Biotechnology Innovation Center (FIZ), Frankfurt am Main, Germany. rotter@genxpro.de.
Björn
B
Rotter
Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany. joerg.vogel@uni-wuerzburg.de.
Jörg
J
Vogel
GenXPro GmbH, Frankfurt Biotechnology Innovation Center (FIZ), Frankfurt am Main, Germany. pwinter@genxpro.de.
Peter
P
Winter
Institute for Molecular BioSciences, Goethe University Frankfurt am Main, Frankfurt am Main, Germany. kahl@em.uni-frankfurt.de.|GenXPro GmbH, Frankfurt Biotechnology Innovation Center (FIZ), Frankfurt am Main, Germany. kahl@em.uni-frankfurt.de.
Günter
G
Kahl
D000818:Animals; D001483:Base Sequence; D059014:High-Throughput Nucleotide Sequencing; D054884:Host-Pathogen Interactions / Q000235:genetics*; D006801:Humans; D012481:Salmonella Infections, Animal / Q000235:genetics* / Q000382:microbiology; D012485:Salmonella typhi / Q000235:genetics* / Q000472:pathogenicity; D059467:Transcriptome / Q000235:genetics*