TY - JOUR A1 - Afonso-Grunz, Fabian A1 - Hoffmeier, Klaus A1 - Müller, Sören A1 - Westermann, Alexander J. A1 - Rotter, Björn A1 - Vogel, Jörg A1 - Winter, Peter A1 - Kahl, Günter T1 - Dual 3'Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells JF - BMC Genomics N2 - 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. KW - complete genome sequence KW - secretion systems KW - RNA-Seq KW - deepSuperSAGE KW - transcriptome KW - gene expression KW - serovar Typhimurium KW - human macrophages KW - epithelial cells KW - infection KW - SuperSAGE KW - receptors KW - Dual 3'seq KW - MACE KW - tag based KW - simultaneous KW - genome wide KW - gene expression profiling KW - host pathogen interaction KW - Salmonella enterica Typhimurium strain SL1344 Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143230 VL - 16 IS - 323 ER - TY - JOUR A1 - Schmidtke, Cornelius A1 - Findeiß, Sven A1 - Sharma, Cynthia M. A1 - Kuhfuss, Juliane A1 - Hoffmann, Steve A1 - Vogel, Jörg A1 - Stadler, Peter F. A1 - Bonas, Ulla T1 - Genome-wide transcriptome analysis of the plant pathogen Xanthomonas identifies sRNAs with putative virulence functions JF - Nucleic Acids Research N2 - The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is an important model to elucidate the mechanisms involved in the interaction with the host. To gain insight into the transcriptome of the Xcv strain 85-10, we took a differential RNA sequencing (dRNA-seq) approach. Using a novel method to automatically generate comprehensive transcription start site (TSS) maps we report 1421 putative TSSs in the Xcv genome. Genes in Xcv exhibit a poorly conserved -10 promoter element and no consensus Shine-Dalgarno sequence. Moreover, 14% of all mRNAs are leaderless and 13% of them have unusually long 5'-UTRs. Northern blot analyses confirmed 16 intergenic small RNAs and seven cis-encoded antisense RNAs in Xcv. Expression of eight intergenic transcripts was controlled by HrpG and HrpX, key regulators of the Xcv type III secretion system. More detailed characterization identified sX12 as a small RNA that controls virulence of Xcv by affecting the interaction of the pathogen and its host plants. The transcriptional landscape of Xcv is unexpectedly complex, featuring abundant antisense transcripts, alternative TSSs and clade-specific small RNAs. KW - SUBSP carotovora KW - regulatory RNA KW - gene-cluster KW - campestris PV vesicatoria KW - escherichia coli KW - determines pathgenicity KW - hypersensitive response KW - ralstonia solanacearum KW - extracellular enzymes KW - secretion systems KW - transcription initiation site KW - RNA sequence analyses KW - messanger RNA KW - plants KW - libraries KW - genome KW - genes KW - gene expression profiling KW - genetic transcription KW - northern blotting KW - untranslated regions KW - xanthomonas KW - xanthomonas campestris KW - bacteria KW - virulence KW - pathogenetic organism KW - RNA KW - small RNA KW - pathogenicity KW - type III secretion system pathways KW - maps KW - consesus KW - host (organism) KW - type III protein secretion system complex Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131781 VL - 40 IS - 5 SP - 2020 EP - 2031 ER -