Afonso-Grunz Fabian, Hoffmeier Klaus, Müller Sören, Westermann Alexander J, Rotter Björn, Vogel Jörg, Winter Peter, Kahl Günter
Institute for Molecular BioSciences, Goethe University Frankfurt am Main, Frankfurt am Main, Germany.
GenXPro GmbH, Frankfurt Biotechnology Innovation Center (FIZ), Frankfurt am Main, Germany.
BMC Genomics. 2015 Apr 19;16(1):323. doi: 10.1186/s12864-015-1489-1.
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.
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.
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.
真核宿主细胞与原核病原体细胞的相互作用与细胞蛋白质组的特定变化相关联，进而与所涉及细胞的感染相关基因表达模式相关联。为了在两者相互作用期间同时评估这两种生物体的转录组，我们开发了双3'Seq，这是一种基于标签的测序方案，可在不预先固定或物理破坏相互作用的情况下，对相互作用的原核和真核细胞中差异表达的转录本进行精确量化。
作为肠道上皮侵袭的模型系统，用人上皮细胞感染鼠伤寒沙门氏菌，通过双3'Seq结合基于下一代测序的转录组分析技术deepSuperSAGE（深度基因表达序列分析），确定受感染宿主细胞的转录反应以及侵袭性和细胞内病原体细胞的差异表达。对包含所用鼠伤寒沙门氏菌菌株操纵子结构的参考转录组进行注释，可在计算机上分离相互作用的细胞，包括多顺反子RNA的定量。发现89%的已知基因座在宿主感染之前或之后在原核细胞中被转录，而所有蛋白质编码基因座的75%存在于人类宿主细胞的多聚腺苷酸化转录组中。
双3'Seq可交替与MACE（cDNA末端大规模分析）结合，以评估允许对更长片段进行测序的文库制备程序 的优缺点。此外，使用三个独立的生物学重复通过qRT-PCR验证了两种生物体的已鉴定表达模式，这证实了RELB以及NFKB1和NFKB2参与鼠伤寒沙门氏菌感染后上皮细胞的初始免疫反应。
