Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.
Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.
Methods. 2020 Apr 1;176:71-81. doi: 10.1016/j.ymeth.2019.03.017. Epub 2019 Mar 21.
Shigella spp. are enterobacteria that invade human colonic mucosal cells using their Type Three Secretion Apparatus (T3SA). Shigella spp. possess a large plasmid that encodes most of its virulence factors and has been the focus of seminal work that defined the T3SA regulon. Thus, a global assessment of the transcriptional response regulated by the T3SA has been lacking. Herein we used RNA-Seq to identify genes that are differentially expressed when the T3SA is active (on-state) versus inactive (off-state). The quality of the RNA-Seq dataset was validated by its correlation with a prior microarray study. Using novel insights about the expression of non-coding regions, bioinformatic tools and experimentations, we demonstrated the existence of six operons and evidence that ipaH2.5 is a pseudogene. In addition, 86 chromosomal genes were downregulated in the on-state including several non-coding transcripts corresponding to short antisense RNA embedded in the 16S and 23S RNA genes, and 40 coding transcripts, whose cognate proteins were highly connected at the genetic and biochemical levels. Finally, we identified two novel chromosomal genes dubbed gem1 and gem3, which were upregulated in the on-state similarly to genes belonging to the T3SA regulon. The latter findings were validated on biological triplicates by droplet digital PCR. To our knowledge gem1 and gem3 are the first chromosomal members of the T3SA regulon that have no homologs on the plasmid. Our approach provides a path to optimizing RNA-Seq studies in case of bacterial models that had previously been the subject of medium to large scale studies.
志贺氏菌属是一种侵袭人类结肠黏膜细胞的肠杆菌，利用其 III 型分泌装置（T3SA）。志贺氏菌属拥有一个大型质粒，其中编码了其大部分毒力因子，并且一直是定义 T3SA 调控子的开创性工作的重点。因此，缺乏对 T3SA 调节的转录反应的全面评估。在此，我们使用 RNA-Seq 来鉴定在 T3SA 活跃（开启状态）与不活跃（关闭状态）时差异表达的基因。通过与先前的微阵列研究的相关性，验证了 RNA-Seq 数据集的质量。利用关于非编码区表达的新见解、生物信息学工具和实验，我们证明了六个操纵子的存在，并证明了 ipaH2.5 是一个假基因。此外，在开启状态下，86 个染色体基因下调，包括一些非编码转录本，这些转录本对应于嵌入 16S 和 23S RNA 基因中的短反义 RNA，以及 40 个编码转录本，其同源蛋白在遗传和生化水平上高度连接。最后，我们鉴定了两个新的染色体基因 gem1 和 gem3，它们在开启状态下与 T3SA 调控子的基因类似地上调。这些发现通过液滴数字 PCR 在生物重复实验中得到了验证。据我们所知，gem1 和 gem3 是 T3SA 调控子中第一个没有质粒同源物的染色体成员。我们的方法为优化 RNA-Seq 研究提供了一种途径，适用于先前曾进行过中等规模至大规模研究的细菌模型。
