RNA-seq to profile the H-NS transcriptome in EHEC O157:H7 strain EDL933

In order to define the role of H-NS in regulating gene transcription and further find out the biological significance of this protein in EHEC, we conducted RNA-seq and then analyzed the transcriptome data, using EHEC O157:H7 strain EDL933 and Δhns. A total of 983 genes were found to be regulated by H-NS. 213 and 770 genes exhibited lower and higher transcript levels in Δhns than in WT, respectively. For instance, chemotaxis and flagellar associated genes were down-regulated in Δhns. Besides, 34 genes on virulence plasmid pO157 were down-regulated by H-NS. The outcome of RNA-seq were verified by real time quantitative PCR. As reported in Salmonella Typhimurium, Δhns showed a growth deficiency and altered fitness. We first detected that both stx1 and stx2 in EDL933 were repressed by H-NS. Although no survival difference between EDL933 and Δhns was detected when phagocytized by macrophage, we characterized the higher ability of colonization and in consequence the higher virulence of Δhns to BALB/c mice by experimental analyses than those of WT, especially when intact commensal flora of mice existed. This might indeed help us understand the core role of H-NS in depth. RNA sequencing of EHEC O157:H7 strain EDL933 and Δhns in 2 biological repeats.

为明确H-NS在基因转录调控中的作用，并进一步阐明该蛋白在肠出血性大肠杆菌（Enterohemorrhagic Escherichia coli, EHEC）中的生物学意义，我们以EHEC O157:H7菌株EDL933及其Δhns突变株为实验材料，开展RNA测序（RNA-seq）并对转录组数据进行系统分析。结果共鉴定出983个受H-NS调控的基因：其中213个基因在Δhns突变株中的转录水平较野生型（Wild Type, WT）更低，770个基因则呈现更高的转录丰度。举例而言，趋化及鞭毛相关基因在Δhns突变株中发生下调表达。此外，致病性质粒pO157上的34个基因同样受H-NS负调控。我们通过实时定量PCR（real-time quantitative PCR）对RNA测序结果进行了实验验证。正如鼠伤寒沙门氏菌（Salmonella Typhimurium）中的既往研究报道，Δhns突变株存在生长缺陷且适应度发生改变。我们首次发现EDL933中的stx1与stx2基因均受H-NS的转录抑制。尽管巨噬细胞吞噬实验未检测到EDL933与Δhns的胞内生存差异，但实验分析表明，相较于野生型菌株，Δhns突变株对BALB/c小鼠的定殖能力更强，由此导致的致病力也更高，这一差异在小鼠肠道正常菌群完整的条件下尤为显著。该研究结果有助于我们深入理解H-NS的核心调控功能。本研究对EHEC O157:H7菌株EDL933及其Δhns突变株开展了2次生物学重复的RNA测序。