大肠杆菌三型分泌系统2转录调节子EtrA对禽致病性大肠杆菌致病性的影响

Effect of ETT2 transcriptional regulator EtrA on the pathogenicity of avian pathogenic Escherichia coli

摘要:

【背景】禽致病性大肠杆菌(avian pathogenic Escherichia coli，APEC)是禽类主要病原菌之一，大肠杆菌三型分泌系统2 (Escherichia coli type III secretion system 2，ETT2)可通过转录调节子调控其致病性，但在APEC中转录调节子EtrA对其致病性的影响目前尚不清楚。【目的】研究ETT2中转录调节子EtrA对APEC致病性的影响。【方法】利用Red同源重组技术构建ETT2-etrA基因缺失株及回复株。比较生长性能、生物被膜形成、运动性及对血清敏感性的差异，基于RNA-Seq测序及Real-time PCR技术比较野生株和缺失株中与生物被膜形成、运动性以及毒力因子相关基因的转录水平。【结果】与野生株相比，缺失株及回复株生长特性无显著变化(P>0.05)，但APEC40-ΔetrA生物被膜形成能力和对血清敏感性明显增强(P<0.001)，运动性较野生株明显下降(P<0.01)，回复株的表型有所回复。转录组学筛选出7个毒力差异基因，生物被膜形成相关基因显著上调，参与影响细菌运动性的基因显著下调。qRT-PCR验证与转录组学结果一致。【结论】etrA缺失可以显著影响APEC的生物被膜形成、运动性及对血清的敏感性，这可为进一步探讨ETT2对APEC的致病作用提供参考。

Abstract:

[Background] Avian pathogenic Escherichia coli (APEC) is one of the main pathogens of poultry. ETT2 (Escherichia coli type III secretion system 2) regulates its pathogenicity through transcriptional regulators. Currently, the effect of the transcriptional regulators EtrA on its pathogenicity in APEC is unclear. [Objective] To study the effect of ETT2 transcriptional regulator EtrA on the pathogenicity of APEC. [Methods] APEC40-ΔetrA and APEC40-CΔetrA were constructed by the lambda Red recombinase system. Then the biological characteristics, including growth characteristics, biofilm formation ability, motility and sensitivity to serum was compared between APEC40 and APEC40-ΔetrA. Besides, the transcriptional levels of virulence genes, biofilm formation related genes and flagellin synthesis genes of wild and complementation strain were compared by RNA-Seq and real-time PCR. [Results] The growth characteristics did not significantly change between APEC40 and APEC40-ΔetrA (P>0.05). However, the biofilm formation and sensitivity to serum were significantly enhanced (P<0.001), while the motility was significantly decreased (P<0.01) compared with the wild strain APEC40. There was some recovery in the phenotype of the complemented strains. Furthermore, transcriptomics screened seven virulence difference genes, which showed that the genes related to biofilm formation were significantly up-regulated, and the genes involved in motility were significantly down-regulated. The results of qRT-PCR were consistent with those of RNA-Seq. [Conclusion] Loss of etrA can significantly affect the biofilm formation, motility, and sensitivity to serum in APEC. This study provides a reference for further study on the pathogenicity of ETT2 in APEC.