禽致病性大肠杆菌Hcp2b对雏鸡气管黏膜细胞因子-细胞因子受体相互作用通路的影响

Effect of Avian Pathogenic Escherichia coli Hcp2b on the Cytokine-Cytokine Receptor Interaction Pathway in Chick Tracheal Mucosa

T6SS(type VI secretion system)是革兰阴性菌中常见的一种分泌系统,其效应蛋白Hcp2b作用机制迄今仍未明晰。本研究以禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)Hcp2b蛋白为研究主体,旨在探究Hcp2b蛋白在APEC感染鸡气管黏膜过程中发挥的作用及机制。采用Red重组方法以质粒pKD3为模板构建hcp 2 b缺失株,pSTV-28质粒连接hcp2b目的片段构建重组载体,导入感受态Δhcp 2 b菌株构建回复株,并对Δhcp 2 b菌株的生长曲线进行测定。7日龄雏鸡气管感染hcp2b缺失株及野生株,感染后12、24 h收集鸡气管黏膜细胞进行转录组学测序及生物信息学分析。结果表明,hcp 2 b缺失株及回复株构建成功,hcp 2 b基因缺失对菌株的生长性能无影响。hcp 2 b基因缺失株感染气管黏膜后,mRNA的表达谱发生变化,感染后12 h,有144个基因表达量发生变化(上调差异基因87个,下调57个);感染后24 h,有135个基因表达量发生变化(上调差异基因79个,下调56个)。生物信息学分析发现差异基因富集在Cytokine-cytokine receptor interaction、Protein processing in endoplasmic reticulum等信号通路。hcp 2 b基因缺失未影响APEC的生长特性,hcp 2 b基因缺失后影响雏鸡气管黏膜Cytokine-cytokine receptor interaction通路基因mRNA转录水平的变化,IL-1β、IL-12b的表达均上调。该结果为APEC的致病机制研究提供了理论依据。

T6SS(type VI secretion system)is a common secretion system in Gram-negative bacteria.And its mechanism of effector protein Hcp2b is still unclear.The Hcp2b protein of avian pathogenic Escherichia coli(APEC)is the main subject of this study,which aims to explore the role and mechanism of Hcp2b protein in the process of APEC infection of chicken tracheal mucosa.The Red recombination method was used to construct the Hcp2b-deleted strain by using the plasmid pKD3 as the template.And the pSTV-28 plasmid was connected to the Hcp2b target fragment to construct the recombinant vector,which was introduced to the competentΔhcp2b strain to construct the Hcp2b-reverted strain.Then,we measured the growth curve ofΔHcp2b strain.Seven-day-old chicks were infected with the Hcp2b-deleted strain and the wild strain by tracheal injection.The infected tracheal mucosal cells were collected at 12 and 24 h after infection and subjected to transcriptomics sequencing and bioinformatics analysis.The results showed that theΔHcp2b strain and C Hcp2b strain were successfully constructed.And the deletion of Hcp2b gene did not affect the growth performance of the strain.The expression profile of the mRNA of the tracheal mucosa changed after the Hcp2b gene-deleted strain infected.Twelve hours after infection,the expression of 144 genes changed(87 differential genes were up-regulated and 57 were down-regulated);Twenty-four hours after infection,135 gene expressions changed(79 differential genes up-regulated and 56 down-regulated).Bioinformatics analysis found that differential genes were enriched in signaling pathways such as Cytokine-cytokine receptor interaction,Protein processing in endoplasmic reticulum,etc.The deletion of Hcp2b gene does not affect the growth characteristics of APEC.After the deletion of Hcp2b gene,it affects the change of mRNA transcription level of Cytokine-cytokine receptor interaction pathway gene in chick tracheal mucosa.And the expression of IL-1β and IL-12b are all up-regulated.This study provides a theoretical basis for the study of the pathogenic mechanism of APEC.