毒力基因ompA在禽致病性大肠埃希菌外膜囊泡诱导鸡气管黏膜上皮细胞凋亡中的功能

Role of virulence gene ompA in apoptosis of chicken trachea epithelium cells induced by outer membrane vesicles of avian pathogenic Escherichia coli

摘要:

【目的】探究毒力基因ompA在禽致病性大肠埃希菌(avian pathogenic Escherichia coli,APEC)分泌外膜囊泡(outer membrane vesicle,OMV)诱导鸡气管黏膜上皮细胞(chicken trachea epithelium cells,CTECs)凋亡中的功能，为后期深入研究APEC-OMV的致病机制奠定基础。【方法】以APEC分离株AE17为野生株，利用CRISPR/Cas9系统构建ompA基因缺失株，利用表达载体pET-28a构建ompA基因过表达株，并分别提取野生株、缺失株、pET-28a空载株及过表达株的OMV。通过透射电镜、纳米颗粒分析、annexin V-FITC/PI双染检测及超微病理切片等实验探究毒力基因ompA在APEC-OMV诱导CTECs细胞凋亡中的功能。【结果】成功构建缺失株AE17△ompA、空载株AE17-pET-28a及过表达株AE17-pET-28a-OmpA。与AE17的OMV(AE17-OMV)相比，AE17 △ompA的OMV(AE17△ompA-OMV)颗粒浓度显著减少且平均粒径显著降低(P<0.05)，而AE17-pET-28a-OmpA的OMV(AE17-pET-28a-OmpA-OMV)颗粒浓度显著增加且平均粒径显著增大(P<0.05)。与AE17-OMV处理组相比，AE17△ompA-OMV对CTECs损伤程度降低，凋亡率下降，仅有部分CTECs线粒体轻微肿胀；而AE17-pET-28a-OmpA-OMV感染CTECs后细胞凋亡率升高且出现显著细胞病变，如部分线粒体基质变淡、嵴消失甚至转化为空泡状结构等。【结论】ompA基因对APEC-OMV的平均粒径和颗粒浓度具有正调控作用，且促进APEC-OMV诱导CTECs细胞凋亡。

关键词:禽致病性大肠埃希菌;外膜囊泡;ompA;鸡气管黏膜上皮细胞;细胞凋亡

Abstract:

[Objective] To explore the role of the virulence gene ompA in apoptosis of chicken trachea epithelium cells (CTECs) induced by outer membrane vesicles (OMV) of avian pathogenic Escherichia coli (APEC), so as to lay a foundation for further research on the pathogenesis of APEC-OMV. [Methods] On the basis of the wild-type APEC strain AE17, the ompA-deleted strain was constructed via the CRISPR/Cas9 system and the ompA-overexpressing strain via the expression plasmid pET-28a. OMV was respectively extracted from the wild-type strain, the ompA-deleted strain, the empty plasmid-transformed strain, and the ompA-overexpressing strain. The role of ompA in the apoptosis of CTECs induced by APEC-OMV was explored by transmission electron microscopy, nanoparticle tracking analysis, annexin V-FITC/PI double staining assay, and ultramicroscopic pathological sections. [Results] The ompA-deleted strain AE17△ompA, the empty plasmid-transformed strain AE17-pET-28a, and the ompA-overexpressing strain AE17-pET-28a-OmpA were successfully constructed. Compared with those of AE17-OMV, the particle density and size of OMV decreased in AE17△ompA-OMV (P<0.05) and increased in AE17-pET-28a-OmpA-OMV (P<0.05). Compared with the CTECs induced by AE17-OMV, those induced by AE17△ompA-OMV showcased reduced apoptosis rate and damage degree, with slightly swelling of only some mitochondria. However, the CTECs infected by AE17-pET-28a-OmpA-OMV presented increased apoptosis rate and damage degree as manifested by the matrix fading of some mitochondrion and disappearance or even transformation into vacuolated structures of mitochondrial cristae. [Conclusion] The virulence gene ompA positively regulated the particle density and size of APEC-OMV and promoted the apoptosis of CTECs induced by APEC-OMV.

Key words:avian pathogenic Escherichia coli;outer membrane vesicle;ompA;chicken trachea epithelium cells;apoptosis