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

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.