禽致病性大肠杆菌外膜蛋白OmpA对DF-1细胞自噬的影响

doi:10.16431/j.cnki.1671-7236.2023.05.024

Abstract

Abstract: 【Objective】 This study was aimed to investigate the effect of the outer membrane protein OmpA of avian pathogenic Escherichia coli (APEC) on autophagy in DF-1 cells and thus provide a basis for understanding whether OmpA helped APEC evade the autophagy mediated clearance in host cells.【Method】 The genomic DNA of APEC E058 strain was used as a template to recover the ompA gene fragment by PCR amplification,and then cloned into the eukaryotic expression vector pEGFP-N1 after double digestion with EcoR Ⅰ and Xho Ⅰ.The positive recombinant plasmid pEGFP-N1-ompA was screened and confirmed by enzyme digestion and sequencing.After transfected into DF-1 cells,the expression of OmpA was detected by Western blotting and immunofluorescence assay.The effects of OmpA on autophagy in DF-1 cells were detected by transmission electron microscopy,immunofluorescence assay and Western blotting.【Result】 After EcoR Ⅰ/Xho Ⅰ enzyme digestion of the sequencing identified recombinant plasmid,two bands with size of 1 038 and 4 700 bp were obtained,which were consistent with the vector and the target fragment,indicating that the eukaryotic expression plasmid pEGFP-N1-ompA was successfully constructed.After transfection of recombinant plasmid with liposome into DF-1 cells,a large amount of green fluorescence could be observed by immunofluorescence assay and 65 ku protein band was detected by Western blotting,indicating that pEGFP-N1-ompA was successfully transfected into DF-1 cells and ompA-EGFP fusion protein was expressed in large amounts.Meanwhile,Western blotting results showed that overexpression of OmpA caused an increase in the expression of autophagy marker protein LC3 Ⅱ.The autophagsomes in DF-1 cells were observed by transmission electron microscopy.The single fluorescent GFP-LC3 plasmid was transfected into DF-1 cells,and the OmpA protein treated group showed aggregation of green fluorescent spots,indicating that OmpA induced autophagy of DF-1 cells.Furthermore,OmpA affected the degradation of autophagy marker protein p62, and the transfection of double fluorescent mRFP-GFP-LC3 plasmid showed that OmpA could block the oceurrence of autophagic flux.【Conclusion】 The APEC outer membrane protein OmpA could cause autophagy in DF-1 cells,but it blocked the autophagic flux and induced incomplete autophagy in DF-1 cells,which would help to better understand the escape mechanism of APEC from the host cells clearance.

Key words: avian pathogenic Escherichia coli; OmpA; DF-1 cells; incomplete autophagy

CLC Number:

S852.61

CHENG Qian, GAO Qingqing, WANG Yuhe, HUAN Changchao, GAO Song. Effects of Avian Pathogenic Escherichia coli Outer Membrane Protein OmpA on Autophagy of DF-1 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1971-1980.

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Effects of Avian Pathogenic Escherichia coli Outer Membrane Protein OmpA on Autophagy of DF-1 Cells

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