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

摘要/Abstract

摘要： 【目的】探讨禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)外膜蛋白OmpA 对DF-1细胞自噬的影响,为研究OmpA是否协助APEC逃逸宿主细胞自噬介导的清除作用提供依据。【方法】以APEC E058 株基因组DNA为模版,通过PCR扩增回收ompA基因片段,经EcoRⅠ和XhoⅠ双酶切后克隆至真核表达载体pEGFP-N1,通过酶切及测序鉴定筛选阳性的重组表达质粒pEGFP-N1-ompA;随后将质粒转染至鸡胚成纤维细胞DF-1并通过Western blotting和免疫荧光试验检测OmpA蛋白的表达情况;通过透射电子显微镜、免疫荧光试验及Western blotting方法检测OmpA对DF-1细胞自噬的影响。【结果】测序鉴定正确的重组质粒pEGFP-N1-ompA经EcoR Ⅰ和Xho Ⅰ双酶切,获得大小分别为1 038和4 700 bp的条带,大小与载体和目的片段相符,表明真核表达质粒pEGFP-N1-ompA构建成功。将重组质粒经脂质体转染DF-1细胞后,通过免疫荧光试验可观察到大量绿色荧光,Western blotting检测到大小为65 ku的蛋白条带,表明成功转染pEGFP-N1-ompA至DF-1细胞中并大量表达ompA-EGFP融合蛋白。同时Western blotting结果显示,过表达OmpA可引起自噬标志性蛋白LC3 Ⅱ的表达量增加;通过透射电子显微镜可观察到DF-1细胞中的自噬小体。将单荧光GFP-LC3质粒转染DF-1细胞,OmpA蛋白处理组呈现绿色荧光点的聚集,说明OmpA引发DF-1细胞自噬。进一步研究发现,OmpA影响自噬标志蛋白p62降解,转染双荧光mRFP-GFP-LC3质粒检测显示OmpA可阻断自噬流的发生。【结论】APEC 外膜蛋白OmpA可引起DF-1细胞发生自噬,但其抑制自噬流的发生,引发DF-1细胞的不完全自噬,这将为进一步探讨APEC逃逸宿主细胞的清除作用提供参考。

关键词: 禽致病性大肠杆菌; OmpA; DF-1细胞系; 不完全自噬

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

中图分类号:

S852.61

程前, 高清清, 王雨禾, 郇长超, 高崧. 禽致病性大肠杆菌外膜蛋白OmpA对DF-1细胞自噬的影响[J]. 中国畜牧兽医, 2023, 50(5): 1971-1980.

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.

参考文献

[1] DHO-MOULIN M,FAIRBROTHER J M.Avian pathogenic Escherichia coli(APEC)[J].Veterinary Research,1999,30(2-3):299-316.
[2] 甘孟侯.中国禽病学[M].北京:中国农业出版社,1999. GAN M H.China Poultry Diseases[M].Beijing:China Agriculture Press,1999.(in Chinese)
[3] JOHNSON J R,STELL A L.Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise[J].Journal of Infectious Diseases,2000,181(1):261-271.
[4] 李芳果.APEC-OMV关键毒力因子OmpA对鸡气管上皮细胞天然免疫的影响[D].合肥:安徽农业大学,2021. LI F G.The effect of APEC-OMV virulence factor OmpA on the innate immunity of chicken tracheal epithelial cells[D].Hefei:Anhui Agricultural University,2021.(in Chinese)
[5] CONFER A W,AYALEW S.The OmpA family of proteins:Roles in bacterial pathogenesis and immunity[J].Veterinary Microbiology,2013,163(3-4):207-222.
[6] ELLIS T N,KUEHN M J.Virulence and immunomodulatory roles of bacterial outer membrane vesicles[J].Microbiology and Molecular Biology Reviews,2010,74(1):81-94.
[7] GALLUZZI L,BAEHRECKE E H,BALLABIO A,et al.Molecular definitions of autophagy and related processes[J].EMBO Journal,2017,36(13):1811-1836.
[8] CASEY A E,LIU W,HEIN L K,et al.Transcriptional targets of senataxin and E2 promoter binding factors are associated with neuro-degenerative pathways during increased autophagic flux[J].Scientific Reports,2022,12(1):17665.
[9] 冯婷婷.VapA蛋白在马红球菌逃逸巨噬细胞自噬中的作用机制[D].银川:宁夏大学,2021. FENG T T.The mechanism of VapA protein within Rhodococcus equi escaped macrophages autophagy[D].Yinchuan:Ningxia University,2021.(in Chinese)
[10] 安志远,黄骁舾,丁文一.鲍曼不动杆菌外膜蛋白A真核表达载体构建及其诱导HeLa细胞不完全自噬[J].免疫学杂志,2017,33(11):996-1002. AN Z Y,HUANG X X,DING W Y.Eukaryotic expression vector construction of outer membrane protein A from Acinetobacter baumannii and the incomplete autophagy in HeLa cells induced by the protein[J].Immunological Journal,2017,33(11):996-1002.(in Chinese)
[11] CHEN X,GAO S,JIAO X,et al.Prevalence of serogroups and virulence factors of Escherichia coli strains isolated from pigs with postweaning diarrhoea in Eastern China[J].Veterinary Microbiology,2004,103(1-2):13-20.
[12] 周业菲.禽病原性大肠杆菌ompA、ompT基因和肠炎沙门氏菌ompA、pgtE基因突变株的构建及其生物学特性研究[D].扬州:扬州大学,2013. ZHOU Y F.Construction and characterization of ompA,ompT mutants of avian pathogenic Escherichia coli and ompA,pgtE mutants of Salmonella Enteritidis[D].Yangzhou:Yangzhou University,2013.(in Chinese)
[13] 任海燕.肠道外致病性大肠杆菌感染小鼠脾脏转录组学分析和COX-2抑制大肠杆菌感染机制的研究[D].南京:南京农业大学,2020. REN H Y,Transcriptomic analysis of mice spleen infected with extraintestinal pathogenic Escherichia coli and studies on the mechanism of COX-2 in inhibiting Escherichia coli infection[D].Nanjing:Nanjing Agricultural University,2020.(in Chinese)
[14] AN Z Y,HUANG X X,ZHENG C M. Acinetobacter baumannii outer membrane protein A induces HeLa cell autophagy via MAPK/JNK signaling pathway[J]. International Journal of Medical Microbiology,2019,309(2):97-107.
[15] 戴晓玥,吴亮,夏雯.鲍曼不动杆菌OmpA经ROS/NLRP3信号通路调控THP-1细胞自噬和凋亡[J].江苏大学学报(医学版),2020,30(5):423-429. DAI X X,WU L,XIA W.Autophagy and apoptosis induced by OmpA of Acinetobacter baumannii via ROS/NLRP3 signaling pathway in THP-1 cells[J].Journal of Jiangsu University(Medicine Edition),2020,30(5):423-429.(in Chinese)
[16] ZHAO D,LI Y,PENG C,et al.Outer membrane protein a in Acinetobacter baumannii induces autophagy through mTOR signaling pathway in the lung of SD rats[J].Biomedicine and Pharmacotherapy,2021,135:111034.
[17] HIURA F,KAWABATA Y,AOKI T,et al.Inhibition of the ATG4-LC3 pathway suppressed osteoclast maturation[J].Biochemical and Biophysical Research Communication,2022,632:40-47.
[18] ICHIMURA Y,KIRISAKO T,TAKAO T,et al.A ubiquitin-like system mediates protein lipidation[J]. Nature,2000,408(6811):488-492.
[19] ZHANG H,CHANG J T,GUO B,et al.Guidelines for monitoring autophagy in Caenorhabditis elegans[J].Autophagy,2015,11(1):9-27.
[20] FULVIO R,KLIONSKY D J.Autophagic processes in yeast:Mechanism,machinery and regulation[J].Genetics,2013,194(2):341-361.
[21] RUSSELL R C,YUAN H X,GUAN K L.Autophagy regulation by nutrient signaling[J].Cell Research,2014,24(1):42-57.
[22] DUNN W A.Studies on the mechanisms of autophagy:Formation of the autophagic vacuole[J].Journal of Cell Biology,1990,110(6):1923-1933.
[23] GLICK D,BARTH S,MACLEOD K F.Autophagy:Cellular and molecular mechanisms[J].Journal of Pathology,2010,221(1):3-12.
[24] KATSURAGI Y,ICHIMURA Y,KOMATSU M.p62/SQSTM1 functions as a signaling hub and an autophagy adaptor[J].FEBS Journal,2015,282(24):4672-4678.
[25] KIMURA S,NODA T,YOSHIMORI T.Dissection of the autophagosome maturation process by a novel reporter protein,tandem fluorescent-tagged LC3[J].Autophagy,2007,3(5):452-460.
[26] KLIONSKY D J,ABDALLA F C,ABELIOVICH H,et al.Guidelines for the use and interpretation of assays for monitoring autophagy[J].Autophagy,2012,8(4):445-544.