hipA对禽致病性大肠杆菌生物学特性和致病性的影响

Effects of toxin gene hipA on biological characteristics of avian pathogenic Escherichia coli

【目的】探究毒素-抗毒素(TA)系统中毒素基因hipA对禽致病性大肠杆菌(APEC)生物学特性和致病性的影响,为深入研究TA系统和APEC的致病机制提供理论依据。【方法】利用Red同源重组方法,构建禽致病性大肠杆菌野生株AE81的hipA基因缺失株AE81ΔhipA及回复株C-AE81ΔhipA,测定其生长曲线、运动性、环境耐受能力、细胞黏附能力,并采用实时荧光定量PCR检测环境耐受相关基因(hdeA、hdeB)、黏附相关基因(fimF、fimH)、毒力相关基因(hcp、ompR、ompC)的转录水平,探究毒素蛋白hipA对APEC的生物学功能及致病性的影响。【结果】成功构建hipA基因的缺失株和回复株。野生株AE81、缺失株AE81ΔhipA和回复株C-AE81ΔhipA的生长情况无明显差异。与野生株AE81相比,hipA基因缺失株AE81ΔhipA的运动能力降低,对酸性、碱性、高渗和氧化应激环境的耐受能力减弱,鞭毛和菌毛的数量和长度明显减小,对鸡胚成纤维细胞(DF-1)的黏附能力极显著减弱(P<0.01),回复株C-AE81ΔhipA各指标基本恢复到野生株水平。实时荧光定量PCR结果显示,与野生株AE81相比,AE81ΔhipA菌株的hdeA、hdeB、fimF、ompR、ompC基因转录水平显著降低(P<0.05),hcp基因转录水平差异不显著,但fimH转录水平显著升高(P<0.05)。【结论】TA系统毒素蛋白hipA影响APEC的运动性、环境耐受能力和对细胞的黏附能力,参与APEC的致病过程。

【Objective】This study explored the effects of toxin gene hipA of toxin/antitoxin(TA)system on the biological characteristics and pathogenicity of avian pathogenic Escherichia coli(APEC)to provide basis for in-depth research on the pathogenesis of TA system and APEC.【Method】The hipA gene mutant strain AE81ΔhipA and complemented strain C-AE81ΔhipA of the wild-type strain AE81 were constructed by Red homologous recombination method.Through the determination of growth curve,motility,environmental tolerance test,cell adhesion ability and transcription levels of environmental tolerance related genes(hdeA and hdeB),adhesion related genes(fimF and fimH)and virulence related genes(hcp,ompR and ompC),the biological functions and pathogenic effects of hipA on APEC were explored.【Result】The mutant and complemented strains of hipA gene were successfully constructed.There was no significant difference in the growth of wild-type strain AE81,mutant strain AE81ΔhipA and complemented strain C-AE81ΔhipA.Compared with the wild-type strain AE81,the motility of the deletion strain was reduced,and the tolerance to acidic,alkaline,hypertonic and oxidative stress was weakened.The number and length of flagella and pilus were significantly decreased,and the adhesion ability to chicken embryo fibroblast(DF-1)was significantly weakened(P<0.01).The complemented strain basically recovered to the level of wild-type strain.The transcription levels of hdeA,hdeB,fimF,ompR and ompC in AE81ΔhipA were significantly decreased(P<0.05),the transcription levels of hcp in AE81ΔhipA had no significant differences,but the transcription levels of fimH significantly increased(P<0.05).【Conclusion】The toxin protein hipA in TA system affected motility,environmental tolerance and cell adhesion of APEC and participated in the pathogenesis of APEC.