毒力基因yqeH功能分析及对禽致病性大肠杆菌致病性的影响

Functional analysis and pathogenicity of avian pathogenic Escherichia coli of virulence gene yqeH

【背景】禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)可引起禽类急性或亚急性感染,在近年新发现的大肠杆菌Ⅲ型分泌系统2(Escherichia coli type III secretion system 2,ETT2)中,毒力基因yqeH对其致病性的影响尚不明确。【目的】探究yqeH在APEC致病过程中的作用,为后期深入研究ETT2致病机制奠定基础。【方法】利用Red同源重组技术构建yqeH缺失株ΔyqeH及其回复株CΔyqeH,通过运动性、生物被膜形成能力、抗逆性、抗血清杀菌能力等试验分析yqeH对APEC生物学功能的影响,并通过细胞黏附、侵袭试验、致病力测定及荧光定量PCR检测细胞炎性因子转录水平,探究yqeH对APEC感染宿主的影响。【结果】构建了缺失株ΔyqeH和回复株CΔyqeH;生物学特性试验结果表明,与野生株APEC81相比,缺失株ΔyqeH生物被膜形成能力、运动能力降低,对酸、碱、渗透压、氧化休克的耐受力降低,抗血清杀菌能力及致病力显著降低;与野生株APEC81相比,缺失株ΔyqeH对鸡气管黏膜上皮细胞的黏附及侵袭能力显著下降;同时,经ΔyqeH感染的鸡气管黏膜上皮细胞炎性因子转录水平显著降低。【结论】yqeH可调控APEC的生物被膜形成、运动性、抗逆性、黏附侵袭能力、细胞炎性因子转录水平及对血清的敏感性等,进而调控APEC对宿主细胞的致病力。

[Background]Avian pathogenic Escherichia coli(APEC)can cause avian acute or subacute infection,E.coli type III secretion system 2(ETT2)is a new type III secretion system found in recent years.The effect of virulence gene yqeH on APEC pathogenicity in APEC is unclear.[Objective]To explore the role of yqeH in the pathogenesis of APEC,so as to lay a foundation for further research on the pathogenesis of ETT2.[Methods]The yqeH deletion strainΔyqeH and its complementary strain CΔyqeH were constructed by Red homologous recombination technology.The effects of yqeH on the biological function of APEC were analyzed by motility test,biofilm formation ability,stress resistance tests and serum resistance test.The effect of yqeH on host infection of APEC was investigated by cell adhesion and invasion test,pathogenicity test and inflammatory factor expression level detected by fluorescence quantitative PCR.[Results]The deletion strainΔyqeH and the complementary strain CΔyqeH were successfully constructed.Compared with the wild APEC81 strain,ΔyqeH strain showed lower biofilm formation ability and movement ability,lower tolerance to acid,alkali,osmotic pressure and oxidative shock,lower serum resistance and lower pathogenicity.The adhesion and invasion ability ofΔyqeH to the epithelial cells of chicken trachea mucosa were significantly decreased.Compared with wild strain APEC81,the transcription level of inflammatory factors in chicken tracheal epithelial cells infected byΔyqeH significantly reduced.[Conclusion]yqeH can regulate biofilm formation,motility,stress resistance,adhesion and invasion ability,inflammatory factor expression,and serum resistance of APEC,thereby regulate the pathogenicity of APEC.