ygeG基因对禽致病性大肠杆菌生物学特性及致病性的影响

Effect of ygeG on biological characteristics and the pathogenicity of avian pathogenic Escherichia coli

摘要:

【目的】本研究构建禽致病性大肠杆菌（avian pathogenic Escherichia coli，APEC）ygeG基因缺失株，并对其进行生物学特性及致病性分析，探究ygeG在APEC致病过程中的作用，为后期深入研究其所在的Ⅲ型分泌系统2（type three secretion systems 2，ETT2）毒力岛的致病机制奠定基础。【方法】利用Red同源重组技术构建ygeG缺失株APEC81-ΔygeG及其回复株APEC81-CΔygeG，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ygeG对APEC致病性相关的生物学功能的影响，并通过细胞黏附、侵袭试验及荧光定量PCR检测细胞炎性因子转录水平探究ygeG对APEC感染宿主过程的影响。【结果】成功构建了缺失株APEC81-ΔygeG和回复株APEC81-CΔygeG；与野生株APEC81相比，缺失株APEC81-ΔygeG生长特性无显著变化（P>0.05），生物被膜形成能力显著降低（P<0.01），运动能力极显著升高（P<0.001），对酸休克和氧化休克的耐受力极显著降低（P<0.001），对碱、渗透压和热休克的耐受力显著降低（P<0.01）；血清杀菌实验表明，ygeG缺失能显著降低其抗血清杀菌作用（P<0.01），血清浓度为100%、30%时，差异极显著（P<0.001）；与野生株APEC81相比，缺失株APEC81-ΔygeG对鸡气管粘膜上皮细胞的黏附能力极显著下降（P<0.001），侵袭能力显著升高（P<0.01）；同时，经APEC81-ΔygeG感染的鸡气管粘膜上皮细胞炎性因子转录水平显著升高（P<0.01）。【结论】ygeG影响APEC的生物被膜形成、运动性、抗逆性、黏附侵袭能力、以及对血清的敏感性等，并可抑制细胞炎性因子表达。

关键词:禽致病性大肠杆菌;ETT2;ygeG;生物学特性;致病性

Abstract:

[Objective] To construct the ygeG deletion strain of avian pathogenic Escherichia coli (APEC), and analyze its biological characteristics and pathogenicity to explore the role of ygeG plays on the pathogenesis of APEC, so as to lay a foundation for further research on the pathogenesis of type three secretion system 2 (ETT2), which ygeG locates in.[Methods] The ygeG deletion strain (APEC81-ΔygeG) and complementary strain (APEC81-CΔygeG) were constructed by Red homologous recombination technology. Then, the growth curve, motility, biofilm formation, stress resistance, serum resistance among APEC81, APEC81-ΔygeG and APEC81-CΔygeG were compared and analyzed. The effect on host infection of ygeG was investigated by cell adhesion, invasion test and inflammatory factor expression level detection by fluorescence quantitative PCR.[Results] We successfully constructed APEC81-ΔygeG and APEC81-CΔygeG. Compared with APEC81, there was no significant changes in growth characteristics in APEC81-CΔygeG (P>0.05). However, biofilm formation ability of APEC81-ΔygeG significantly decreased (P<0.01), motility of APEC81-ΔygeG extremely significantly improved (P<0.001). Compared with APEC81, APEC81-ΔygeG showed lower tolerance to acid (P<0.001) and oxidative shock (P<0.001), and higer tolerance to alkali (P<0.01), osmotic pressur (P<0.01) and heat shock (P<0.01). Furthermore, serum survival experiments results showed that deletion of ygeG significantly decreased survival abilities of APEC81 in serum (P<0.01), and extremely significantly decreased at serum concentrations of 100% and 30% (P<0.001); The adhesion of APEC81-ΔygeG to epithelial cells of chicken trachea mucosa was extremely significantly decreased (P<0.001), and the invasion ability was significantly increased (P<0.01). Furthermore, qPCR results also showed that APEC81-ΔygeG significantly up-regulated the transcription level of of inflammatory factors in chicken tracheal epithelial cells (P<0.01).[Conclusion] These data indicated that ygeG plays roles in regulation of biofilm formation, motility, stress resistance, adhesion and invasion ability, and serum resistance of APEC, it also can inhibit cell inflammatory factor expression.

Key words:avian pathogenic E. coli;ETT2;ygeG;biological characteristics;pathogenicity