yeaI基因对奶牛源大肠杆菌NJ17生物学特性的影响

Effect of yeaI gene on biological characteristics of Escherichia coli of cattle origin

摘要:

c-di-GMP是细菌中广泛存在的第二信使，可通过效应蛋白参与调控细菌的生物被膜形成、运动性和毒力等生物学特性。YeaI因含有能结合c-di-GMP分子的EGEVF基序，可能作为c-di-GMP效应蛋白发挥作用。[目的] 研究yeaI基因缺失对奶牛源大肠杆菌临床分离株NJ17生物学特性的影响。[方法] 构建NJ17的yeaI缺失株（NJ17ΔyeaI）及回复株cNJ17ΔyeaI，分析yeaI对NJ17生物学特性（如生长特性、生物被膜形成能力和对小鼠乳腺上皮细胞（EpH₄-Ev）的黏附）的影响。[结果] 成功构建NJ17的yeaI缺失株（NJ17ΔyeaI）及其回复株（cNJ17ΔyeaI）；与野生株NJ17相比，缺失株NJ17ΔyeaI生长特性及耐药性无显著变化，生物被膜形成能力显著下降，运动性显著升高（P<0.05）；透射电镜检测结果表明，yeaI缺失影响NJ17菌毛和鞭毛的形成；实时定量PCR（qPCR）结果显示，yeaI基因显著抑制NJ17鞭毛基因filG和motB的转录水平（P<0.05）；血清杀菌实验表明，yeaI缺失能显著增强其抵抗血清杀菌作用（P<0.05）；对EpH₄-Ev细胞黏附实验表明，yeaI缺失对NJ17黏附性无显著影响（P>0.05）。[结论] yeaI对奶牛源大肠杆菌NJ17的生物学特性具有重要的调控作用。

Abstract:

c-di-GMP is a widespread second messenger in bacteria and regulates many important bacterial biological characteristics (such as biofilm, motility and virulence) by binding effector proteins. YeaI protein containing EGEVF motifs can bind c-di-GMP and may regulate bacterial performance. [Objective] To study the roles of yeaI in Escherichia coli NJ17 strain. [Methods] The yeaI mutant (NJ17ΔyeaI) and complementary strain (cNJ17ΔyeaI) were constructed. Growth characteristics, biofilm formation ability, adhesion and other biological characteristics were analyzed in NJ17, NJ17ΔyeaI and cNJ17ΔyeaI. [Results] We successfully constructed NJ17ΔyeaI and cNJ17ΔyeaI. Compared with NJ17, there was no significant changes in growth characteristics and drug resistance of NJ17ΔyeaI (P>0.05). However, biofilm formation ability of NJ17ΔyeaI significantly decreased and motility of NJ17ΔyeaI significantly improved (P<0.05). Transmission electron micrographs shows that yeaI deletion affected the formation of NJ17 fimbriae and flagella. qPCR results also showthat the yeaI gene significantly inhibited the transcription level of the flagella-related genes motB and filG in NJ17. Furthermore, serum survival experiments results show that deletion of yeaI significantly enhanced survival abilities of NJ17 in rat serum (P<0.05). However, compared with NJ17, there was no significant change in adhesion rate of yeaI mutant to EpH₄-Ev (P<0.05). [Conclusion] The yeaI plays an important role in regulating the biological characteristics of E. coli of cattle origin.