ppk1基因缺失对脑膜炎大肠杆菌K1株生物学功能的影响

Changes of biological behavioral of E. coli K1 after ppk1 gene deletion

目的比较大肠杆菌K1株E44敲除ppk1基因后与野生株之间差异并探讨ppk1基因在E.coli K1株致脑膜炎机制中的作用。方法(1)将野生株与敲除株置于56℃2、4、6 min,以比较二者对热刺激的抵抗力差异;(2)利用电子显微镜直接观察以及采用经典的粘附、侵袭率定量实验比较二者对脑微血管内皮细胞(HBMEC)的粘附和侵袭能力;(3)将细菌与脑微血管内皮细胞共同孵育后,利用激光共聚焦观察二者诱导脑微血管内皮细胞的细胞骨架重排现象。结果与野生株E44相比,ppk1敲除株对于56℃热刺激的抵抗力明显下降;电子显微镜可以观察到,敲除株粘附和侵袭入HBMEC的数量均少于野生株,定量粘附、侵袭实验也进一步证实;借助激光共聚焦发现敲除株诱导HBMEC细胞骨架重排的能力要弱于野生株。结论 ppk1对于脑膜炎大肠杆菌K1株抵抗热刺激、粘附和侵袭HBMEC以及诱导HBMEC的细胞骨架重排具有重要作用。

Objective To study the changes in biological behaviors of meningitis E. coli K1 strain E44 after deletion of polyphosphate kinase 1 （ppk1） gene and explore the role of ppk1 in the pathogenesis of E. coli K1-induced meningitis. Methods The wild-type strain E. coli K1 and ppk1 deletion mutant were exposed to heat at 56℃for 6 min, and their survival rates were determined. The adhesion and invasion of the bacteria to human brain microvascular endothelial cells （HBMECs） were observed using electron microscopy and quantitative tests. HBMECs were co-incubated with wild-type strain or ppk1 deletion mutant, and the cytoskeleton rearrangement was observed under laser scanning confocal microscope. Results The survival rate of the ppk1 deletion mutant was significantly lower than that of the wild-type strain after heat exposure. The ppk1 deletion mutant also showed lowered cell adhesion and invasion abilities and weakened ability to induce cytoskeleton rearrangement in HBMECs. Conclusion ppk1 gene is important for E.coli K1 for heat resistance, cell adhesion and invasion, and for inducing cytoskeletal rearrangement in HBMECs.