尿道致病性大肠杆菌U17株rstA缺失株降低对小鼠的致病性

Deletion of the rstA gene of uropathogenic Escherichia coli strain U17 led to attenuation of virulence in mouse

摘要:

[目的]探究双组份系统RstA/RstB中效应基因rstA对尿道致病性大肠杆菌毒力的影响。[方法]利用λ Red重组系统构建UPEC U17 rstA缺失株U17ΔrstA，并构建相应的回复株，通过体内外试验评价rstA基因缺失对UPEC毒力的影响。[结果]生长曲线的测定结果显示，在LB普通培养基中，缺失株生长速度与野生株相似，但在LB贫铁培养基中，缺失株生长速度较野生株相比明显下降；体外环境应激试验结果显示，缺失株在强酸、强碱、高渗透压、尿素、氧化应激等环境压力下的生存能力与野生株相似；菌株生物被膜检测结果显示，缺失株的生物被膜形成能力与野生株相当；荧光定量PCR检测结果显示，rstA基因在贫铁环境下的表达水平较正常条件下显著上调，暗示贫铁环境可能是rstA发挥效应的刺激信号。6周龄BALB/c小鼠尿道感染试验结果显示，rstA缺失株在尿液、膀胱、肾脏中的带菌量显著低于野生株，而回复株毒力恢复至野生株水平，表明rstA缺失能显著降低UPEC U17的毒力。[结论]rstA与尿道致病性大肠杆菌的致病性相关，为潜在的毒力因子。

关键词:尿道致病性大肠杆菌;双组份系统;rstA;缺失株;毒力

Abstract:

[Objective] To study the role of response regulator rstA of TCS RstA/RstB in pathogenesis of uropathogenic Escherichia coli (UPEC). [Methods] By using λ Red recombination system, we generated the rstA knockout mutant U17ΔrstA and the complementation strain ReU17ΔrstA. We then compared and analyzed the characterization of the mutant strain and the wild type strain in vivo and in vitro.[Results] The growth curves in LB showed that the deletion of rstA did not affect growth kinetics of mutant, whereas in LB-Fe medium, the growth rate of U17ΔrstA was lower than that of the wild-type strain U17. Under the selected environmental stress conditions in vitro, the bacterial survival experimental results showed that the mutant was not sensitive to acid, alkali, high osmotic pressure, urea and oxidants. Strain biofilm assay showed that the biofilm formation ability of the mutant was similar to that of the wild-type strain. qRT-PCR results showed that the rstA gene was significantly upregulated in LB-Fe medium, indicating that the iron-deficiency environment may be the stimulus signal evoking the RstA regulator. The mouse model of ascending urinary tract infection demonstrated that the deletion of rstA led to attenuation of virulence, because the mutant showed significantly decreased colonization compared with the wild type strain in urine, bladder and kidney, whereas the complementation strain restored the virulence to resemble that of wild-type strain. [Conclusion] The rstA gene was a potential virulence factor and associated with the pathogenesis of UPEC.

Key words:Uropathogenic Escherichia coli;two-component regulatory system;rstA;mutant;virulence