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

作　　者：高清清[1] 邵启文 叶正琴 夏乐[1] 高崧[1] 焦新安[1] 刘秀梵[1] 

机构地区：[1]扬州大学兽医学院,江苏省动物重要疫病与人兽共患病防控协同创新中心,农业部禽用生物制剂创制重点实验室,江苏扬州225009

出　　处：《微生物学报》2018年第3期501-510,共10页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31272559,30972196,30771604,30471281,31672553,31602059);农业部公益性行业专项(201303044);中国博士后科学基金(2015M580477);江苏省博士后基金(1501076C);江苏省自然科学基金(BK20140485,BK20151308);江苏省高校自然科学基金(14KJB230001);江苏高校优势学科建设工程资助项目(PAPD);扬州大学科技创新培育基金(2014CXJ051,2015CXJ057)

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λRed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UPECU17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。[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 U17△rstA and the complementation strain ReU17△rstA. 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 U17△rstA 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.

关 键 词：尿道致病性大肠杆菌 双组份系统 rstA 缺失株 毒力 

分 类 号：S852.61[农业科学—基础兽医学]