摘要
目的 确定肺炎链球菌丝氨酸/苏氨酸激酶StkP与耐药相关性及其胞外区与β-内酰胺类抗生素结合的能力.方法 采用插入失活法构建肺炎链球菌ATCC6306株stkP基因敲除（ΔstkP）突变株.采用E-test检测青霉素（PCN）和头孢噻肟（CTX）对Δstkp突变株及其野生株的最低抑菌浓度（MIC）.采用生物信息学软件确定肺炎链球菌ATCC6306株丝氨酸/苏氨酸激酶编码基因（stkP）胞外区（EC-StkP）、构建EC-StkP三维空间结构模型、分析EC-StkP结构与功能关系.采用PCR扩增stkP基因胞外区片段（EC-stkP）,T-A克隆后测序.构建EC-stkP原核表达系统,SDS-PAGE联合凝胶图像分析系统检查目的重组蛋白（EC-rStkP）表达情况,Ni-NTA亲和层析法提纯EC-rStkP.采用等温滴定量热法（VT-ITC）和表面等离子共振法（Biacore）检测EC-rStkP与PCN和CTX结合能力.结果 肺炎链球菌ATCC6306株对PCN和CTX敏感（MIC=0.06和0.12 μg/ml）,但ΔstkP突变株对PCN和CTX高度耐药（MIC=16和32 μg/ml）.肺炎链球菌ATCC6306株StkP C端295 aa片段为胞外区,该胞外区有4个青霉素结合蛋白和丝氨酸/苏氨酸激酶相关（PASTA）功能结构域.克隆的stkP胞外区序列与GenBank中相应基因核苷酸和氨基酸序列相似性分别为99.6%和100%.构建的EC-stkP原核表达系统可表达可溶性EC-rStkP.PCN和CTX均能与EC-rStkP结合,但后者结合EC-rStkP能力强于前者.结论 肺炎链球菌stkP基因与β-内酰胺类抗生素耐药性密切相关,其表达产物丝氨酸/苏氨酸激酶StkP具有识别并结合β-内酰胺类抗生素的能力.
Objective To investigate the correlation between Streptococcus pneumoniae （S.pneumoniae） StkP kinase and drug resistance and to analyze the binding ability of StkP extracellular region （EC-StkP） to β-lactam antibiotics.Methods A stkP gene knockout （ΔstkP） mutant was constructed from S.pneumoniae strain ATCC6306 by insertional inactivation method.E-test was performed to detect the minimum inhibitory concentrations （MIC） of penicillin （PCN） and cefotaxime （CTX） against ΔstkP mutant and its wild-type strain.Bioinformatic softwares were used to predict the EC-StkP of S.pneumonia strain ATCC6306,to generate the three-dimensional structure model of EC-StkP and to analyze the correlation between the structure and functions of EC-StkP.PCR was performed to amplify the extracellular segment of stkP （EC-stkP） gene and the product of it was sequenced after T-A cloning.A prokaryotic expression system of EC-stkP gene was constructed.SDS-PAGE in combination with a gel image analysis system was used to detect the expression of the recombinant EC-StkP （EC-rStkP）.The expressed EC-rStkP was extracted by Ni-NTA affinity chromatography.The binding abilities of EC-rStkP to PCN and CTX were detected by isothermal titration calorimetry （VT-ITC） and surface plasmon resonance （Biacore）.Results S.pneumonia strain ATCC6306 was sensitive to PCN （MIC=0.06 μg/ml） and CTX （MIC=0.12 μg/ml）,but its ΔstkP mutant was resistant to the two antibiotics （PCN MIC=16 μg/ml,CTX MIC=32 μg/ml）.The 295 aa segment was predicted as the extracellular region at C-end of StkP of S.pneumoniae strain ATCC6306,containing four penicillin-binding proteins and Ser/Thr kinase-associated （PASTA） domains.The cloned EC-stkP segment and the EC-stkP segment in GenBank shared 99.6% similarity in nucleotide sequence and 100% in amino acid sequence.The constructed prokaryotic expression system for EC-stkP gene expressed EC-rStkP in soluble form.Both PCN and CTX could bind to EC-rStkP and CTX was better than PCN in term of binding ability.Conclusion The stkP gene of S.pneumonia is closely related to drug resistance and the encoded protein,Ser/Thr kinase StkP,can recognize and bind to β-lactam antibiotics.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2017年第6期424-430,共7页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金项目（81271893）
浙江省卫生高层次创新人才培养工程项目（2012-241）
