肺炎链球菌CiaR调控pbps和csRNAs基因表达作用及其与耐药相关性  

Roles of Streptococcus pneumoniae CiaR in regulating the expression of pbps genes and csRNAs and its correlation with drug resistance

作　　者：张新蔚 黄燕颖[1,2] 楼永良[1] 严杰[3] 孙爱华[1,2] 

机构地区：[1]温州医科大学检验医学院,325035 [2]杭州医学院,310053 [3]浙江大学医学院病原生物学系,杭州310058

出　　处：《中华微生物学和免疫学杂志》2017年第1期48-56,共9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）

摘　　要：目的构建肺炎链球菌ciaR基因敲除（ΔciaR）突变株，了解该菌二元信号传导系统CiaH/CiaR中反应调节蛋白CiaR对青霉素结合蛋白基因（pbps）和cia-依赖小RNA基因（csRNAs）表达的作用。方法采用电泳迁移率试验（ESMA）确定与重组表达CiaR蛋白（rCiaR）结合的pbps基因。构建用于敲除肺炎链球菌ciaR基因的自杀质粒pEVP3ciaR，通过自杀质粒同源重组、插入失活及氯霉素筛选获得ΔciaR突变株。采用PCR及测序鉴定ΔciaR突变株。采用E-test测定青霉素（PCN）和头孢噻肟（CTX）对肺炎链球菌菌株最低抑菌浓度（MIC）。采用实时荧光定量RT-PCR，检测ΔciaR突变株及其野生株1/4 MIC PCN或CTX作用前后pbps-mRNAs和csRNAs水平变化与差异。结果CiaR能与肺炎链球菌pbp1a、pbp1b和pbp2b基因启动子区结合。PCR和测序结果证实ΔciaR突变株中ciaR基因被插入失活。1/4 MIC PCN或CTX作用后，肺炎链球菌ΔciaR突变株pbps-mRNAs水平明显升高（P〈0.05）、csRNAs水平明显降低（P〈0.05），但其野生株pbps-mRNAs水平明显降低（P〈0.05）、csRNAs水平明显升高（P〈0.05）。E-test结果显示，PCN和CTX对ΔciaR突变株MIC均较野生株增加250倍。结论肺炎链球菌CiaR可通过下调PBP1a、PBP1b和PBP2b表达或上调csRNAs表达抑制PBPs表达，使该菌对PCN和CTX耐药性增强。ObjectiveTo construct a ciaR gene-knockout （ΔciaR） mutant of Streptococcus pneumoniae （S.pneumoniae） and to investigate the effects of CiaR in CiaH/CiaR, a streptococcal two-component signal-transducing system, on the expression of genes encoding penicillin-binding proteins （pbps genes） and cia-dependent small RNAs （csRNAs）.MethodsElectrophoretic mobility shift assay （ESMA） was performed to detect the recombinant CiaR （rCiaR）-binding pbps genes. A suicide plasmid pEVP3ciaR for ciaR gene knockout was constructed and then a ΔciaR mutant was obtained through homologous recombination and insertion inactivation of the suicide plasmid, and screening with chloromycin. The mutant was identified using PCR and sequencing analysis. E-test was used to detect the minimal inhibitory concentrations （MIC） of penicillin （PCN） and cefotaxime （CTX） against S. pneumoniae strains. Changes and differences in the expression of pbps genes and csRNAs in the ΔciaR mutant and its wild-type strain before and after treatment with 1/4 MIC of PCN or CTX were detected using real-time quantitative RT-PCR.ResultsThe rCiaR could bind to the promoter regions in pbp1a, pbp1b and pbp2b genes of S. pneumoniae. The ciaR gene in ΔciaR mutant was inactivated by insertion according to the results of PCR and sequencing analysis. After treatment with 1/4 MIC of PCN or CTX, the expression of pbps genes at mRNA level （pbps-mRNAs） in the ΔciaR mutant was significantly increased （P〈0.05）, but the levels of csRNAs were significantly decreased （P〈0.05）; whereas a significantly decreased pbps-mRNAs （P〈0.05） and increased csRNAs （P〈0.05） were observed in its wild-type strain. The result of E-test showed that the MICs of PCN and CTX against ΔciaR mutant were increased by 250-fold as compared with those against its wild-type strain.ConclusionThe CiaR can enhance the drug resistance of S. pneumoniae to PCN and CTX through down-regulating the expression of PBP1a, PBP1b and PBP2b and up-regulating the

关 键 词：肺炎链球菌 耐药 反应调节蛋~/CiaR蛋白 基因敲除 青霉素结合蛋白/cia-依 赖小RNA 

分 类 号：R440[医药卫生—诊断学]