细菌功能性第2类整合子自身结构及SOS反应系统对其捕获耐药性基因盒调控机制的研究

The capture of exogenous antimicrobial resistance genes through lateral gene transfer is an important method for the emergence and spread of antimicrobial resistant strains. By capturing antimicrobial resistance gene cassettes through site-specific recombination and ensuring the expression of the genes within gene cassettes, integrons act as "Antimicrobial Agent Decoder" in emergence and dissemination of antimicrobial resistance in bacteria. However, the mechanism for integron excising and integrating gene cassettes remained to be determined. We previously showed that in class 1 integron, the structure of integron plays important roles in regulating integrase catalyzed gene cassettes integration. Functional class 2 integron has a similar structure to class 1 integron and also has its own characteristics, moreover, class 2 integron integrase shows only 40% homology with that of class 1 integron integrase, indicated functional class 2 integron integase has a different mode in catalyzing gene cassettes excision and integration when compared with that of class 1 integron integrase. For the above reasons, our study will focus on the structure of functional class 2 integron, to determine the relationships between the different common promoters of variable regions and the expression of class 2 integron integrase, as well as integrase catalyzed site-specific recombination. Whether the SOS response system can regulate the expression of class 2 integron integrase will also be clarified. By constructing random mutation library of class 2 integron integrase and screened high throughputly with "papillation assay", the key animo acids or domains in class 2 integron integase in catalyzing site-specific recombination will be determined. The results of this research will provide the theory basis for control the emergence and dissemination of antimicrobial resistance among clinical isolates.

通过基因的水平转移，获得外源性耐药基因是细菌耐药性产生与播散的重要原因，整合子可通过位点特异性重组捕获与表达耐药性基因盒，在细菌耐药性产生和播散中起到“抗菌药物解码器”的作用，但其剪切与整合基因盒的机制仍未明了。我们前期研究表明第1类整合子自身结构对其捕获耐药性基因盒起着重要的调控作用，功能性第2类整合子与第1类整合子结构类似但又有其自身的特点，同时第2类整合酶氨基酸序列与第1类整合酶同源性较低(40%)，提示其具有不同于第1类整合酶剪切与整合耐药性基因盒的方式。我们拟从功能性第2类整合子自身结构出发，明确不同可变区启动子与第2类整合酶表达及其催化的位点特异性重组之间的关系，确定细菌SOS反应系统在第2类整合酶表达中的调控作用，通过建立第2类整合酶随机突变文库，结合“乳头状试验”高通量筛查第2类整合酶催化位点特异性重组的关键氨基酸或结构域，研究结果为控制细菌耐药性的产生和播散提供理论依据。

已按计划基本完成了本项目的所有研究内容。发现第2类整合子不同可变区启动子下游基因盒表达水平差别无统计学意义，与近期法国巴斯德研究所发表的相关研究论文结论一致。首次通过实验验证5种天然存在的功能性第2类整合酶蛋白均能介导基因盒的剪切和整合，其多态性位点限于172, 174, 175, 176 和256位氨基酸残基，该多态性对于第2类整合酶蛋白的活性影响较小。发现T23、Y26、 F34、 L72、L88、V111、L129、L134、R135、I154、F272、T274、R283、G290、T296、 Y301位点氨基酸的改变可使第2类整合酶的活性显著降低，结合生物信息学分析，301 位酪氨酸可能为第2类整合酶催化活性氨基酸。发现临床分离的38株变形杆菌中的功能性第2类整合子均插入细菌基因组DNA中基因fbpc与glmS之间，其周围的转座酶基因tnsE, tnsD, tnsC, tnsB, tnsA与大肠埃希菌第2类整合子周围转座酶基因相比，同源性分别为91% (1362/1492)、 91% (1384/1528) 、93% (1552/1668)、95% (2014/2111)和97% (801/822)。在变形杆菌临床分离株中首次检出基因盒psp及基因盒组合estX-psp-aadA2-cmlA1-aadA1a -qacI-tnpA-sul3、estX-psp-aadA2-cmlA。变形杆菌临床分离株中第1类整合子共检出3种可变区启动子PcS、PcWTGN-10和PcH1。携带较强的PcS或PcWTGN-10启动子的菌株与携带较弱的PcH1启动子的菌株相比，对复方新诺明、庆大霉素和妥布霉素的耐药率较高，而携带较弱的PcH1启动子的菌株较携带较强的PcS或PcWTGN-10启动子的菌株携带较多的抗菌药物耐药基因。在血流感染大肠埃希菌中首次检出blaADC-162 和blaCMY-2。临床分离的耐碳青霉烯类肺炎克雷伯菌首次发现携带blaKPC-2的ST290型流行株，携带blaNDM-5 的ST340型菌株，以及同时携带blaKPC-2和blaNDM-5菌株。上述研究结果为第2类整合酶催化基因盒剪切与整合反应机制的阐明奠定基础，为控制细菌耐药性的产生和播散提供理论依据。本项目截止目前发表SCI收录论文2篇，修回1篇，审稿中1篇，另有2篇撰写整理中。达到了预期目标。