亚MIC抗生素作用下铜绿假单胞菌阻遏蛋白LexA调控接合相关基因的研究

With the application of antibiotics, clinical isolate rate of drug-resistant Pseudomonas aeruginosa (PA) was increasing. In addition to gene mutation, horizontal gene transfer (HGT) has played a major role in enhanced bacterial antibiotic-resistance. Bacterial conjugation is a genetic transfer that involves cell-to-cell between donor and recipient, which was considered a main path of HGT. To research the mechanism how HSL signaling molecules regulating conjugation reaction of PA, our team have done many test in a project founded by National Natural Science Foundation of China (NSFC) ( NO.81071397). We found that HSL could interfere with the conjugation process throuth regulating the activity of conjugation-related genes. By the way, we also found that antibiotics have effect on PA conjugation. This phenomenon was associated with the activation of SOS response. To explore the molecular mechanisms behind this phenomenon, firstly, we intend to research the optimal concentration of sub-MIC antibiotics promoting the conjugation response. Secondly, we'll analyze the gene expression profile of PA under the treatment of sub-MIC antibiotics by cDNA microarray, and analyze the LexA repressor regulating gene of the PAO1 by using bioinformatics methods and discover the repressor LexA binding genes by CHIP-on-chip experiment also. Finally, We'll screen positive genes from these three databases and analyze gene functions in the conjugation reaction by gene mutation technology. So, this project is aim to fully clarify the regulatory mechanism between bacterial conjugation reaction and the SOS response under the treatment of sub-MIC antibiotics.

随着抗生素的应用，临床检出的耐药细菌日益增多。细菌耐药性增强的原因除自身基因的突变外，更主要的是由耐药基因水平转移引起。目前发现接合（conjugation）是耐药基因水平转移的主要途径。为此，课题组前期在NSFC资助下进行了HSL信号分子对铜绿假单胞菌(PA)接合反应的调节机制研究，结果发现HSL能通过调节多个接合相关基因的活性而干预接合过程。实验还发现抗生素也具有调节PA接合反应的作用，该作用与激活SOS反应相关。为探究这一现象的分子机制，本课题拟首先摸索亚MIC浓度抗生素促进接合反应的最适浓度，再通过生物信息学分析PAO1基因组上LexA识别的基因谱，cDNA芯片和CHIP-on-chip试验研究亚MIC抗生素作用下LexA蛋白阻遏调控的接合反应相关基因，筛选阳性基因，最后通过基因突变技术分析这些基因在接合反应中的作用，从而全面阐明抗生素作用下细菌接合反应与SOS反应之间的作用机制。

抗生素应用以来，临床检测出的耐药性细菌逐渐增多。细菌耐药性增强的原因除了自身基因的突变外，更重要的是耐药基因的水平转移。目前发现接合（conjugation）是耐药基因水平转移的主要途径。课题组在前一国家自然科学基金(NO.81071397)资助课题中发现HSL信号分子能通过调节多个接合相关基因的表达而影响接合过程，同时发现抗生素也具有调节E.coli-PA接合反应的作用。为阐明抗生素对E.coli-PA接合反应的影响机制，本课题首先根据CLSI临床指南先后尝试多种抗生素，发现环丙沙星(cip)和左旋氧氟沙星(lev)对E.coli-PA接合反应频率有较大影响，在明确接合反应中供体菌的作用较受体菌更大后，先优化cip、lev处理最适浓度与时间，并通过qPCR检测到接合相关基因traI、traG表达亦有增强；此外，通过生物信息学分析供体菌E.coli基因组上LexA识别位点涉及的基因谱；以二代测序技术(next generation sequencing, NGS) 获得亚MIC抗生素作用下供体菌sm10λpir的转录组差异基因谱，从中筛选接合反应相关基因并进行GO聚类、KEGG pathway分析等，从而阐明亚MIC抗生素对细菌接合反应的影响。