环丙沙星作用下大肠埃希菌sRNA对接合反应的调控作用

With the widespread use of antibiotics, multi-drug resistance strains are more frequently encountered in clinical practice, which bring great hardship to the anti-infective treatment. The resistance mechanisms of bacteria are very complex. There are two major mechanisms, firstly, the point mutation, insertion or deletion on chromosome cause the change of amino acid sequence, such as the missing of outer membrane porin OprD2, the modification of penicillin binding protein, resulting in the reduction or loss of antimicrobial susceptibility; secondly, the horizontal gene transfer (HGT) of antibiotic resistance determinants is the other of the mechanisms. Conjugation is the most important mechanisms of HGT in the environment, which is a genetic transfer that involves cell-to-cell between donor and recipient. In our preliminary study, we found that ciprofloxacin could promote the conjugation process between Escherichia coli and Pseudomonas aeruginosa at sub-minimum inhibitory concentration (sub-MIC) of antibiotics. In addition, quantifying donor cells' transcriptome by RNA sequencing had revealed that conjugation related genes had undergone differential expression under the treatment of ciprofloxacin. Compared to control group, the expression of 624 small non-coding RNA (sRNA) at sub-MIC ciprofloxacin treatment group up-regulated or down-regulated. Meanwhile, the bioinformatics data mining showed that several sRNA may target at these conjugation related genes. In this project, to illuminate the mechanisms of transcriptional regulation of conjugation related gene by sRNA in Escherichia coli under the treatment of ciprofloxacin, we will verify the differential expression of the candidate sRNA of Escherichia coli under the treatment of ciprofloxacin. Then, illuminate the function of the candidate sRNA in conjugation reaction by overexpression or knockout the candidate sRNA. We will study the interaction between the candidate sRNA and target genes or proteins also. At last, we will explore the mechanism of expression regulation of ciprofloxacin on sRNA.

耐药基因通过接合反应发生水平转移是细菌获得耐药性的重要方式，但其调控机制尚不清楚。前期实验发现亚抑菌浓度环丙沙星促进大肠埃希菌-铜绿假单胞菌接合反应。通过转录组测序分析供体菌E.coli的基因表达变化，发现接合相关基因和非编码小RNA(sRNA)表达发生显著变化，生物信息学分析发现多个针对接合相关基因的候选sRNA。鉴于sRNA具有转录调控作用，我们推测在抗生素胁迫下，供体菌sRNA表达发生变化，并通过调节接合相关基因的表达而促进接合反应，是耐药基因水平转移的重要机制。本课题拟通过①验证环丙沙星作用下E.coli的sRNA表达变化；②过表达和基因敲除候选sRNA，明确其对接合反应的调控功能；③研究sRNA与靶基因或蛋白的作用机制；④探讨抗生素对sRNA的表达调控机制，从而阐明抗生素作用下sRNA的表达变化和对接合反应的调控作用，为阻断耐药基因水平转移和指导临床合理应用抗生素提供实验室依据。

细菌耐药对全球公共卫生健康造成巨大威胁。深入研究细菌耐药机制，阻断耐药基因播散具有重大意义。接合（conjugation）是细菌以性菌毛为桥梁进行基因水平转移的过程，是质粒介导耐药基因传播的主要机制。尽管抗生素的广泛使用被认为是导致细菌耐药性迅速播散主要原因，但有关抗生素对接合反应影响的报道并不一致，对其背后的机制更是知之甚少。本课题以细菌适应环境变化的sRNA为切入点，以基因组整合了接合性质粒RP4的大肠埃希菌（E. coli）菌株SM10λπ为供体菌、铜绿假单胞菌（P. aeruginosa）标准菌株PAO1或大肠埃希菌菌株EC600为受体菌建立接合模型，采用RNA-seq、Real-time PCR、基因敲除与过表达、报告系统等一系列细胞和分子生物学手段，深入探讨了抗生素（环丙沙星，CIP）-sRNA-SdiA信号通路对耐药基因接合转移的调控作用及其具体机制。研究结果发现：1）亚MIC浓度CIP能促进E. coli-P. aeruginosa、E. coli-E. coli接合反应、加快耐药质粒水平播散；2）通过转录组测序结合生物信息学分析筛选出CIP作用下表达上调且可能参与接合调控的sRNA GadY；3）过表达GadY具有促进接合的功能，其具体机制为通过靶向SdiA进而抑制结合关键基因TraI表达；4）与部分sRNA和靶基因相互调控的现象一致，GadY的表达受到SdiA的调控。我们的研究结果丰富了抗生素诱导耐药基因接合转移的分子机制，拓展了sRNA调控网络及其生物学功能，为抗菌治疗提供了新的策略，具有较高的科学意义和潜在的临床价值。