SOS应答协同核糖开关aac/aad调控耐药基因表达机制研究

Our previous work demonstrated that riboswitch aac/aad regulates the expression of aminoglycoside antibiotic resistance genes, while the conservative sequences of SOS response exists in the promoter region of riboswitch aac/aad. Ciprofloxacin,one of quinolone antibiotics, induces SOS response. So two kinds of antibiotics could induce overexpression of the reporter gene lacZ. Here we hypothesize that the SOS response and riboswitch aac/aad regulate the expression of antibiotic resistant genes synergistically. In the current study, we will investigate the interaction between LexA protein and SOS box by EMSA. Then real-time PCR and miller assay will be employed to detect LacZ activity in the strains such as lexA mutant strains, the key site modification of SOS box, recA and riboswitch aac/aad.Therefore, SOS response at the transcriptional level, the riboswitch aac/aad at the translation level, both synergistically control antibiotic resistance. Furthermore, in vivo SHAPE will be used to detect the structural change model of interactions between riboswitch aac/aad and aminoglycoside antibiotics. By revealing the mechanism of bacterial multiple drug resistance, it will provide a novel idea for the development of new targets for antibiotics, and also lay a theoretical basis for the rational use of aminoglycoside antibiotics and other antibiotics as well。

课题组前期研究发现核糖开关aac/aad调控氨基糖苷类抗生素耐药基因表达；而核糖开关aac/aad启动子区域存在SOS应答保守序列，喹诺酮类抗生素环丙沙星诱导SOS应答，两类抗生素协同诱导报告基因lacZ高表达，因此提出假说：SOS应答协同核糖开关aac/aad调控耐药基因表达。本课题拟进行EMSA实验验证LexA蛋白与SOS box的相互作用；构建lexA突变菌株，报告载体SOS box，recA及核糖开关aac/aad关键位点修饰，real-time PCR与LacZ酶活性实验检测SOS应答在转录水平，核糖开关aac/aad在翻译水平介导细菌耐药；通过in vivo SHAPE检测细菌体内核糖开关aac/aad与氨基糖苷类抗生素的相互作用结构变化模型。通过揭示细菌多重耐药调控机制，为抗生素新靶点的研发提供新的思路，也为合理利用氨基糖苷类抗生素与其他抗生素的联合用药提供理论依据。

本项目在前期研究发现核糖开关aac/aad调控氨基糖苷类抗生素耐药基因表达；而核糖开关aac/aad启动子区域存在SOS应答保守序列，喹诺酮类抗生素环丙沙星诱导SOS应答，两类抗生素协同诱导报告基因lacZ高表达。因此进一步通过EMSA实验验证LexA蛋白与SOS box的相互作用；构建lexA突变菌株，报告载体SOS box，recA及核糖开关aac/aad关键位点修饰，real-time PCR与LacZ酶活性实验检测SOS应答在转录水平，核糖开关aac/aad在翻译水平介导细菌耐药；通过in vivo SHAPE检测细菌体内核糖开关aac/aad与氨基糖苷类抗生素的相互作用结构变化模型。同时喹诺酮类抗生素环丙沙星和氨基糖苷类抗生素 Kanamycin B共同处理菌株 BW26444/ΔrecA/pGEX-recA-SOS box-riboswitch aac/aad-lacZ，检测lacZ酶活性及mRNA转录水平，以及核糖开关aac/aad转录水平，明确了SOS应答在转录水平，核糖开关 aac/aad在翻译水平协同调控耐药基因表达，完成了SOS应答机制验证。通过揭示细菌多重耐药调控机制，为抗生素新靶点的研发提供新的思路，也为合理利用氨基糖苷类抗生素与其他抗生素的联合用药提供理论依据。