cbs/cse核糖开关调控金黄色葡萄球菌H2S生成的耐药机制研究

诱导性耐药是细菌产生耐药性的主要原因之一。氨基糖苷类抗生素诱导金黄色葡萄球菌H2S合酶(CBS/CSE)表达，导致大量H2S生成，从而使细菌产生广谱性耐药，但抗生素诱导H2S 生成的调控机制未知。课题组前期工作表明，H2S生成的诱导表达与cbs/cse基因5'端前导序列RNA结构变化相关，证实此RNA为核糖开关。本课题拟构建报告基因表达系统，通过平板扩散实验、lacZ 酶活性、实时荧光定量PCR，检测cbs/cse核糖开关在细菌体内对H2S生成的调控作用；通过非变性凝胶电泳、表面等离子共振SPR、紫外交联、SHAPE、in-line probing、DMS等技术及突变分析明确氨基糖苷类抗生素的结合位点，建立cbs/cse核糖开关结构变化模型。通过BLAST,clustal W等生物信息学方法，研究核糖开关模型调控耐药H2S生成的普遍性，为抗生素新靶点的研究提供新的思路。

Antibiotic resistance of bacterium commonly occurs through induction by the antibiotics. The aminoglycoside antibiotics can induce high level of expressions,such as cystathionine-β-synthase/cystathionine-γ-1yase(cbs/cse) in Staphylococcus aureus, which results in antibiotic resistance for the production of hydrogen sulfide(H2S).However,the induction mechanism remains unclear.Our previous work showed that induction of H2S expression is related to the RNA structure of 5'-UTR. We therefore confirmed that this RNA could be a novel riboswitch. This study intends to construct the reporter gene expression system in vivo by disc-diffusion method,lacZ activity and real-time PCR, and verify the riboswitch biochemically by surface plasmon resonance(SPR), cross-link, selective 2-hydroxyl acylation analyzed by primer extension(SHAPE), in-line probing，DMS and mutational analysis. We will show that aminoglycosides bind to cbs/cse riboswitch and cause a significant structural transition in the conformation of the RNA and thus induce the production of H2S. We will propose a model of riboswitch which regulates the expression of the H2S resistance genes. Understanding the mechanism of inducible H2S antibiotic resistance would provide newways for developing new antibiotic targets.