核糖开关调控抗生素抗药性基因的研究

Antibiotic resistance has increasingly become one of the biggest problems in the world. Riboswitches are regulatory RNAs that bind small molecule metabolites and cofactors; they exploit specific interactions between low molecular weight metabolites and non-coding regions of messenger RNAs to regulate the biosynthetic pathway of the metabolite. They utilize a simple feedback mechanism whereby the interplay between two distinct structures in the mRNA controls the level of gene expression, in response to cellular conditions. As corresponding author, the applicant has published a paper titled “Riboswitch Control of Aminoglycoside Antibiotic Resistance” in CELL January, 2013. The paper reported the discovery of a new aminoglycoside binding riboswitch that is widely distributed amongst antibiotic resistant bacterial pathogens. This riboswitch is present in the leader RNA of the resistance genes that encode the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes that confer resistance to aminoglycoside antibiotics through modification of the drugs. We show that expression of the AAC and AAD resistance genes is regulated by aminoglycoside binding to a secondary structure in their 5’ leader RNA. Reporter gene expression, direct measurements of drug RNA binding, chemical probing and UV cross-linking combined with mutational analysis demonstrate that the leader RNA functions as an aminoglycoside sensing riboswitch in which drug binding to the leader RNA leads to the induction of aminoglycosides antibiotic resistance. Based on this work, we will continue to search for new riboswitches that control other class of antibiotic resistance. This study will shed light on the understanding and mechanism of the antibiotic resistance and pave foundation for solving the problem of antibiotic resistance.

抗生素耐药性日益巨增，已发展成世界范围内的一大公共健康威胁，因此，抗生素耐药性产生机制的研究至关重要。核糖开关是存在于mRNA非编码区域的RNA序列，通过与小分子配体结合改变自身结构从而调控基因表达。申请人以通讯作者身份于2013年1月在CELL杂志上（复旦为第一单位）首次报道了氨基糖苷类抗生素耐药性核糖开关，该核糖开关通过结合氨基糖苷类抗生素引起结构改变从而调控下游抗药性基因的表达。本项目将利用已有的实验体系重点研究除氨基糖苷类以外的其他几大类抗生素的耐药性产生机制，利用体内报告基因系统，体外RNA和抗生素相互作用检测技术以及RNA化学修饰和突变等生化方法寻找调控这几大类抗生素耐药性基因的新型核糖开关。本项目是已发表工作的延伸和拓展，将从一个全新角度深入阐明耐药性产生的分子机理，开创耐药性机理研究的新领域，引领和占据世界耐药性研究的学科前沿，为人类最终认识并战胜致病菌耐药性提供理论基础。

抗生素在临床，商业养殖和兽医中的广泛使用使它们充满了环境。这种环境污染导致对共生和致病细菌的选择压力，导致抗生素抗性基因的进化和增殖。抗生素耐药性是对当前医疗实践的威胁。. 1类整合子通过aatI 1位点的位点特异性重组积累抗生素抗性基因。捕获的基因从位于整合子内的启动子转录，对于1类整合子，第一个转录和翻译的基因正常编码氨基糖苷类抗生素抗性蛋白（乙酰 - （AAC）或腺苷 - （AAD）转移酶）。来自荧光假单胞菌1类整合子的前导RNA含有氨基糖苷类感应核糖开关RNA，控制下游氨基糖苷类抗性基因的表达。在这里，我们探讨了整合子依赖性DNA重组与潜在的氨基糖苷类感应核糖开关产物之间的关系，这些产物来自一系列氨基糖苷类抗性临床菌株。临床菌株的序列分析鉴定了一系列与I类整合子衍生的氨基糖苷类抗性（aac和aad）重组体相关的序列变体。对于aac／aad重组体，代表性序列显示高达6倍氨基糖苷类依赖性的报告基因表达调节。 MicroScale Thermophoresis（MST）证实了RNA结合。协方差分析产生RNA的二级结构模型，其是对来自诱变和化学探测数据的先前模型的独立验证，并且类似于荧光假单胞菌核糖开关RNA。氨基糖甙类药物是临床上首批使用的抗生素之一，数据表明，在富含氨基糖苷类的环境中，在整合子介导的重组过程中选择功能性核糖开关重组体来调节氨基糖苷类抗性。通过整合子重组掺入功能性氨基糖苷类感应核糖开关赋予了不同来源的抗性基因表达的选择性优势。