多粘菌素耐药鲍曼不动杆菌中TnpISAba11核糖开关的分子机制研究

Antibiotics-resistance Acinetobacter baumannii is pandemic along with the abuse of antibiotics and the treatment of its infection has become a great challenge in the filed of anti-infection. Polymyxin has been considered as the last-line antibiotic the treatment of infections caused by A. baumannii,while recent study shows that there is an upward trend in polymyxin-resistance A. baumannii. So it of great urgency to study on the mechanism of polymyxin resistance in A. baumannii. In previous study, We discovered that polymyxin B can bind to leader RNA of ISAba11 which coding TnpISAba11 transposase in polymyxin resistance A. baumannii, and regulate the expression of transposase in translation level, result in the resistance of polymyxin. additionally the leader RNA contains variable secondary structure predicted by mfold and riboswitch prediction software of our lab. Therefore,We propose that the leader RNA in TnpISAba11 transposase is an brand new riboswitch-- TnpISAba11 riboswitch. In this study we will systemically investigate the mechanism of TnpISAba11 riboswitch. Firstly,we will prove the biological activity of TnpISAba11 riboswitch by LacZ reporter system and GFP- TnpISAba11transposase fusion system in JM109; secondly, using SPR,SHAPE,In-Line Probing,DMS and mutational analysis，we will explose the binding site between polymyxin and TnpISAba11 riboswitch, anlyse the TnpISAba11 riboswitch structural changes in the condition of with or without binding of polymyxin. Our study will not only identify a new riboswitch associated with polymyxin resistance in A. baumannii, but also provide a new polymyxin resistance mechanism and provide an newway in polymyxin improvement.

耐药鲍曼不动杆菌已成为全球抗感染领域的挑战。多粘菌素曾被看做鲍曼不动杆菌治疗的最后一道防线。但文献表明鲍曼不动杆菌对多粘菌素耐药也日趋严重，因此多粘菌素耐药机制的研究工作迫在眉睫。申请者前期发现编码TnpISAba11转座酶前导序列RNA结合多粘菌素B进而介导多粘菌素耐药。多粘菌素B结合转座酶前导RNA，且在翻译水平调控转座酶的表达，mfold和本实验室核糖开关预测软件分析发现该前导RNA存在可变二级结构，表明该前导序列从功能上发挥核糖开关的作用。本研究拟进一步在体内利用LacZ酶报告系统等证明TnpISAba11核糖开关的生物学活性；在体外利用表面等离子共振（SPR）、SHAPE、In-Line Probing、DMS等技术证明多粘菌素与核糖开关的结合位点及结合前后RNA二级结构变化。总之，本研究发现一种新的核糖开关，将阐明鲍曼不动杆菌多粘菌素耐药机制，为多粘菌素的结构改造提供理论基础。

耐药鲍曼不动杆菌已成为全球抗感染领域的挑战。本研究通过对成都医学院附属医院的110株临床分离鲍曼不动杆菌的流行病学调查结果显示60.9%的鲍曼不动杆菌对碳青霉烯抗生素耐药，76.23%的菌株喹诺酮耐药。临床鲍曼不动杆菌的耐药形势非常严重。多粘菌素曾被看做鲍曼不动杆菌治疗的最后一道防线。本研究中110株临床菌株中虽未发现多粘菌素耐药菌株，但多粘菌素耐药机制的研究工作仍不可忽视。多粘菌素B结合转座酶前导RNA，且在翻译水平调控转座酶的表达，mfold和核糖开关预测软件分析发现该前导RNA存在可变二级结构，表明该前导序列从功能上发挥核糖开关的作用，体内实验利用LacZ酶报告系统等证明TnpISAba11核糖开关的并不存在生物学活性；在体外利用SHAPE、DMS等技术证明多粘菌素与核糖开关的结合位点及结合前后RNA二级结构无变化。上述结果表明TnpISAba11可能不是一个核糖开关。在此实验过程中还发现了2株感染同一株多重耐药鲍曼不动杆菌的噬菌体，为研究噬菌体与宿主多重耐药鲍曼不动杆菌的之间的相互作用提供新的实验材料。