转运蛋白PM1526介导禽多杀性巴氏杆菌多重耐药的分子机制研究

Fowl Pasteurella Pasteurella is a strong infectious disease causing acute death of many birds. The increasing drug resistance is one of the main factors that cause the acute death of birds. The active external drainage system composed of transporters is one of the important mechanisms for bacteria to promote drug outflow. The early stage of the project team found that PM1526 gene in drug-resistant strains induced by three kinds of drug, respectively, expressed highly. It impled that PM1526 on pasteurella multocida play an important role in multiple drug resistance. The multidrug-resistant strains obtained from this project were compared with those of clinical isolates, and the relationship between the expression of PM1526 and drug resistance and the type of drug resistance was studied. To identify the biological characteristics of PM1526 and its promoter sequence and to explore the key transcriptional regulatory factors in the upstream, the transcriptional regulation analysis will be carried out by constructing PM1526 and upstream regulatory factor gene deletion strains. The expression of PM1526 will be cloned, and its influence on biofilm formation ability and drug type will be analyzed. We will clarify the molecular basis of pm1526-mediated multidrug resistance, and provides a new way for the design of the drug targets for the pasteurella multocida.

禽多杀性巴氏杆菌是引起多种禽急性死亡的一种烈性传染病病原，其耐药性不断增强是该病难以控制的主要因素之一。由转运蛋白组成的主动外排系统是细菌促进药物外流而产生耐药性的重要机制之一。项目组前期通过耐药菌株诱导和RNA-seq发现，编码转运蛋白PM1526基因在三种药物分别诱导形成的耐药菌株中均呈显著的上调表达，提示PM1526在多杀性巴氏杆菌多重耐药中具有重要的作用。本项目拟诱导获得的多重耐药菌株与临床分离的多重耐药株进行比较分析，研究PM1526基因表达情况与耐药程度和耐药种类的关系；鉴定PM1526及其启动子序列的生物学特征，挖掘其上游关键转录调控因子；通过构建PM1526和上游调控因子基因缺失株，开展转录调控分析；克隆表达PM1526，检测分析其影响生物被膜形成能力和外排药物种类。系统阐明PM1526介导多杀性巴氏杆菌多重耐药的分子基础，为多杀性巴氏杆菌药物靶标的设计提供新的思路。

禽多杀性巴氏杆菌（Pasteurella multocida，PM）可引起禽类急性、接触性、败血性传染病。耐药性的不断增强是该病难以控制的主要因素之一。项目组前期通过RNA-seq发现转运蛋白PM1526在人工诱导的耐药菌株中菌显著上调表达，但其介导PM耐药的分子机制尚不清楚。本课题围绕PM耐药的分子机制完成以下研究：（1）阐明了PM1526介导禽多杀性巴氏杆菌多重耐药的分子机制。发现PM1526蛋白为TRAP转运体组件，非ABC转运蛋白，通过C4-羧酸盐的代谢产物影响菌株的耐药性；（2）发现转录调控因子PM_RS07615与菌株对链霉素的耐药性相关，并证明了其通过正调控TolC家族TdeA蛋白的表达，影响菌株对药物的积累和外排而介导对链霉素的耐药性；（3）发现并揭示黄连碱对禽多杀性巴氏杆菌的抑菌效应及机制，利用表型测定与RNA-seq试验相结合，揭示黄连碱主要通过抑制DNA和可溶性物质合成、呼吸系统相关酶类活性，破坏细胞壁等多个方面发挥抑菌效应。本课题为揭示禽多杀性巴氏杆菌耐药机制和临床药物治疗理论支撑。