维氏气单胞菌SmpB及AcrR蛋白介导持留菌耐药的分子机制

Aeromonas veronii is a kind of important pathogens which infect aquatic products, because it can form the persisters with heterogenic bacterial populations exhibiting drug tolerances toward antibiotic stress. However, the comprehensive understanding of its drug tolerances mechanism was ambiguous. The research findings reveal that both Small Protein B (SmpB) and Multidrug Efflux Pump subunit AcrB are crucial for drug tolerances. Our preliminary results demonstrated that SmpB could regulate the repressor AcrR expression, while the latter might regulate AcrB，which is to say SmpB and AcrR and AcrB might construct a drug-tolerance signal pathway. On the basis of our previous research, we propose a novel molecular model for drug resistances. SmpB and AcrR senses the variations of antibiotic concentrations, subsequently upregulates AcrB expression which resulting in antibiotics excretion. In this study, expression levels of three .proteins were evaluated by Real-time PCR and Western Blot and site-directed mutagenesis under antibiotic stress ,thereby confirming signal transmission schedule. The regulation circuit mode between SmpB and AcrR, or the controlling mode of AcrR regulating AcrB were identified using agarose gel retardation assay, circular dichroism spectrum and surface plasma resonance, leading to construct the drug tolerance model mediated by SmpB and AcrR. The coming results will uncover the molecular mechanism of drug tolerances in Aeromonas veronii, seek.for the therapeutic targets for bacterial diseases and provide technique supports for drugs screening and designing. The current research also has practical significances for the improvement of disinfection method and the reduction of recurrent infections in aquaculture process.

维氏气单胞菌是重要的水产致病细菌。它可产生异质化持留菌，以逃避抗生素胁迫并存活，其耐药机理不清楚。研究发现SmpB及外排泵AcrB在持留菌耐药中均起重要作用。我们近期结果提示，SmpB调节外排泵抑制因子AcrR表达，而AcrR可能调控AcrB表达，即SmpB、AcrR、AcrB构成一条信号通路。我们提出新的耐药分子模型，SmpB和AcrR介导抗生素压力，上调AcrB表达，主动外排抗生素以增加耐药性。本项目拟用实时定量PCR、免疫印迹、定点突变等方法，追溯抗生素胁迫下三者表达水平变化，确证信号传递时序性；用凝胶迁移、圆二色谱、表面等离子体共振等技术，解析SmpB、AcrR、AcrB的调节方式和关键位点，用耐药行为动力学验证AcrB外排泵功能。这为阐明维氏气单胞菌耐药机制奠定基础，为寻找细菌性疾病治疗靶点、筛选和设计抗生素增效剂提供技术支持，同样对解决水产养殖过程复发性微生物感染具有实际意义。

维氏气单胞菌是重要的致病细菌。它可产生异质化持留菌，以逃避抗生素胁迫并存活，其耐药机理不清楚。研究发现SmpB及外排泵AcrB在持留菌耐药中均起重要作用。我们研究证实了一种新的耐药分子模型，SmpB调节外排泵抑制因子AcrR表达，而后者AcrR调控AcrB表达，主动外排抗生素以增加耐药性。探索了不同抗生素浓度对SmpB、AcrR及AcrB表达的影响，确证了SmpB、AcrR、AcrB信号传递过程的时序性；用EMSA验证SmpB与acrR启动子的结合，用定点突变、细菌单杂交等找出互作位点；类似的我们证明了下游AcrR和acrB启动子的结合及互作位点。在野生菌及ΔacrB突变株中，验证AcrB外排头孢菌素的功能。在保质保量完成研究内容基础上，我们拓宽项目发展空间，利用机器学习卷积神经网络，初步鉴定转录因子CysB结合tmRNA启动子，在转录水平进行调控。tmRNA 在转录、翻译水平对丙氨酸脱氢酶双重调控，进而影响病原菌由高代谢活性进入持留菌状态。这为阐明维氏气单胞菌耐药机制奠定基础，为寻找细菌性疾病治疗靶点、筛选和设计抗生素增效剂提供技术支持。.相关研究在国内外刊物发表论文27篇，其中SCI源刊物15篇;授权专利8项。在项目执行期间，项目组成员参加国内外学术会议14人次，提交会议摘要7篇，做大会报告1次，分会场报告2 次。培养博士生1人，硕士生16人。项目主持人获奖4次，研究生获奖2次。研究成果超过合同书所拟定的在国际刊物发表 4-6 篇较高学术影响力的 SCI 论文，申请专利 1 项以上，培养研究生 4-8 名的预定目标。