迟缓爱德华氏菌抗生素耐受期基因突变机制的研究

Bacterial resistance is a serious threat to human health and food safety. It is of great significance to deepen the study of bacterial resistance mechanism. Recently，it has been found that，to become antibiotic resistance under the selective condition of antibiotics, bacteria should experience a process which is called antibiotic tolerance. However，the reason how the antibiotic- tolerant bacterial became antibiotic-resistant bacterial is unknown. During the research about antibiotic resistant bacterial, we found that：1) glucose is a key biomarker in the evolution of tolerance to resistance, 2) the level of reactive oxygen species (ROS) which leads to gene mutation had significant changed during the process of evolution；3) loss of CRP，the tolerance period was 1.5 times longer than that of wild type. It is known that glucose inhibits the global regulator cAMP/CRP complex which plays an important role in metabolic regulation. Therefore, a scientific hypothesis that glucose-mediated cAMP/CRP complex regulates gene mutation in evolution is proposed. Based on the results of the changes of cAMP and CRP, and their relationship with glucose level, clarify the effects of deleted CRP in the evolution of tolerance to resistance. Based on the results of the effects of the promoters and inhibitors of ROS to evolution, reveal how cAMP/CRP complex regulates the ROS level. Based on the relationship between gene mutations and gene repair systems, explore how the cAMP/CRP complex regulates the gene repair systems. The aim of these studies is to reveal the mechanism of gene mutation in the process of resistance evolution, and will control the antibiotic-resistant bacterial during the evolution of tolerance to resistance.

细菌耐药严重危害人类健康和食品安全, 深化耐药机制研究具有重要意义。近年发现细菌耐药需先经历耐受过程，期间发生基因突变然后出现耐药表型，但机制未知。本项目前期研究发现，在耐受进化为耐药过程中，葡萄糖为关键生物标志物，导致基因突变的活性氧分子(ROS)水平发生明显变化，缺失crp使耐受期延长1.5倍。已知葡萄糖抑制具有代谢调节作用的全局调节子cAMP/CRP复合物，因此提出葡萄糖介导的cAMP/CRP复合物调控这一进化中基因突变的科学假设。拟检测传代菌cAMP和CRP变化及其与葡萄糖水平关系的基础上，明确缺失crp对转化的影响；通过ROS的促进剂和抑制剂确认其作用的基础上，揭示cAMP/CRP复合物对ROS的调节；基于基因突变与基因修复的关系，探讨cAMP/CRP复合物对基因修复系统的调节；旨在通过这些深入系统的研究，揭示耐药转变过程中基因突变的发生机制，为从源头上防治细菌耐药提供基础。

细菌耐药在世界范围内变得日益严重，危害人类健康和食品安全, 深化耐药机制研究意义重大。近年发现细菌耐药需先经历耐受过程，期间发生基因突变然后出现耐药表型，但机制未知。文献报道，代谢状态影响细菌对抗生素的敏感性，细菌抗生素耐药性与代谢转变有关。然而，代谢状态如何决定细菌的抗生素耐受性和获得性耐药尚未报道。本项目拟开展迟缓爱德华氏菌抗生素耐受期基因突变机制的研究。在前期研究中基于代谢组发现葡萄糖为耐受转变耐药关键生物标志物且其浓度与导致基因突变的活性氧分子(ROS)相关。已知葡萄糖抑制有代谢调节作用的全局调节子cAMP/CRP复合物。基于此发现系统开展了细菌耐受向耐药转变过程中与葡萄糖相关的代谢物调节机制研究，探讨代谢物葡萄糖对细菌耐受向耐药转变的影响，揭示耐药转换过程中包括氨苄青霉素、磷酸转移系统(PTS)、丙酮酸脱氢酶(PDH)、葡萄糖、cAMP/CRP复合物以及活性氧分子(ROS)的级联调节机制，以及证明这一级联调节机制在细菌从耐受到耐药的转变中的普遍性。研究结果发现：①外源氨苄青霉素靶向PTS的启动子P1b和PDH的E1亚基可以分别激活pts和抑制PDH活性，来调节葡萄糖的胞内浓度引发耐受向耐药的转变。②葡萄糖代谢流向改变引起胞内ROS水平的变化，外源ROS促进剂以及抑制剂可以促进或延迟细菌耐受向耐药转变过程，显示代谢调节对抗细菌耐药的潜在可能。③胞内细菌迟缓爱德华氏菌与胞外菌大肠杆菌在抗生素耐受到耐药的转变过程中具有相同的代谢调节机制，说明这一机制在细菌中可能普遍存在。.从通过本研究，我们建立了迟缓爱德华氏菌与大肠杆菌氨苄青霉素耐受向耐药转变代谢图谱，以差异代谢物葡萄糖为线索，深入了解了迟缓爱德华氏菌和大肠杆菌普遍存在的抗生素耐受向耐药转变的代谢调节机制，有助于我们开发有效的工具来对抗细菌抗生素的耐受和耐药性。