营养胁迫下铜绿假单胞菌耐药性相关转录调控和信号通路的研究

The extensive use and discharge of antibiotics facilitate the ecological environment where antibiotics and native environmental factors coexist in some areas. Nutritional stress is one of the most common stress factors faced by bacteria in natural environment. In previous studies, we found that nutritional conditions had effect on resistance to a variety of antibiotics in drug resistant bacteria Pseudomonas aeruginosa, but the molecular regulatory mechanism is unknown. Based on this, this project aims to elucidate the variation characteristics of resistance of P. aeruginosa to various antibiotics under nutritional stress, and to reveal the correlation between response to nutritional stress and drug resistance by comparative transcriptome method. By analyzing gene expression profiles under different nutritional conditions and treatment of ceftazidime or tobramycin, the coupling regulatory pathways of nutritional stress and drug resistance are explored. The regulatory role of the pathways on key genes related to drug resistance will be further clarified at the level of molecular biology in order to demonstrate the molecular mechanism of how the response mechanism to nutritional stress changes the resistance to ceftazidime and tobramycin in P. aeruginosa. This study can provide a scientific basis for the adaptation mechanism of the adverse environment and the drug resistance regulatory network of P. aeruginosa.

抗生素的大量使用和排放促成了部分地域抗生素与原生环境因素并存的生态环境。营养胁迫是细菌最常面临的环境胁迫因素之一，前期实验发现营养条件能够改变耐药细菌铜绿假单胞菌对多种抗生素的耐药性，但其中的分子调控机制未知。基于此，本项目旨在阐明营养胁迫条件下铜绿假单胞菌对各类抗生素耐受能力的变化特征，采用比较转录组的方法揭示营养胁迫响应与耐药性间的关联。通过分析在不同营养条件和头孢他啶或妥布霉素处理下的基因表达谱，挖掘营养胁迫与耐药性的偶联调控通路，在分子生物学层面明确调控通路对耐药性关键基因的调控作用，阐述营养胁迫应答机制改变铜绿假单胞菌对头孢他啶和妥布霉素耐受能力的分子机理。该研究可为铜绿假单胞菌的逆境环境适应机制与耐药性调控网络提供相关科学依据。

铜绿假单胞菌是一种重要的条件致病菌，而且容易对抗生素产生耐药性。我们前期发现该菌株在不同营养条件下对抗生素的耐受性具有差异，本项目通过生理表型观察和转录组分析的手段研究其中的调控机制。我们发现铜绿假单胞菌在无营养物的生理盐水中对头孢他啶、氯霉素和四环素的耐受性比在富营养的LB培养基中弱，但是在生理盐水中对氟喹诺酮类和氨基糖苷类的耐受性明显更强。转录组分析显示该菌株通过提高四环素靶向的核糖体蛋白和多药外排泵的表达来提高对四环素的耐受性，富营养条件更有利于激活这种抗性机制。相比之下，环丙沙星处理主要诱导DNA损伤修复和绿脓杆菌素合成有关的基因，同时还发现降低ATP水平可提高菌株对环丙沙星的耐受性，推测低营养水平引起的低代谢速率是铜绿假单胞菌在生理盐水中对环丙沙星的耐受性更高的主要原因。另外，我们以恶臭假单胞菌为研究对象还发现：（1）四环素通过诱导胞外粘附蛋白LapA的表达来显著增强恶臭假单胞菌的生物被膜形成能力，环丙沙星同样对生物被膜具有强烈的诱导作用，生物被膜的形成显著降低了菌株对抗生素的敏感性。（2）重金属外排系统Czc和Cad存在直接的联系，调控因子CadR在Cd2+存在下调控czcRS3的表达，进一步激活两个CzcCBA外排泵的表达，赋予菌株对镉的抗性。