原子力显微镜研究铜绿假单胞菌噬菌体的结合与感染

Bacteriophage or phage, which is among the most common and diverse entities in the biosphere, is a virus that infects and replicates within a bacterium. Pseudomonas aeruginosa, which is an opportunistic pathogen of humans, animals and plants, is very easy to produce antibiotics-resistance, leading to difficultly treat antibiotics-resistant Pseudomonas aeruginosa involved infections. Multi-antibiotics-resistant pathogenic bacteria might lead to a situation of no drug to cure pathogenic bacteria involved infections, which is an international problem. Phages can evolve with bacteria and its activity is not affected by antibiotics-resistance mechanisms. Therefore, phage is an effective candidate to treat antibiotics resistant bacteria involved infections. Infections of phages to bacteria are initiated by specifically binding of phages to host bacteria surface, about the process of which lots of problems have not been clearly understood. For instance, is the binding strength of phages and host bacteria related to infection activity? Are the effects of environment directly associated to phage adsorption to host bacteria? In this project, atomic force microscopy (AFM) based techniques will be developed to study the binding of phages to host bacteria at single bacterium and the molecular mechanisms of the interactions between phages and host bacteria, which will provide theoretical foundation for phage therapy of infections caused by antibiotics-resistant Pseudomonas aeruginosa and provide effective approach to isolating phages to effectively infect Pseudomonas aeruginosa. This project will provide guidance for phage therapy to antibiotics-resistant bacteria.

噬菌体是一种能特异感染细菌的病毒。铜绿假单胞菌是一种常见的机会致病菌，极易产生耐药和多耐药，多耐药病原菌的广泛存在将使我们面临无药可治的局面，寻找更有效持久的治疗方法是我国乃至全球亟待解决的难题。噬菌体由于与细菌共进化，不受耐药机制的影响，是一种治疗耐药菌感染的有效途径。噬菌体感染细菌始于噬菌体特异地结合到宿主表面，然而关于的这一过程的许多问题尚未弄清，如噬菌体与宿主的结合能力是否与其感染能力直接相关？环境对噬菌体感染能力的影响是否与对噬菌体吸附能力的影响直接相关？本项目拟以铜绿假单胞菌为对象，采用原子力显微镜力谱和成像技术在单细菌水平研究噬菌体与宿主菌的相互作用，阐明噬菌体与宿主菌结合与感染的关系，为噬菌体治疗铜绿假单胞菌引起的感染提供理论依据，并为分离更有效感染铜绿假单胞菌的噬菌体提供有效途径。项目的研究成果将为临床上耐药菌的噬菌体治疗提供有效的理论指导。

随着抗生素的广泛使用，常见的很多机会致病菌如铜绿假单胞菌、鲍曼不懂杆菌等对抗生素耐药越来越普遍。多耐药病原菌的广泛存在将使我们面临无药可治的局面，寻找更有效持久的治疗方法是我国乃至全球亟待解决的难题。对多耐药菌进行防治，一是发展新型的抗菌物质；二是发展新型的检测方法实现病原菌快速准确检测。噬菌体是一种能特异感染细菌的病毒，由于与细菌共进化，不受耐药机制的影响，是一种治疗耐药菌感染的有效途径。噬菌体感染细菌始于噬菌体特异地结合到宿主表面，然而关于的这一过程的许多问题尚未弄清，如噬菌体与宿主的结合能力是否与其感染能力直接相关？环境对噬菌体感染能力的影响是否与对噬菌体吸附能力的影响直接相关？首先我们以铜绿假单胞菌为对象，采用SPR技术和荧光成像技术从分子水平研究了噬菌体的尾丝蛋白与宿主菌的相互作用，为噬菌体治疗铜绿假单胞菌引起的感染提供理论依据，并为分离更有效感染铜绿假单胞菌的噬菌体提供有效途径。针对病原菌的快速检测，这里我们利用噬菌体的宿主特异性发展了几种新型的病原菌检测方法。首先利用噬菌体能特异识别宿主菌，在磁珠表面修饰噬菌体，用噬菌体捕获相应的病原菌，再通过与抗体连接的显色分子进行显色来能在2 h内实现病原菌的快速检测，检测灵敏度为800 CFU，并将该方法成功用于牛奶或苹果汁等样本中病原菌的快速检测。其次，通过噬菌体对宿主菌特异性裂解并对噬菌体进行扩增，通过直接PCR扩增血清样本，检测噬菌体的核酸拷贝数的变化来确定血清样本中是否含有目标菌，该方法能在4 h内能检测到血清中的100 CFU的菌，该方法适合临床上败血症样本中病原菌的血流感染。再次利用噬菌体的扩增性能我们发展了一种短暂培养联合噬菌体PCR的方法对痰液中病原菌进行检测，该方法无需对痰液样本进行提取，在6 h内能检测到痰液中1 CFU的菌。本项目的研究成果为临床上耐药菌的防治提供了有效的理论指导并有着实际的应用价值。