【作者】 路新枝；

【导师】 于文功；

【作者基本信息】 中国海洋大学 ， 药物化学， 2009， 博士

【摘要】 随着抗菌药物的广泛使用，细菌耐药性越来越严重，已成为人类生存的最大危机。我国是世界上滥用抗生素最为严重的国家之一，每年约有8万人死于抗生素的泛滥使用，细菌耐药问题与其它国家相比更加严峻，专家预言，我国有可能率先进入“后抗生素时代”，亦即抗生素发现之前的时代。因此，如何解决细菌耐药的难题和研发新型抗生素已经迫在眉睫。铜绿假单胞菌(Pseudomonas aeruginosa,PA)是一种临床上最常见的引起严重获得性感染的条件致病菌，能伺机感染任何部位，如烧伤处伤口、角膜、尿道及肺部等，并引起细菌性心内膜炎及肠胃炎。研究表明，PA生物膜(Biofilm，BF)的形成是其产生抗生素耐药的主要诱因。生物膜是由细菌和其分泌的胞外基质在物体表面形成的高度组织化的多细胞结构，一旦形成生物膜，细菌将具有极强的抗生素耐药性(比浮游细菌高100—1000倍)。对细菌生物膜的研究将为新型抗生素的研发开辟新的途径。生物膜的形成受复杂的网络调控，以往的研究阐明了群体感应(QuorumSensing,QS)和细菌的第二信使环二鸟苷酸(Cyclic Diguanylic Acid，c-di-GMP)均能调控PA的BF形成。然而，目前QS和c-di-GMP之间的关系尚不清楚。本研究拟通过构建QS缺失突变株、c-di-GMP合成或降解酶过表达株，利用激光共聚焦、定量PCR等技术研究PA中QS和c-di-GMP信号通路之间的内在关系。构建了PAO1群体感应单缺失菌株JP1(△lasI)、PD0100(△rhlI)和双缺失菌株MW(△lasI△rhlI)，通过对群体感应单缺失和双缺失菌株在运动能力、致病因子的产生和生物膜形成能力的比较，表明Rhl-QS系统对菌株影响最为明显，Rhl-QS缺失以后，菌株的运动能力降低，不能产生鼠李糖酯和绿脓菌素等致病因子。利用静态和动态两种模型评价了群体感应对生物膜的影响，结果显示在静态条件下群体感应缺失有利于生物膜形成，但是在动态模型下群体感应缺失生物膜形成减少。测定了野生型和群体感应缺失菌株对氨基糖苷类抗生素阿米卡星的敏感性，结果表明，生物膜形成后菌株的抗生素耐药性比浮游状态提高了约100倍，但是群体感应缺失对细菌的抗生素敏感性没有明显影响。以pMF54载体为骨架构建了组成型表达载体pMF54-A，利用绿色荧光蛋白检验了载体在PAO1中的稳定性，通过对PAO1基因组的分析选择了6个c-di-GMP合成酶基因和4个降解酶基因，目的基因重组入载体pMF54-A后转化PAO1，静态造膜显示降解酶PA2133和合成酶PA3702对菌株生物膜的形成影响最为明显。定量PCR显示PA2133过表达株目的基因的转录水平是野生型的270倍，而PA3702过表达株目的基因的转录是野生型的50余倍。在此基础上系统比较了二鸟苷酸环化酶和磷酸二酯酶过表达对野生型和群体感应缺失突变株运动能力、毒性因子产生、抗生素敏感性和生物膜形成能力的影响，结果表明c-di-GMP增加可以抑制野生型和Las-QS缺失突变株的运动、促进鼠李糖酯的产生，但是对于Rhl-QS缺失株和双缺失菌株，c-di-GMP的减少并不能恢复其运动能力和鼠李糖酯的产生。静态造膜结果显示，c-di-GMP的增加促进了生物膜的形成，反之则抑制生物膜的形成，这种作用是不依赖于群体感应的。细胞毒性结果表明c-di-GMP的增加可以增加菌株的毒性。在抗生素敏感性方面，虽然c-di-GMP不能改变浮游菌的抗生素敏感性，但是可以明显改变抗生素对生物膜的清除作用(MBEC)，其敏感性提高了8倍之多。与内源性的c-di-GMP不同外源加入的c-di-GMP抑制了生物膜的形成，说明c-di-GMP不能自由穿越细菌的细胞膜。评价了c-di-GMP在基因水平上对群体感应信号系统的影响，以及信号分子对c-di-GMP作用的影响，结果显示，c-di-GMP增加可以从转录水平上促进Rhl-QS，但是对Las-QS没有作用，而外源加入的信号分子不能改变c-di-GMP对生物膜形成的影响。对群体感应和c-di-GMP影响菌株生物膜形成的机制进行了探讨，结果显示群体感应系统缺失尤其是Rhl-QS的缺失可以抑制菌株的初始黏附，但是同时群体感应缺失菌株的微克隆形成能力高于野生株；在初期阶段c-di-GMP的减少可以明显抑制细菌与介质的黏附能力，而c-di-GMP增加后不能提高菌株的黏附能力但是菌株的生存状态发生了改变，由短杆状的增殖体状态转变为细丝状的潜生体状态，随着时间的延长c-di-GMP的增加有利于细菌微克隆的形成。比较了二尿苷酸环化酶和磷酸二酯酶过表达后菌株胞外多糖、细胞表面疏水性、细胞表面黏附器官的变化，结果表明c-di-GMP增加后细胞表面的疏水性降低，多糖合成酶基因pelA和pelD的转录上调；原子力显微镜和投射电镜结果未观察到群体感应缺失和环二鸟苷酸改变对细菌表面运动器官的影响。综上所述，在铜绿假单胞菌中环二鸟苷酸和群体感应两条信号通路并不是完全孤立的，环二鸟苷酸能调控Rhl-QS群体感应系统，但是其对生物膜的影响并不完全依赖于群体感应。更多还原

【Abstract】 With the widespread appearance of antibiotic-resistant bacteria, there is anincreasing demand for novel strategies to control infectious diseases. Every year morethan 80,000 Chinese die from antibiotics abuse, making China one of the worstoffenders in the word. It is presumed that we will be the first to enter the’post-antibiotic era’ in comparison with other countries. Therefore, how to solve theproblem of bacterial resistance and development of new antibiotics have beenimminent.Pseudomonas aeruginosa (PA) is an opportunistic pathogen and it is a commoncause of serious hospital acquired infection. Infections can occur in any part of thebody, such as the wounds of bum, cornea, urinary tract and the lungs, etc. It is saidthat biofilm formation is the mean reason of antibiotic resistance. Biofilm is amulticellular, matrix-enclosed assemblies formed by bacteria and extracellular matrix(ECM). Once the biofilm formed, the antibiotic resistance will be increased more than100-1000 folds. Therefore, the interference of biofilm formation is of greatsignificance for the research and development of new antimicrobial drugs.Bacterial biofilm formation is controlled by complex net work. In the previousstudy, quorum sensing and the second messenger Cyclic-di-GMP have been reportedto relevant for biofilm formation. But the relationship of quorum sensing andC-di-GMP is unclear and this is the aim of our study. In this thesis, quorum sensingdeficient strain and C-di-GMP overexpression or degradation strain were used tostudy the relationship between both.The moving motility, virulence production and biofilm formation ability of quorumsensing mutant strains JP1 (△lasI) , pDO100 (△rhlI) , and MW (△lasI△rhlI)were compared. The result indicated that the effect of Rhl-QS is more importantcompared with Las-QS. Rhl-QS dificient strain has lost the moving motility,rhamnolipid and pyocyanin production ability. Static and flow-cell models were used to investigate the effect of quorum sensing on biofilm formation. Contrast to flow-cellmodel, quorum sensing muatnt strain showed increased biofilm formation ability instatic condition. Biofilm formtion had increased the antibiotic resistance for about 100folds, but their was no obvious difference between the wild type and the quorumsensing mutant strain.A constitutive expression vector pMF-54A was constructed for constitutive expressof Diguanylate Cyclases (DGC) and Phosphodiesterases (PDE) and its stability was testedwith green fluorescent protein gene. After a full analysis of the whole genome ofPseudomonas aeruginosa PAO1, six proteins with GGDEF domain and four proteinswith EAL domain were chosen for next study. Target genes were recombinant intopMF-54A and transformed into PAO1. Static biofilm evaluation indicated thatphosphodiesterase PA2133 and diguanylate cyclase PA3702 significantly influence thebiofilm formation ability.Real time PCR analysis indicated that the transcription level increased 270 folds forPA2133 and 50 folds for PA3702. The virulence production, moving motility andbiofilm formation of wild type and quorum sensing muant strain with thoseoverexpress of PDE or DGC were observed. The results showed that the increase ofc-di-GMP inhibibited the moving motility of PAO1 and JP1 but improve theproduction of rhamnolipid. While the decrease of c-di-GMP can’t restore the movingmotility and rhamnolipid production of both pDO100 and MW. In the static model,c-di-GMP increase the biofilm formation independent of quorum sensing.Furthermore, the increase of c-di-GMP level had improved the toxity of the strain.C-di-GMP can’t change the antibiotic sensitivity of planktonic bacteria but theminimum biofilm eleberate concentration (MBEC) decreased for more than eightfolds after the transformation of PDE. Exogenous c-di-GMP inhibit the biofilmformation of PAO1. Real time PCR result indicated that c-di-GMP improved bothRhlI and RhlR transcription but there was no effect for LasI and LasR.The mechanisms of quorum sensing and c-di-GMP on biofilm formation wereinvestigated. The results indicated that quorum sensing deficiency strain, especiallyfor Rhl-QS deficient, inhibited the initial attachment but the microclone formation ability increased with long term incubation. The decrease of c-di-GMP cansignificantly inhibit the attachment ability, while the increase of c-di-GMP can’timprove the attachment. Strains with overexpress of DGC changed from vegetativegrowth phase to cryptic growth phase and with the elongation of the incubation timethey formed more microclones compared to wild type. We also evaluated the effect ofc-di-GMP on the exopolysaccharide production, hydophobicity and the cell surfacemotion organs. The result indicated that the hydrophobicity decreased, while thetranscription level of polysaccharide production gene pelA and pelD increased, withthe increase of c-di-GMP. Atomic Force Microscope and Transmission ElectronMicroscope observation doesn’t show any differences in the motion organs.All in all, the two signal pathway was not completely isolated. The secondmessenger c-di-GMP can regulate Rhl-QS quorum sensing but its effect on biofilmformation was only partially depended on quorum sensing.更多还原