【作者】 刘湛军；

【导师】 于文功；

【作者基本信息】 中国海洋大学 ， 生药学， 2013， 博士

【摘要】 上世纪20年代末青霉素的发现，开辟了抗生素治疗的新时代，许多感染性疾病从此得到了有效控制。但是，随着抗生素的广泛使用、错用和滥用，细菌耐药性越来越严重，涌现出了大量的耐药菌、多重耐药菌甚至超级耐药菌株，这些问题已成为制约人类生存和发展的极大危机。我们国家是世界上抗生素滥用最严重的国家之一，每年约有8万人死于抗生素泛滥使用导致的不良反应，且死亡数量每年都在递增，细菌耐药问题和其他国家相比更加严峻。因此，如何解决细菌耐药难题和发现新的药物靶点研发新型抗生素已经迫在眉睫。研究表明抗生素在低于治疗剂量即亚抑菌浓度(subinhibitory concentrations)条件下引起的生物学效应是临床上引起细菌耐药的一个重要因素。因此研究抗生素亚抑菌效应不仅对分析细菌耐药机理、发现新的药物靶点具有重要作用，而且抗生素在亚抑菌浓度下产生的生理生化反应对临床给药剂量和给药时间都有非常重要的指导意义。本研究中，我们发现在紫色杆菌培养平板上卡那霉素抑菌圈外围紫色菌素合成明显增强。实验证明这种增强作用是由亚抑菌浓度卡那霉素引起，且其在1/4、1/6、1/8MIC时均明显促进紫色杆菌紫色菌素合成，其中1/6MIC促进效果高达25%。这种现象在临床常见抗生素阿米卡星、庆大霉素、红霉素、四环素中同样存在，但促进效果均不如卡那霉素明显。由于紫色杆菌中紫色菌素合成受群体感应系统密切调控，我们采用荧光定量PCR和生物报告菌株分析了亚抑菌浓度抗生素作用下紫色杆菌信号分子合成酶基因转录水平和信号分子产量，实验结果表明亚抑菌卡那霉素明显提高了群体感应信号分子合成酶基因的转录水平和信号分子的产生。此外，群体感应增强同时促进了与其相关的几丁质酶产生和细菌生物被膜形成。进一步研究显示，在抗生素刺激下与紫色杆菌生物被膜形成相关的swimming, swarmming运动行为和聚集能力明显增强。上述结果表明，亚抑菌浓度抗生素促进紫色杆菌群体感应行为发生。c-di-GMP是细菌中普遍存在的第二信使，其调控的多种细菌行为如：细胞运动、生物被膜形成等均与群体感应系统相互交叉，暗示c-di-GMP信号系统和群体感应信号系统相互关联。在本论文中，我们采用双亲接合转化方式成功将来自大肠杆菌的c-di-GMP合成基因adrA导入紫色杆菌中。当过表达adrA时，紫色杆菌群体感应行为明显受到抑制。荧光定量PCR实验结果显示adrA转入后紫色杆菌群体感应信号分子合成基因的转录水平下调。信号分子定量分析也得到了一致的实验结果。同时，受群体感应系统调节的紫色菌素合成和几丁质酶产生都受到明显抑制。这些结果表明，adrA可能通过增加c-di-GMP合成量来调节紫色杆菌群体感应行为。传统的细胞内c-di-GMP监测方法是利用放射性同位素对c-di-GMP合成底物GTP进行标记，然后利用薄层层析和放射自显影监测胞内的同位素量，该方法对实验条件要求高、操作繁琐而且很难实时反映胞内c-di-GMP含量。为了解决这一问题，我们利用c-di-GMP能够作为核糖开关配体分子调控基因表达原理构建了c-di-GMP核糖开关监测模型并以此模型监测抗生素作用下细胞内c-di-GMP水平变化，从而确定抗生素是否通过第二信使调控细菌群体感应系统。首先，我们分别从霍乱弧菌VC16961、腊样芽孢杆菌和嗜水气单胞菌基因组上克隆了六个c-di-GMP核糖开关(riboswitch)基因。然后分别利用sacB和lacZ报告基因成功构建了6组c-di-GMP含量变化体内检测模型。通过结构优化和功能验证，我们获得了一个特异性强，灵敏度高的c-di-GMP核糖开关生物检测模型Ahy-1。利用此模型我们检测了亚抑菌浓度抗生素作用下紫色杆菌胞内c-di-GMP含量变化，结果表明亚抑菌浓度抗生素下调了紫色杆菌胞内c-di-GMP水平。由于c-di-GMP负调控紫色杆菌群体感应系统，因此我们认为亚抑菌浓度抗生素能通过c-di-GMP信号通路调节紫色杆菌群体感应行为。综上所述：本文我们发现并证明了亚抑菌浓度抗生素促进紫色杆菌群体感应发生，探究了c-di-GMP合成酶基因adrA转入后对紫色杆菌群体感应行为的调控，构建并优化了c-di-GMP核糖开关生物检测模型，证明了亚抑菌浓度抗生素信号能通过影响紫色杆菌c-di-GMP信号通路调节群体感应行为。本论文的研究为解决临床细菌耐药和发现新的药物作用靶点、开发新型抗感染药物提供了新的理论依据。同时，为抗菌药物在临床使用时趋利避害，合理应用提供了有价值的参考。更多还原

【Abstract】 In the late1920s, the discovery of penicillin opened up a new era of antibiotictreatment and many infectious diseases have been effectively controlled from then on.However, with the misuse and abuse of antibiotics, bacterial resistance is more andmore serious and a large number of drug-resistant bacteria, multi-resistant bacteriaand even super-resistant strains emergence. These problems have restricted thesurvival and development of human. Our country is one of the most seriousantibiotics-abuse countries where the problem of bacterial resistance is more severecompared to other countries. Each year about80,000people died due to the adversereaction of antibiotic abuse, and the number is increasing progressively every year.Therefore, how to solve the problem of bacterial resistance and find novel drug targetsis imminent. It has been proposed that the biological effect caused by antibiotics atsubinhibitory concentrations (less than therapeutic dose) is an important factor inclinical bacterial resistance. The study of antibiotic-effect at subinhibitoryconcentrations will not only play an important role in the analysis of bacterialresistance mechanism, but also have great significance for guiding clinical dosage andadministration time.In this study, we found a clear enhancement of violacein production at the edge ofthe kanamycin inhibition zone when strain CV12472was grown on plates. Our datashowed that this enhancement was caused by kanamycin at subinhibitoryconcentrations and violacein production was promoted when the concentration ofantibiotics was1/4,1/6and1/8of MICs. A peak induction occurred in the presence ofkanamycin at1/6MIC. The amount of violacein was increased by25%at thisconcentration. Similar results were observed in1/4MIC of amikacin,1/2MIC ofgentamycin,1/16MIC of tetracycline, and1/8MIC of erythromycin, but the effect is not as obvious as1/6MIC of kanamycin. In Chromobacterium violaceum, thesynthesis of violacein is under the control of QS. Thus, we analyzed the violaceinsignal molecule synthase gene and signal molecule production with quantitativereal-time PCR and signal molecule report strain, respectively. The results showed thatkanamycin at1/6MIC enhanced the transcription of signal molecule synthetase geneand the production of QS signaling molecules N-acyl-L-homoserine lactones. At thesame time, antibiotics promoted the QS controlled virulence, including chitinaseproduction and biofilm formation. Besides, a positive flagellar activity and increasedbacterial clustering ability were found to be related to the antibiotic-induced biofilmformation. These results indicated that antibiotics at subinhibitory concentrationsimproved the QS behavior of C. violaceum.Cyclic di-guanosine monophosphate (c-di-GMP) is the second messenger inbacteria and triggers wide-ranging physiological changes interdisciplinary with QS,such as cell movement and biofilm formation, which suggests that c-di-GMP signalsystem and QS signal system are not separated. In this thesis, we transferred thec-di-GMP synthetase gene adrA of E. coli into C. violaceum successfully withconjugal transfer and the QS behavior of it was inhibited obviously when overexpressed adrA. Quantitative real-time PCR analysis showed that adrA decreased thegene transcription level of the violacein signal molecule synthase. Signal moleculequantitative analysis was consistent with these results. At the same time, violaceinsynthesis and chitinase production were both inhibited. These results indicated thatadrA regulate QS behavior of C. violaceum by increasing the c-di-GMP synthesis.The traditional cell c-di-GMP monitoring method is using radioisotopes ofc-di-GMP synthesis substrate GTP tag, and then detecting the intracellular amount ofisotope with thin layer chromatography and autoradiography. However, this methodrequests high experimental condition, operation is complicated and it is difficult toreflect intracellular c-di-GMP content in real-time. To solve this problem, we built thec-di-GMP riboswitch monitoring model according the principle of c-di-GMP acting asriboswitch ligand molecular regulates gene expression. Then, we used this model tomonitor the intracellular level of c-di-GMP changes and determine whether antibiotics regulate bacterial QS system through c-di-GMP. Here we cloned six c-di-GMPriboswitch genes from the genomes of Vibrio cholera VC16961, Aeromonashydrophila and Bacillus cereus, and constructed six c-di-GMP content-changedetection models in vivo using sacB and lacZ biological reporter gene successfully.Through function verification and structure optimization, we got a strong specificityand high sensitivity c-di-GMP riboswitch biological detection model Ahy-1. Usingthis model, we found that antibiotics at subinhibitory concentrations decreased thelevel of c-di-GMP and antibiotics regulated QS behavior through c-di-GMP signalpathway in C. violaceum.In summary, we proved that antibiotics at subinhibitory concentrations improvedthe QS behavior of C. violaceum, demonstrated c-di-GMP regulation of QS behavior,constructed a strong specificity and high sensitivity c-di-GMP riboswitch biologicaldetection model and found that antibiotics at subinhibitory concentrations regulatedQS behavior through c-di-GMP signal pathway in C. violaceum. Our findings in thisthesis provided a new theoretical basis to solve clinical bacterial resistance anddiscover new drug targets for developing new anti-infective drugs. Meanwhile, itprovided valuable reference for antibacterial drugs in clinical use.更多还原