【作者】 史晓翀；

【导师】 于文功；

【作者基本信息】 中国海洋大学 ， 水产品加工及贮藏工程， 2008， 博士

【摘要】 随着抗菌药物的广泛应用,细菌的耐药性变得越来越严重,已成为全球关注的危害人类健康的重大难题。目前临床上使用的抗生素都是以细菌的蛋白质合成、细胞壁合成、DNA超螺旋等为靶点,通过干扰这些重要生命过程直接杀死微生物或抑制微生物生长来实现抗感染目的的。在这种生存压力下,细菌很容易产生耐药性。显然以传统的靶点和筛选方法获得的抗生素无法从根本上解决细菌的耐药问题。因此,寻找治疗细菌感染的新途径已成为当今生命科学研究的前沿和热点。近几年来,细菌群体感应(Quorum-Sensing,QS)研究的发展,给我们带来了新的机遇和挑战。群体感应是细菌根据细胞密度变化进行基因表达调控的一种群体行为。研究发现,细菌通过群体感应系统调控致病过程的一些重要环节,如致病因子产生、生物膜(Biofilm)形成等。根据美国NIH的调查报告,80%以上的细菌性感染与生物膜有关,而生物膜的形成是细菌产生抗生素耐药性和免疫逃逸的主要原因之一。因此,细菌群体感应系统干扰的研究对于新型抗菌药物的研究与开发具有重要意义。本文以革兰氏阴性菌的酰基高丝氨酸内酯(N-acyl- homoserine lactones, AHL)介导的群体感应系统为靶标,从海洋微生物中筛选群体感应抑制因子,并对其活性和作用机理进行研究。综合利用唯一能源法和报告菌株法,建立了一种从环境微生物中快速、高效地筛选能产生AHL群体感应抑制因子菌株的方法;利用该方法从海洋微生物中筛选得到多株具有AHL降解能力和竞争性抑制AHL群体感应能力的细菌,并对其中活性最高的一株进行了菌种鉴定,命名为Stenotrophomonas sp.A8。构建了菌株A8基因组DNA文库,利用AHL唯一能源培养基从文库中筛选得到一个具有AHL降解活性的克隆,并利用亚克隆试验确定了具有AHL降解酶活性基因的部分读码框;进一步通过反向PCR克隆得到该基因全长序列,最终得到的降解酶AQ1基因序列全长747bp,编码248个氨基酸,分子量为31.2kD。与Pseudomonas aeruginosa中的AHL降解酶PvdQ同源性为12%,与QuiP同源性为13%,可能属于酰氨酶家族。该酶对多种不同碳链长度的AHL分子均具有降解活性,尤其对3OC6HSL降解活性最强。采用层析技术结合多种AHL报告菌株,从A8的发酵液上清中分离纯化了三个具有AHL竞争性抑制作用的化合物,NMR和MS分析表明这三个化合物均为环肽,命名为:CP1、CP2和CP3。这三个化合物都能竞争性抑制AHL分子与其受体的结合;其中,CP2还能够抑制革兰氏阳性菌金黄色葡萄球菌、表皮葡萄球菌生物膜的形成;同时S.aureus RN4220中QS调控基因agrA、SarA、Hld的定量PCR结果表明,其mRNA转录水平均受到CP2的抑制。因此,CP2的抗细菌生物膜形成作用可能是通过抑制S.aureus RN4220的QS系统来实现的。综上所述,我们建立了一种高效的从环境微生物中筛选产生AHL群体感应抑制因子菌株的方法;从活性菌株Stenotrophomonas sp.A8中克隆了一个AHL降解酶基因,其重组酶具有降解多种不同碳链长度AHL分子的活性,该类型的酶在Stenotrophomonas属细菌中还未见报道;同时还从A8菌株发酵液上清中分离得到三个具有AHL竞争性抑制作用的环肽CP1、2和3,首次发现环肽化合物CP2对革兰氏阳性菌表皮葡萄球菌、金黄色葡萄球菌生物膜的形成具有显著抑制作用,并初步证明CP2的抗生物膜作用与QS系统有关。与传统的抗生素的作用机制完全不同,细菌群体感应抑制因子通过干扰细菌的群体感应系统实现抗感染的目的,而不影响细菌的生长,因此不会产生细菌耐药性。群体感应抑制因子作为新一代抗生素,在多个领域具有广泛的应用前景。本研究工作为开发海洋微生物资源和寻找以QS为靶点的新型抗生素奠定了一定基础。更多还原

【Abstract】 With the widespread appearance of antibiotic-resistant bacteria, there is an increasing demand for novel strategies to control infectious diseases. Conventional antibiotics possess broad-spectrum toxic or growth-inhibitory effects on organisms. Therefore, an increased frequency of bacterial mutations has resulted in a significantly increased incidence of antibiotic resistance. Obviously, the problem of the increasing bacterial antibiotic-resistance could not be resolved absolutely by antibiotics against traditional targets. Hence, the development of new approaches to the treatment of bacterial infections constitutes a focal point of research. The discovery of bacterial communication systems (Quorum-Sensing, QS), which orchestrate important temporal events in the infection process, have afforded a novel opportunity to ameliorate bacterial infection by means of methods other than growth inhibition. Recent research showed that Quorum-Sensing system is widespread and involves complex networks that serve as fine-tuner of the performance of diverse behaviors, such as pathogenic factor production, biofilm formation, and so on. According to the estimation of National Institutes of Health (NIH), biofilms may be involved in up to 80 percent of human infections. Studies showed that biofilms play important roles in drug resistance and many chronic infectious diseases relapse repeatedly due to the biofilm formation. Therefore, the interference with QS is of great significance for the research and development of new antimicrobial drugs.In the present study, an effective method was developed for screening of marine bacteria isolates capable of inhibiting the N-acyl- homoserine lactones (AHL) mediated quorum-sensing process of many gram-negative bacteria. The action mechanism of such inhibition was also investigated.Our protocol is based on sole source of energy method and AHL reporter strains method, which was used for the rapid and sensitive screening of quorum-sensing inhibitor from marine bacteria. Many bacterial strains with QS inhibiting activities were isolated, and one of them, which showed the highest activity, was identified as Stenotrophomonas sp.A8.The partial sequence of an AHL quenching enzyme AQ1 was cloned by screening A8 genomic library with AHL sole source of energy method. The complete AQ1 gene was cloned by inverse PCR. The gene is 747bp in length, and is predicated to encode a protein of 248 amino acids with a molecular mass of 31.2 kDa, which is 12% and 13% amino acid sequence similar with those of Pseudomonas aeruginosa AHL-quenching enzyme PvdQ and QuiP, respectively. The AQ1 showed quenching activities to many AHL molecules with different side chain lengths, especially to the 3OC6HSL.Interestingly, from the bioactive partition of A8 fermentation broth, three compounds, CP1, CP2 and CP3, was isolated by reverse-phase high performance liquid chromatography and their activities were assayed by AHL reporter strains. By means of spectroscopic methods (MS, 1H NMR, 13C NMR), the structures of compounds CP1, CP2 and CP3 were elucidated. All these three compounds showed activities of inhibiting the binding of AHLs to their receptors. CP2 can also inhibit biofilm formation of gram-positive bacteria, Staphylococcus aureus and Staphylococcus epidermidis. The mRNAs of agrA, SarA and Hld gene were analyzed with real-time quantitative polymerase chain reaction. All of them were regulated by QS system in S.aureus RN4220. The results showed that mRNAs of these three genes decreased by CP1, so the effect of attenuating biofilm formation can be attributed to the depression of QS system.In conclusion, an effective method was developed for screening of environmental bacterial isolates capable of inhibiting the AHL-mediated quorum-sensing system. The AHL-quenching enzyme AQ1 gene was cloned from strain Stenotrophomonas sp.A8. Recombinant AQ1 showed activities to many AHL molecules with different side chain lengths. Meanwhile, three Antagonistic cyclic peptides of Quorum-Sensing were isolated from the bioactive partition of fermentation broth of A8. And it was reported for the first time that the cyclic peptide CP2 can inhibit biofilm formation of gram-positive bacteria, S.aureus and S.epidermidis. Analysis of three QS regulated genes demonstrated that the reduction of biofilm formation is related to the depression of QS system.Research into quorum sensing and its inhibition, may provide a mean of treating many common and damaging chronic infections without the use of growth-inhibitory agents, such as antibiotics, preservatives and disinfectants which unavoidably select for resistant organisms. To the large number of bacteria that employ quorum-sensing communication systems, attenuation of unwanted bacterial activities rather than bactericidal or bacteriostatic strategies may find application in many different fields, e.g., medicine, agriculture and food technology.更多还原