【作者】 Djakpo Odilon （奥德伦）；

【导师】 姚卫蓉； 钱和；

【作者基本信息】 江南大学 ， Food Science and Technology， 2010， 硕士

【摘要】 群体感应(OS)或细菌之间的信息交流是细菌根据细胞密度进行自我协调的一种反应,是通过一种被称作白诱导剂的激素样物质介导进行的。基因表达调控系统依赖于OS,控制着多种原核表型。考虑到QS系统也控制着多种有害的细菌表型,包括生物膜的形成、致病因子的表达、运动黏附性,因此抑制QS被视为一种抗菌治疗的新方法。本论文对传统中药植物中的群体感应抑制剂(QSl)进行了探索性研究。用紫色色杆菌(chromobacterium violaceum)和根癌农根菌(Agrobacterium tumefaciens)两种菌株为报告菌,从29种植物中筛选具有抗QS活性的植物。发现五倍子和黄连两种植物显示了对QS的抑制活性。这2种植物对紫色色杆菌有强烈的QSl活性,且在一定剂量组中抑制紫色色杆菌CV026菌株产生紫色菌素。对于根癌农根菌A136,黄连不影响其QS介导的β-一半乳糖苷酶的表达,其QSl的活性范围狭窄,而五倍予则有很强的活性。以铜绿假单胞菌(Pseudomonas aeruginosa)和大肠杆菌(Escherichia coli)为报告菌株,进一步检验了这些植物对QS调控的致病因子(模型菌株的群体运动能力和生物膜形成)的影响作用。实验发现一定剂量级别下,五倍子和黄连会明显抑制铜绿假单胞菌PAO1和大肠杆菌的群体运动能力。100μg/rnL浓度的提取物,五倍了分别拟制PAO1和大肠杆菌群体运动力的84%和90%,而黄连分别抑制73%和63%。200μg/mL时,黄连和五倍子分别使PA01的生物膜厚度减少了37%和23%。此外,优化了无细胞裂解液系统,并首次将其应用到QsI活性筛选过程,将活性筛选过程将原来的16-18h降低到l到2h,建立了QSl快速选择系统。采用反相高效液相色谱法与新建立的QSI快速选择系统联用技术进行鉴定,首次发现五没食了酰葡萄糖(Pentagalloylglucose)是五倍了中的QSI活性物质之一。该物质作为五倍予鞣酸(gallotanin)的前体物质,在抑制绿脓杆菌PA01群体运动力方面,表现了与五倍_了相同的抗菌活性。5μg/mL、lOμg/mL和25μg/mL浓度的五没食子酰葡萄糖存在的条件下培养PAO1,致病因子绿脓菌素,色素产量被强烈抑制了,分别降低了27.95%、42.25%和58.53%。本研究从具有抗菌活性的传统中药中发现两种抗群体感应的中药,具有靶向QS系统,而不是杀死细菌,为解决抗生素耐药性问题提供一种方案。这一研究结果可能会为传统中药活性机理研究开辟出一个新领域,并有望为细菌性感染的治疗提供一个新发展方向。更多还原

【Abstract】 The emergency of multi-resistant bacteria due to indiscriminant use of antibiotic led to the search for new antibacterial approaches, for new classes of compounds that are not affected by resistance mechanisms already present in the bacterial. Quorum sensing (QS) or cell to cell communication is a cell-density dependent bacterial response mediated by hormone-like compounds called autoinducers. QS-dependent regulation of gene expression controls a wide variety of prokaryotic phenotypes including biofilm formation, virulence factor expression, and motility. Therefore the inhibition of QS is considered as a new approach of antimicrobial chemotherapy as anti-QS compounds target genes that are essential for basic metabolism in vitro, rather than the microorganisms itself. This study initiates a directed search for QS inhibiting agents in Traditional Chinese Medicinal Herbs. 2medicinal herbs were screened for anti-QS activity using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens. Of these plants, two showed QS inhibition: Galla chinensis and Coptis chinensis. These plants showed both strong QSI activity with Chromobacterium violaceum and inhibited violaceum production by CV026 at dose grade. Coptis chinensis did not affect QS mediated-galactosidase expression in Agrobacterium A136, suggesting a narrow range of QSI activity while Galla chinensis showed a strong activity and thereby wider range of QSI activity. These plants were further examined for their effects on the QS-Regulated virulence factors namely; swarming motility and biofilm formation of model strains Pseudomonas aeruginosa opportunistic pathogens responsible for, morbidity and mortality in the immunocompromised patient and Eschen’chia coli. Galla chinensis and Coptis chinensis were found to significantly inhibit swarming motility of PAOI and E. coli at grade dose. Galla chinensis inhibited 84% and 90% swarming mortility of PAOI and E. coli respectively whereas Coptis chinensis inhibited 73% and 63% respectively at 100g/mL. The thickness of PAOI biofilm was reduced by 37% by Coptis chinensis but was wickedly affected (23%) by Galla chinensis extracts at the concentration of 200g/mL. Furthermore we developed a fast QSI screening system; using a Cell free lysate of the strain Agrobacterium tuMefaciens A136 based on P-galactosidase activity. Typically, an assay with a whole-cell bioreporter such as A. tumefaciens A136 requires 16 to 18 h to culture the bioreporter strain and an additional 16 to 18H for incubation of the assay mixture to detect QSI compounds. In contrast, our QSI screening system takes 1 to 2h in total. Therefore, the cell-free assay system for detecting QSI compounds eliminates the time-consuming cell conditioning step in biosensor cell cultures that is required before each whole-cell bioassay.Activity guided fractionation, identification using Reversed-phase HPLC coupled with the new QSI selector system led to the identification of Pentagalloylglucose, a gallotanin precursor as one of the active QSI compound from Galla chinesis. Moreover this compound as a prototype of Chinese gallotannins, showed the same extent of antipathogenic activity as Galla chinensis in Pseudomonas aeruginosa PAO1 swarm plate. When we co-cultured PAO1 in presence of 5; 10 and 25 ug/mL of Pentagalloylglucose, the virulence factor pyocyanin, pigment production was strongly repressed by 27.95 ; 42.25; 58.53% respectively.This study introduces 2 new anti quorum sensing plants from China possessing a great antipathogenic activity. Targeting the QS system, instead of killing bacteria, provide a solution to antibiotic resistance. The result of this study may open a new area of research on mechanistic activity of traditional Chinese medicinal plant and potentially give a new therapeutic direction for the treatment of bacterial infections.In addition, this is the first report of Pentagallolglucose, pure compound isolated from Galla chinensis, having a revealed inhibition on the QS system and QS-Regulated virulence factors. And the least this is the first report of the time-saving QSI selector system based on free cell lysate to screen and compare QSI compounds.In addition, this is the first report of Pentagallolglucose, pure compound isolated from Galla chinensis, having a revealed inhibition on the QS system and QS-Regulated virulence factors. And the least this is the first report of the time-saving QSI selector system based on free cell lysate to screen and compare QSI compounds.更多还原