迟缓爱德华氏菌(Edwardsiella tarda) LuxS/AI-2群体感应系统研究

IOCAS-IR > 海洋环流与波动重点实验室

	迟缓爱德华氏菌(Edwardsiella tarda) LuxS/AI-2群体感应系统研究
其他题名	LuxS/AI-2-Mediated Quorum Sensing in Edwardsiella tarda
	张敏
学位类型	博士
	2009-05-21
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	迟缓爱德华氏菌 Luxs 基因表达 Rna干扰阻遏子
摘要	从患病牙鲆中分离出迟缓爱德华氏菌株TX1，经报告菌株检测发现TX1有AI-2活性。用梯度PCR和Genome walking的方法克隆了TX1 luxS基因，将luxS基因在大肠杆菌DH5α中表达，证明其具有功能活性。在TX1中，luxS的表达与AI-2的活性基本是一致的，二者均受生长时期和生长条件的调节，即在glucose存在的条件下luxS表达和AI-2活性升高，而在高温条件下luxS表达和AI-2活性降低。glucose对AI-2活性以及luxS表达的影响经过荧光定量PCR，启动子活性检测，AI-2活性检测以及凝胶滞缓等一系列的实验证实是由cAMP-CRP复合物介导的，该复合物可以通过与luxS启动子相互作用而抑制luxS的表达。RNA干扰表明，TX1中luxS表达被干扰以后，对细菌产生了多方面的影响，包括：(1) 降低AI-2水平；(2) 降低细菌的生长能力；(3) 降低Ⅲ型分泌系统相关基因的表达水平以及生物膜的形成能力；(4) 减弱细菌毒力。外源AI-2的添加可以回复Ⅲ型分泌系统相关基因的表达水平以及生物膜的形成，但是并不能修复生长状况，表明LuxS在TX1中具有双重功能，即参与细胞代谢以及群体感应信号传导。基于LuxS/AI-2群体感应系统对细菌毒力的重要性，设计并筛选了一个该系统的阻遏因子5411。Pull-down实验证明5411可以和LuxS特异性结合。研究表明5411在TX1中表达导致细菌毒力显著下降。将5411克隆至牙鲆共生菌FP3中，发现5411可以被分泌到胞外并能被TX1吸收。将表达5411的共生菌导入牙鲆，发现其能够有效阻遏TX1对牙鲆的侵染。这些结果表明：(1) TX1中AI-2的活性受控于LuxS，而后者则受生长时期和生长条件的调控；(2) luxS的正常表达对于细菌的正常生长和侵染是必需的；(3) LuxS/AI-2群体感应系统调控Ⅲ型分泌系统相关毒力因子的表达；(4) 通过阻遏LuxS/AI-2群体感应系统来抑制病原菌侵染是一种具有潜力的新型病害防控方法。
其他摘要	Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed.tarda strain isolated from diseased fish, was found to produce autoinducer 2 (AI-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the AI-2 synthase was cloned from TX1 and designated luxS. LuxS was able to complement the AI-2 mutant phenotype of Escherichia coli strain DH5α. Expression of luxS correlated with AI-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS expression. Overexpression of luxS enhanced AI-2 activity in TX1, whereas disruption of luxS expression by antisense RNA interference (i) reduced the level of AI-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous AI-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. A small peptide 5411 was designed based on the sequence of luxS. 5411 expressed by E.coli DH5α and the commensal strain FP3 could be secreted out of the cells and absorbed by E. tarda. Fish administered with FP3 expressing 5411 showed enhanced resistance against E. tarda infection. Our results (i) demonstrated that the AI-2 activity in TX1 is controlled at least in part at the level of luxS expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS is essential for optimal bacterial infection and survival; (ii) suggested the existence in E. tarda of a LuxS/AI-2-mediated signal transduction pathway that regulates the production of virulence-associated elements. (iii) suggested that LuxS inhibitory peptides might be used as an effective means to control bacterial infections.
页数	102
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1117
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	张敏. 迟缓爱德华氏菌(Edwardsiella tarda) LuxS/AI-2群体感应系统研究[D]. 海洋研究所. 中国科学院海洋研究所,2009.

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