禽致病性大肠杆菌安徽分离株luxS和pfs基因的克隆、表达与细胞外合成AI-2 活性检测

Cloning and expression of luxS and pfs and in vitro biosynthesis autoinducer 2 of avian pathogenic Escherichia coli from Anhui Province

摘要:

摘要:【目的】LuxS /AI-2 型密度感应系统存在于革兰氏阴性和阳性菌中，可产生用于细菌种间交流的通用自诱导信号分子AI-2(Autoinducer-2，AI-2)，细菌许多生理功能都受此系统的调节。本研究开展对禽致病性大肠杆菌(Avian Pathogenic Escherichia coli，APEC)自诱导信号分子AI-2 的检测和建立体外合成、定量的方法，为进一步研究APEC 的AI-2 调控作用奠定基础。【方法】利用哈维弧菌BB170 (Vibrio harveyi BB170) 开展对APEC AI-2 的检测; 利用表达、纯化的LuxS 和Pfs 在体外催化S-腺苷同型半胱氨酸( Sadenosylhomocysteine，SAH)，进行AI-2 的体外合成。【结果】APEC 能产生自诱导信号分子AI-2;成功表达可用于AI-2 合成的可溶性重组蛋白LuxS 和Pfs;纯化的重组蛋白LuxS 和Pfs 与SAH 同时作用后，合成了浓度为300μmol/L的AI-2;运用哈维弧菌BB170对合成的AI-2活性检测表明，其活性是阴性对照的700倍。【结论】APEC 存在LuxS /AI-2 型密度感应系统，APEC 的LuxS 和Pfs 可以在体外催化SAH 生成有活性的AI-2分子。本研究为进一步研究APEC的AI-2的调控作用奠定基础。

Abstract:

Abstract:［Objective］The LuxS /AI-2 quorum sensing (QS) system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2)． In this study，the method of biosynthesis of AI-2 was established using recombinant LuxS and Pfs of avian pathogenic Escherichia coli (APEC)，which will be benefit for future study of the role of AI-2 in APEC． ［Methods］ We investigated AI-2 production in APEC by vibrio harveyi BB170 (BB170)．Furthermore，APEC LuxS and Pfs were expressed，purified and used to investigate the production of AI-2 in vitro． LuxS and Pfs were incubated with S-ribosylhomocysteine (SAH)，the reaction was detected for the production of luminescence of BB170． ［Results］APEC can produce AI-2 by BB170 bioassay． Purified LuxS and Pfs enzymes incubated with SAH and produced 300μmol/L AI-2 in the reaction products． ［Conclusion］The results demonstrated that recombinant Pfs and LuxS synthesize AI-2 in vitro from SAH． These findings will be of benefit to future studies of the role of AI-2 in APEC．