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

【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。

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

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.