信号分子AI-2的体外合成及其对副溶血性弧菌四环素耐药性的调控作用  

In vitro synthesis of AI-2 and its effect on tetracycline resistance of Vibrio parahaemolyticus

作　　者：林才云 江艳华[2] 姚琳[2] 李风铃[2] 曲梦 王联珠[2] 许加超[1] LIN Cai-Yun;JIANG Yan-Hua;YAO Lin;LI Feng-Ling;QU Meng;WANG Lian-Zhu;XU Jia-Chao(College of Food Science and Engineering,Ocean University of China,Qingdao,Shandong 266003,China;Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality,Ministry of Agriculture and Rural Affairs,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,China)

机构地区：[1]中国海洋大学食品科学与工程学院,山东青岛266003 [2]中国水产科学研究院黄海水产研究所农业农村部水产品质量安全检测与评价重点实验室,山东青岛266071

出　　处：《微生物学通报》2020年第5期1321-1331,共11页Microbiology China

基　　金：国家自然科学基金(31601566);中国水产科学研究院黄海水产研究所基本科研业务费(20603022020004);国家重点研发计划(2017YFC1600703)。

摘　　要：【背景】抗菌药的过度使用引起细菌耐药性日益严重,作为重要的食源性致病菌,副溶血性弧菌也表现出一定程度的耐药性。群体感应系统可以调控细菌的耐药性,为研究副溶血性弧菌的耐药机制和控制技术提供新的途径。【目的】探讨群体感应信号分子AI-2(autoinducer-2)对海产品中分离的副溶血性弧菌四环素耐药性的调控作用。【方法】通过原核表达制备AI-2合成关键酶--S-核糖同型半胱氨酸酶(S-ribosylhomocysteinase,LuxS)和S-腺苷同型半胱氨酸核苷酶(S-adenosylhomocysteine nucleosidase,Pfs),体外合成AI-2,通过菌落计数法分析AI-2对副溶血性弧菌在四环素亚抑菌浓度下耐受性的影响,采用逆转录实时荧光定量PCR法测定不同浓度AI-2对副溶血性弧菌四环素耐药基因转录水平的影响。【结果】通过原核表达获得LuxS和Pfs,作用于底物S-腺苷同型半胱氨酸能合成具有生物活性的AI-2,其荧光强度约为阳性对照的6倍。在四环素亚抑菌浓度下,AI-2能显著促进副溶血性弧菌的生长,6、15、30μmol/L浓度AI-2能不同程度地提高副溶血性弧菌四环素耐药基因的转录水平。【结论】AI-2能增强副溶血性弧菌对四环素的耐受作用,为解析副溶血性弧菌的耐药机制、研制以AI-2为靶点的副溶血性弧菌耐药性控制技术提供基础。[Background]Antimicrobial resistance of bacteria has become more and more serious due to the abuse of antimicrobials.As an important foodborne pathogen,Vibrio parahaemolytics also exhibited a certain level of antimicrobial resitance.Quorum sensing system can regulate the antimicrobial resistance of bacteria,which provides a new pathway to study the mechanism and control technique of antimicrobial resitance of V.parahaemolytics.[Objective]To study the effect of signaling molecule autoinducer-2(AI-2)on tetracycline resistance of V.parahaemolyticus strains from seafoods.[Methods]AI-2 was synthesized in vitro by a reaction using the critical enzymes,S-ribosylhomocysteinase(LuxS)and S-adenosylhomocysteine nucleosidase(Pfs),which were prepared by prokaryotic expression.The effect of AI-2 on tetracycline resistance of V.parahaemolyticus was determined by colonies counting method.The effect of AI-2 at different concentrations on the transcriptional levels of tetracycline resistance genes in V.parahaemolyticus was assayed by reverse transcription and real-time quantitative PCR.[Results]LuxS and Pfs enzymes were obtained by prokaryotic expression.The bioactive AI-2 could be synthesized in vitro by adding LuxS and Pfs to the substrate S-adenosylhomocysteine(SAH),with the fluorescence intensity of about 6 times as much as positive control.When treating with tetracycline at subinhibitory concentration,AI-2 could significantly promote the growth of V.parahaemolyticus strains,and AI-2 at the concentrations of 6,15 and 30μmol/L could increase the transcriptional levels of tetracycline resistance genes in V.parahaemolyticus strains to a certain extent.[Conclusion]AI-2 could enhance the tetracycline resistance of V.parahaemolyticus,which provided a reference for further study on the antimicrobial resistance mechanism of V.parahaemolyticus and developing control techniques targeting AI-2 on antimicrobial resistance of V.parahaemolyticus.

关 键 词：副溶血性弧菌 信号分子AI-2 S-核糖同型半胱氨酸酶 S-腺苷同型半胱氨酸核苷酶 四环素耐药性 四环素耐药基因tet 

分 类 号：Q93[生物学—微生物学] TQ455[化学工程—农药化工]