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

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、30 μmol/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。

Abstract:

[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 30 μmol/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.