AI-2通过调节c-di-GMP代谢酶DosC影响类志贺邻单胞菌生物膜形成及运动性

AI-2 affects biofilm formation and motility of Plesiomonas shigelloides by regulating the c-di-GMP-metabolizing enzyme DosC

摘要:

细菌中广泛分布的群体感应信号分子autoinducer-2(AI-2)会影响细菌的生物膜形成及运动性等生理过程，然而该信号对类志贺邻单胞菌相关表型的调控作用及其分子机制尚未有所报道。【目的】揭示AI-2通过影响胞内环二鸟苷单磷酸(c-di-GMP)水平调控类志贺邻单胞菌生物膜形成及运动性的内在机制，为类志贺邻单胞菌感染的防治提供新思路。【方法】首先利用同源重组方法构建luxS基因敲除菌株(ΔluxS)，通过软琼脂平板法和结晶紫染色法分别检测其与野生型泳动能力和生物膜形成水平的差异；之后通过序列比对找到AI-2的潜在受体蛋白DosC (SAMEA2665130_2180)，利用哈维氏弧菌生物发光实验及等温滴定量热实验(ITC)研究DosC的配体结合结构域(ligand-binding domain，LBD)与AI-2的结合能力；通过体外酶活实验、胞内c-di-GMP定量分析研究AI-2对DosC受体活性的影响；最后参照前述方法构建受体DosC编码基因敲除菌株(ΔdosC)并检测其与野生型相比泳动能力和生物膜形成水平的变化。【结果】AI-2与DosC-LBD显示出高亲和作用力；通过高效液相色谱分析发现AI-2会增强受体蛋白DosC的磷酸二酯酶活性，促进c-di-GMP的降解。与野生型相比，ΔluxS和ΔdosC菌株胞内c-di-GMP水平显著升高，泳动与生物膜形成能力均显著降低。【结论】受体蛋白DosC通过响应群体感应信号分子AI-2，增强自身磷酸二酯酶活性，促进c-di-GMP的降解，影响胞内c-di-GMP的含量，进而调控细菌的生物膜形成和泳动能力等表型。

Abstract:

Autoinducer-2 (AI-2), a quorum sensing signal molecule ubiquitous in bacteria, influences a variety of bacterial physiological processes including biofilm formation and motility. However, the role of AI-2 in regulation of these phenotypes and the underlying mechanism have not been reported for Plesiomonas shigelloides. [Objective] To reveal the mechanism through which AI-2 regulates biofilm formation and motility of P.shigelloides by regulating the intracellular cyclodiguanosine monophosphate (c-di-GMP) level, and provide a new idea for the prophylaxis and treatment of P.shigelloides infections. [Methods] Firstly, we constructed the luxS gene knockout strain (ΔluxS) by homologous recombination method. Soft agar plate assays and crystal violet staining assays were employed to compare the swimming motility and biofilm formation between ΔluxS and the wild type. Then, we identified the potential receptor protein DosC (SAMEA2665130_2180) of AI-2 by sequence alignment.Vibrio harveyi MM32 bioluminescence and isothermal titration calorimetry (ITC) were employed to test the binding affinity of the ligand-binding domain of DosC (DosC-LBD) to AI-2. Afterwards, we studied the effect of AI-2 on DosC activity by in vitro enzyme activity assay and intracellular c-di-GMP quantitative assay. Finally, we constructed the dosC knockout strain (ΔdosC) according to the above method and compared its swimming motility and biofilm formation with those of the wild type. [Results] AI-2 bound to DosC-LBD with high affinity. Compared with the wild type, ΔluxS and ΔdosC showed significantly weakened swimming motility and biofilm formation and dramatically increased intracellular c-di-GMP level. HPLC demonstrated that AI-2 enhanced the phosphodiesterase (PDE) activity of DosC. [Conclusion] The c-di-GMP-metabolizing enzyme DosC enhances its PDE activity in response to AI-2, thus affecting the intracellular c-di-GMP level and regulating the biofilm formation and swimming motility of P.shigelloides.