Hfq对溶藻弧菌碱性丝氨酸蛋白酶合成的调控机制研究

Vibrio alginolyticus is the main pathogen causing serious economic loss annually in the aquaculture in South China and its major virulence factor is determined as the exotoxin, alkaline serine protease (Asp) which is under the tight regulation of quorum sensing system. Moreover, the previous work has characterized that the chaperon protein of non-coding small RNA, Hfq is present in V. alginolyticus and indicated that Hfq plays pivotal roles in the modulation of various progresses in this pathogen, such as the extracellular protease avtivity and many other virulence-related phynotypes. Hfq takes effects on the alkaline serine protease production mainly by the post-transcriptional inhibition of the core element in the quorum sensing system, LuxR. This study intends to figure out the underlying mechanisms of the regulation of Hfq on the Asp production in V. alginolyticus. It's going to characterize the sRNAs which are participating in the Asp activity and determine the effects of the sRNAs on the target mRNA stability. Besides, this study also attempts to reveal the roles of Hfq in the base-pairing process between the sRNA and the responding target mRNA and draw the interaction diagram between the Hfq-sRNA and the quorum sensing system in V. alginolyticus. The study will open a brand new frontier for the pathogenecity mechanism of this pathogen and lay a solid theoretical foundation for the development of disease control strategy against V. alginolyticus and other mariculture pathogens in the future.

溶藻弧菌是我国南方水产养殖重要病原菌，主要毒力因子为胞外碱性丝氨酸蛋白酶，其合成受到群体感应系统的紧密调控。前期研究发现，溶藻弧菌中存在非编码小RNA（sRNA）伴侣蛋白Hfq，且Hfq在溶藻弧菌的多种生理活动中起重要调控作用，如碱性丝氨酸蛋白酶的活性及其他毒力相关表型。Hfq对碱性丝氨酸蛋白酶活性的调控主要通过转录后抑制群体感应系统中枢元件LuxR实现。本项目以Hfq对LuxR转录后调控作用为出发点，研究Hfq在溶藻弧菌中调控碱性丝氨酸蛋白酶合成的分子机制，包括鉴定参与Hfq调控碱性丝氨酸蛋白酶活性的相关sRNA及其对LuxR靶标mRNA分子稳定性的影响，阐明Hfq-sRNA-LuxR之间的作用机制，描绘Hfq-sRNA系统与群体感应系统之间的相互作用网络图景。揭示Hfq调控溶藻弧菌碱性丝氨酸蛋白酶活性的作用机制将为病原菌致病机制研究及海水养殖病害防治新技术的开发应用提供一定的理论依据。

溶藻弧菌是我国南方水产养殖重要病原菌，主要毒力因子为胞外碱性丝氨酸蛋白酶，其合成受到群体感应系统的紧密调控。前期研究发现，Hfq在溶藻弧菌的多种生理活动中起重要调控作用，如碱性丝氨酸蛋白酶的活性及其他毒力相关表型。Hfq主要作为sRNA分子伴侣蛋白实现对不同靶标mRNA的转录后调控作用。本项目通过生物信息学和分子生物学手段，在溶藻弧菌体内鉴定了5个sRNA分子，Qrr1-5。研究发现Qrr分子对溶藻弧菌的生长、运动性、生物被膜、碱性丝氨酸蛋白酶合成具有一定的调控作用，其中，Qrr1-5对碱性丝氨酸蛋白酶的抑制作用主要通过对LuxR的负调控实现。探究了Hfq-Qrr-LuxR之间的作用模式，发现Qrr1-5与LuxR的RBS区及上游序列可以互补配对，在Hfq存在的条件下抑制LuxR的表达。同时，对Hfq蛋白结构域进行分析并构建了相应的突变株，结果发现Hfq不同结构域缺失后，LuxR的表达水平上升，表明Qrr对LuxR的调控作用需要Hfq的协助。同时鉴定了群体感应系统低密度调控元件AphA，其可通过对自身和对LuxR的抑制实现对碱性丝氨酸蛋白酶合成的下调作用。此外，研究了Hfq-Qrr与群体感应系统间的作用关系，结果显示Qrr对群体感应系统元件luxMN、luxO、aphA等均具有调控作用，而同时群体感应元件亦对Qrr表达产生影响，LuxO激活Qrr转录，LuxR和AphA则可以直接结合至Qrr分子的启动子区，分别发挥转录激活和抑制作用。通过对Hfq-Qrr对溶藻弧菌毒力因子的调控作用机制的初步研究，可为海水养殖病害防治新技术的开发应用提供一定的理论依据。