无氧胁迫对巴西弧菌应激体下游蛋白磷酸化效应调控研究

The ability of bacteria to adapt to dynamic and diverse environment by altering their cellular behavior is crucial for adaptation and survival. Pathogenic Vibrio bacteria has shown to respond to anaerobiosis by promoting virulence gene induction. In our previous study, a novel stressosome complex from the Gram-negative marine bacterium Vibrio brasiliensis that contained a heme-bound sensor protein and had the ability to sense environmental O2 was described. In limited oxygen condition, stressosome transduces environmental signal by a series phosphorylation events initiated by a kinase, a subunit of the stressosome protein complex. However, the stressosome-modulated downstream signaling pathways have yet to be characterized. The focus of this study is to interrogate the activation mechanism of downstream pathways by a previously identified anaerobically-activated stressosome signaling hub in V. brasiliensis. We plan to investigate the phosphorylation level of the downstream protein; establish the relationship between phosphorylation level and protein activity, and interrogate kinase-mediated activation of protein mechanistically. The proposal presents an opportunity to interrogate the effects of ligand-dependent stressosome signaling both in vitro and in vivo, as well as provides further insights into its potential role in regulating virulence gene expressions in limited O2 condition in the pathogenic vibrio.

细菌的致病力与耐受环境条件的胁迫息息相关。研究致病菌抗逆调控机制，对深入了解其致病机理、防控致病微生物对食品的污染、保障食品安全意义重大。我们前期研究发现革兰氏阴性模式菌株巴西弧菌通过应激体(stressosome)蛋白可逆磷酸化的方式传递无氧胁迫信号的全新抗逆机制。目前，关于无氧胁迫调控巴西弧菌应激体下游通路信号转导的机制尚未有报道。基于此，申请人拟进行以下三方面的研究：1，通过检测无氧胁迫对应激体下游蛋白磷酸化水平的调控，探究无氧胁迫信号的转导方式；2，明确应激体下游蛋白磷酸化水平与其活性的关联性，解析无氧胁迫激活下游蛋白的机制；3，鉴别调节激酶活性关键的氨基酸位点并揭示无氧胁迫激活应激体下游蛋白的分子机理。本项目拟层层阐明无氧胁迫信号通过巴西弧菌应激体转导至下游的动力学过程和机制，为研究环境胁迫对致病菌毒力基因表达的调控提供新思路。

细菌的致病力与耐受环境条件的胁迫的能力息息相关。研究致病菌抗逆调控机制，对深入了解其致病机理、防控致病微生物对食品的污染、保障食品安全意义重大。我们前期研究发现革兰氏阴性模式菌株巴西弧菌应激体(stressosome)蛋白复合体中激酶vbRsbT通过可逆磷酸化的方式传递无氧胁迫信号的全新抗逆机制。本项目通过体外磷酸化活性实验结合分子信息学分析，发现磷酸酶vbRsbU对vbRsbT-P具有一定去磷酸化的能力，但vbRsbT不能磷酸化vbRsbU。结合分子对接，推测vbRsbT可以通过与vbRsbU的N端结合实现互作，将应激信号传递给vbRsbU，通过去磷酸化作用继续转导应激信号。此外，基于蛋白组学与转录组学分析发现，厌氧胁迫时巴西弧菌通过抑制鞭毛合成蛋白、血凝素及菌毛组装蛋白的表达，降低运动能力和生物被膜生成来维持能量代谢平衡。在响应无氧胁迫时共有2487个基因发生差异性表达（1202个上调，1285个下调），其中磷酸二酯酶抑制剂基基因显著上调，而与鞭毛合成相关的蛋白基因显著下调。本项目为阐明巴西弧菌的信号转导通路提供理论依据，进而解析致病性弧菌的逆境生存机制，为提高水产品安全性提供借鉴。