ToxR作为分子开关调控溶藻弧菌进入存活但不可培养状态(VBNC)的分子机制

The viable but nonculturable (VBNC) cells have been accepted as a survival strategy of bacteria exposed to adverse environmental circumstances. Diseases caused by foodborne or waterborne pathogens in the VBNC state are emerging. The VBNC pathogens have the ability to evade conventional microbiological detection methods. Moreover, pathogenic bacteria in the VBNC state can regain virulence after resuscitation into culturable cells under suitable conditions. All these pose a significant and potential environmental and public health risk. So far, not much is in fact known about the molecular mechanisms underlying VBNC state induction and resuscitation. The loss of regulator ToxR, by regulated intramembrane proteolysis (RIP), may be associated with entry of Vibrio into the VBNC state. Toxin-antitoxin (TA) systems were found to be important in regulation of cell growth and dormancy, which is realized by inhibition of translation and protein biosynthesis caused by free toxin. The ppGpp synthesized in response to the environmental stress can controls the activity of TA modules. As an important zoonotic pathogen in marine aquaculture industries, Vibrio alginolyticus can enter into the VBNC state when exposed to conditions adverse to growth in the natural aquatic environment. In order to illuminate the mechanisms and basic regulatory network of VBNC state switched by ToxR, we set to explore cognation and regulating characteristics between the proteolysis of ToxR by RIP and the entry of V. alginolyticus into the VBNC state. Also, the genetic pathways in response to stresses, which undergo RIP signal cascade associated with TA system, will be delve deeply. All these expected results will reveal and illuminate preliminary the molecular mechanism in VBNC state formation, which will helpful to go into more detail on the comprehensive knowledge of the VBNC state induction in V. alginolyticus and other pathogenic bacteria.

VBNC（活的非可培养状态）是细菌在不利环境条件下的一种生存策略。病原菌的VBNC状态对流行病学监测、环境安全和公共健康构成重要的潜在风险。迄今为止，细菌VBNC的形成与复苏机制尚未被揭示。RIP（蛋白质受控膜内水解）信号转导通路触发的调控子ToxR水解可能参与VBNC的形成过程。环境胁迫应答反应可激活TA（毒素-抗毒素）系统活性，通过降解mRNA和抑制DNA复制等来调节细胞的生长与休眠。作为海水养殖病害中的重要人畜共患病原，溶藻弧菌遇到环境胁迫时可以VBNC形式分布于自然水生环境中。本课题以该病原为研究对象，拟研究在不同环境胁迫下由RIP信号通路介导的ToxR蛋白水解并进入VBNC状态的调控规律，同时探究这一环境胁迫应答与TA系统的级联关系及调控机制，从而初步阐明溶藻弧菌以ToxR为分子开关的VBNC形成机制和调控网络框架。

一些不利生长条件会诱导细菌进入一种类似休眠的生存状态，即存活但不可培养(VBNC)状态，然而导致细菌进入VBNC状态的分子机制以及相关调控通路尚未得到阐明。本研究以溶藻弧菌为研究对象，发现其在碱性和营养限制条件下会进入VBNC状态，且其关键毒力调节因子ToxR与此过程相关。通过滴板计数和活细胞染色实验发现，在营养限制和碱性环境下，23.8%的溶藻弧菌形态发生变化，可培养数降低，进入了VBNC状态。进一步发现，ToxR的缺失或在碱性饥饿条件下的水解会影响溶藻弧菌的胆盐耐受、运动性和毒力基因的表达，削弱溶藻弧菌毒力，促进其进入VBNC状态。对在VBNC诱导条件下溶藻弧菌进行RNA-seq和ChIP-seq分析表明，ToxR在VBNC诱导条件下直接控制8个基因的表达，包括与活性氧(ROS)抗性有关的ahpC和dps。凝胶迁移实验证明ToxR直接结合kdgR、ppiC、ahpC和dps的启动子区域，并在氧化应激条件下进一步控制它们各自的表达。ToxR缺失后的细胞会积累大量有害的细胞内活性氧。此外，这些基因有助于细菌保持可培养性，它们的框内缺失菌株显示出显著的可培养数下降，回补菌株则表现出可培养数回升。综上，本项目揭示了ToxR通过感应溶藻弧菌的内源性ROS (过氧化氢)等不利环境信号而诱导进入VBNC的分子机制，将为弧菌在海洋环境中的不同生活方式提供新的角度，有助于开发新的高效安全水产绿色防控方法。