Membrane stress and ToxR-mediated virulence of Vibrio cholerae

革兰氏阴性菌霍乱弧菌是沿海水生和河口环境中的天然居民，是引起胃肠道疾病霍乱的病原体。霍乱弧菌的毒力基因调控是高度复杂的，主要调控成分ToxR/ToxS位于调控级联反应的顶端，负责控制毒力和管家/健康基因。尽管它很重要，但信号输入，特别是ToxS在这个主调控系统中的功能仍然未知。在这个项目中，我们将研究该系统的一种新的激活模式，因为我们发现膜结合的转录调节因子ToxR的活性受到一种蛋白酶(DEGS)的影响，该酶也是SigmaE膜应激反应通路的一部分。此外，ToxR的反应还受到另一个信号转导系统的影响，我们认为该系统是一个双组分调控系统，称为OsmRK。我们还观察到，受ToxR调控的孔蛋白ompU和ompT的表达明显不同，这取决于环境胁迫条件以及toxS、osmRK和degs的等位基因状态。首先，分析表明，这些调节作用是由ToxR介导的，ToxR可能被DEGS引起的蛋白水解性修饰激活，然后可能被未知的蛋白酶进一步降解。此外，ToxS似乎负责稳定ToxR，从而维持其在膜上的活性。因此，我们的数据表明，ToxR的活性是由一种新的、更复杂的膜内蛋白分解(RIP)变体控制的。这项拟议的项目将为这一刚刚出现的新的调控途径提供更多的实验证据，并将解决进一步的问题，如：(I)在接收激活信号时，ToxR内发生了什么变化或修改？(Ii)ToxR是否是副署长的直接目标？(Iii)OsmRK系统对毒素受体活性有何影响？最后，(Iv)DEGS和OsmRK识别的信号的性质是什么，这些系统是否在一条单一的路径上合作？预期的结果将有助于我们理解ToxR介导的调控，这不仅对霍乱弧菌的毒力基因调控和弧菌的其他关键细胞功能至关重要，还可能揭示RIP途径的新的调控潜力。

The Gram-negative bacterium Vibrio cholerae, which is a natural inhabitant of the coastal aquatic and estuarine environment, is the causative agent of the gastrointestinal disease cholera. Virulence gene regulation in V. cholerae is highly complex with the master regulator components ToxR/ToxS acting at the top of a regulation cascade, which is responsible for the control of virulence as well as housekeeping/fitness genes. Despite its importance, signal input and in particular the function of ToxS in this master regulatory system have remained unknown. In this project we will investigate a new mode of activation of this system, as we found that the activity of the membran-bound transcriptional regulator ToxR is influenced by a protease (DegS), which is also part of the sigmaE membrane-stress response pathway. In addition, the ToxR response is influenced by another signal transduction system, which we identified as a two-component regulatory system, termed OsmRK. We also observed, that the expression of the ToxR-regulated porins ompU and ompT varies significantly, depending on environmental stress conditions and the allelic status of toxS, osmRK and degS. First analyses indicate, that these regulatory effects are ToxR-mediated, and that ToxR may be activated by proteolytic modification caused by DegS, and may then be further degraded by yet unknown proteases. In addition, ToxS seems responsible for stabilizing ToxR and thereby maintaining its activity in the membrane. Thus, our data indicate that ToxR activity is conrtolled by a novel and more complex variant of regulated intramembrane proteolysis (RIP). The proposed project will provide additional experimental evidence for this just emerging new regulatory pathway and will address further questions such as: (i) what is changed or modified within ToxR while receiving activating signals? (ii) Is ToxR a direct target for DegS? (iii) How is ToxR activity influenced by the OsmRK system? And finally, (iv) what is the nature of the signals recognized by DegS and OsmRK and do these systems cooperate in a single pathway? The expected results will significantly contribute to our understanding of ToxR-mediated regulation, which is not only crucial for virulence gene regulation in V. cholerae and other pivotal cell functions in Vibrio spp., but may also reveal a novel regulatory potential of the RIP pathway.