Thiol-based switches in Vibrio cholerae pathogenesis

描述(申请人提供)：革兰氏阴性菌霍乱弧菌是霍乱的病原体，是一种兼性病原体，存在于人类和水环境中。广泛的体外研究已经确定了在感染过程中产生疾病所需的一些毒力因子。然而，霍乱弧菌在从海洋生态系统转移到人类宿主以及感染进展过程中如何改变其基因表达在很大程度上尚不清楚。我们发现转录因子AphB具有半胱氨酸残基，该残基可以根据肠道的微氧条件进行修饰，导致毒力的激活。我们现在有进一步的证据表明，AphB中不同的半胱氨酸修饰通过基因转录和蛋白质稳定性的变化对细胞生理产生不同的影响。我们推测AphB是霍乱弧菌感染相关环境信息的中央处理器。具体地说，我们假设AphB使用硫醇修饰来将还原、氧化和亚硝化反应物的存在整合到引导有机体进出人类肠道所必需的基因表达决定中。我们将使用生化和遗传技术的组合来机械地定义AphB如何协调霍乱弧菌的致病生命周期，以及在这个基于半胱氨酸的感觉中枢还涉及哪些其他因素。我们相信，通过全面定义AphB监测霍乱弧菌化学微环境的方式，我们可能能够将基于半胱氨酸的环境传感范例扩展到其他霍乱弧菌蛋白和其他病原体。我们将研究AphB如何被活性氮物种(RNS)修饰，如一氧化氮(NO)，以及体内产生的活性氧物种(ROS)的氧化应激，以及这些修饰如何影响AphB的功能。我们将研究AphB修饰对细胞生理的广泛影响，重点关注ROS/RNS应激管理，记住以下模型：当进入肠道时，降低的AphB激活毒力以响应低氧压；作为对肠道中增加的化学应激的响应，修饰的AphB失活毒力，同时上调ROS和RNS的解毒，从而为细胞在水环境中的生存做好准备。

DESCRIPTION (provided by applicant): The Gram-negative bacterium Vibrio cholerae, the causative agent of cholera, is a facultative pathogen that resides in both human and aquatic environments. Extensive in vitro studies have identified a number of virulence factors required to produce disease during infection. However, how V. cholerae alters its gene expression upon transition from its marine ecosystem to the human host and along progression of infection is largely unknown. We have discovered that the transcription factor AphB possesses a cysteine residue that undergoes modification in response to the microoxic conditions of the intestines, leading to activation of virulence. We now have further evidence that diverse cysteine modifications in AphB lead to diverse effects on cell physiology through changes in gene transcription and protein stability. We hypothesize that AphB is the central processor of environmental information relevant to infection for V. cholerae. Specifically, we hypothesize that AphB uses thiol modifications to integrate the presence of reductive, oxidative and nitrosative reactants into the gene expression decisions necessary to guide the organism in and out of the human gut. We will use a mix of biochemical and genetic techniques to define mechanistically how AphB orchestrates the pathogenic life cycle of V. cholerae and what other factors are involved at this cysteine-based sensory hub. We believe that by comprehensively defining the way AphB monitors the chemical microenvironment for V. cholerae, we then may be able to extend the paradigm of cysteine-based environmental sensing to other V. cholerae proteins and other pathogens. We will examine how AphB is modified by reactive nitrogen species (RNS), such as nitric oxide (NO), and oxidative stress from reactive oxygen species (ROS) generated in vivo and how these modifications affect AphB functionality. We will study the broad effects of AphB modification on cell physiology, focusing on ROS/RNS stress management with the following model in mind: upon entry to the gut, reduced AphB activates virulence in response to low oxygen tension; in response to increasing chemical stress in the gut, modified AphB deactivates virulence while up-regulating ROS and RNS detoxification, thus preparing the cell for survival in the aquatic environment.