sRNA-regulated S-glutathionylation controls Vibrio cholerae virulence

项目摘要 霍乱弧菌是一种寄生于小肠的人类病原体，可导致严重的腹泻 被称为霍乱的疾病。一旦到达小肠，霍乱弧菌通过以下途径建立定植： AraC型主毒力调节因子ToxT的毒力基因激活。此外，霍乱弧菌必须 在感染期间科普许多宿主防御攻击，包括活性氧的氧化应激 （ROS）。小调节RNA（sRNA）是帮助细菌快速生长的关键转录后调节因子。 对刺激的反应。在我们的初步研究中，我们发现两种sRNA可以抑制grxA的表达， 编码谷氧还蛋白，其通过催化二硫键还原和蛋白质脱- 谷胱甘肽化S-谷胱甘肽化，巯基的可逆翻译后蛋白质修饰， 半胱氨酸与谷胱甘肽结合可以改变蛋白质功能和/或稳定性。我们还发现， 调节因子ToxT被谷胱甘肽化（ToxT-SSG），GrxA过量产生显著降低ToxT 谷胱甘肽化此外，grxA的失调降低了体外霍乱弧菌毒力基因的表达， 肠道定植在婴儿小鼠模型，表明sRNA介导的信号级联可能有助于 诉通过蛋白S-谷胱甘肽化的霍乱毒力。因此，我们假设宿主信号，如ROS， 诱导sRNA产生，而sRNA转录后抑制GrxA（一种去- 谷胱甘肽化酶，保护S-谷胱甘肽化毒力激活因子ToxT的活性库，从而 有助于霍乱弧菌发病机制的时空调节。为了验证这一假设，我们将首先 确定sRNA如何调节Aim 1中的grxA。我们将使用以下方法鉴定和表征grxA的sRNA调节剂： 候选方法和无偏系统方法rGRIL-Seq，其依赖于 sRNA与其靶标结合，选择性富集和测序。我们还将研究宿主信号， 激活sRNA进行grxA调节。在目标2中，我们将研究GrxA调节的S-谷胱甘肽化的影响， 关于霍乱弧菌毒力的研究我们的初步研究还发现，外源性谷胱甘肽（GSH）减少， 毒力基因表达GrxA使用GSH作为还原能力来使修饰的半胱氨酸脱谷胱甘肽。我们因此 假设谷胱甘肽化促进了ToxT功能，而霍乱弧菌利用肠内GSH作为空间结合， 时间线索来引导它的殖民。我们将研究谷胱甘肽化如何影响ToxT功能/稳定性。我们 还将使用靶向蛋白质组学研究GrxA对其他蛋白质谷胱甘肽化的影响。最后我们将 用成像质谱仪检测细胞内GSH对毒力的影响。拟议的研究将 揭示了霍乱弧菌氧化还原传感和调节中新的转录后sRNA信号传导，并揭示了 霍乱弧菌毒力控制中的翻译后蛋白S-谷胱甘肽化，扩大霍乱弧菌毒力 控制基因转录的能力。

PROJECT SUMMARY Vibrio cholerae is a human pathogen that colonizes small intestines, resulting in the onset of a severe diarrheal disease known as cholera. Once reaching the small intestine, V. cholerae establishes colonization through virulence gene activation by the AraC-type master virulence regulator ToxT. Additionally, V. cholerae must cope with many host defense attacks during infection, including oxidative stress from reactive oxygen species (ROS). Small regulatory RNAs (sRNAs) are key posttranscriptional regulators that assist bacteria for rapid responses to stimuli. In our preliminary studies, we found that two sRNAs can repress expression of grxA, encoding a glutaredoxin that responds to ROS stress by catalysis of disulfide bond reduction and protein de- glutathionylation. S-glutathionylation, a reversible posttranslational protein modification of thiol groups of cysteine with glutathione, can alter protein function and/or stability. We also found that the master virulence regulator ToxT was glutathionylated (ToxT-SSG) and GrxA overproduction significantly reduced ToxT glutathionylation. Moreover, dysregulation of grxA reduced V. cholerae virulence gene expression in vitro and gut colonization in the infant mouse model, suggesting sRNA-mediated signaling cascades may contribute to V. cholerae virulence via protein S-glutathionylation. We therefore hypothesize that host signals, such as ROS, induce sRNA production, and sRNAs posttranscriptionally inhibit production of GrxA, a protein de- glutathionylation enzyme, protect the active pool of the S-glutathionylated virulence activator ToxT, thus contributing to the spatio-temporal regulation of V. cholerae pathogenesis. To test this hypothesis, we will first determine how sRNAs regulate grxA in Aim 1. We will identify and characterize sRNA regulators of grxA using a candidate approach and an unbiased systems approach rGRIL-Seq that relies on in vivo proximity ligation of sRNAs bound to their targets, selective enrichment and sequencing. We will also investigate host signals that activate sRNAs for grxA regulation. In Aim 2, we will examine the effects of GrxA-regulated S-glutathionylation on V. cholerae virulence. Our preliminary studies also found that exogenous glutathione (GSH) reduced virulence gene expression. GrxA uses GSH as reducing power to deglutathionate modified cysteines. We thus hypothesize that glutathionylation promotes ToxT function and V. cholerae use lumenal GSH as a spatio- temporal cue to guide its colonization. We will examine how glutathionylation affects ToxT function/stability. We will also examine GrxA impacts on glutathionylation of other proteins using targeted proteomics. Finally, we will use imaging mass cytometry to examine effects of the lumenal GSH on virulence. The proposed studies will reveal new posttranscriptional sRNA signaling in V. cholerae redox sensing and regulation and uncover the posttranslational protein S-glutathionylation in V. cholerae virulence control, expanding V. cholerae virulence control beyond the extensively studied gene transcription.