sRNA-regulated S-glutathionylation controls Vibrio cholerae virulence

sRNA 调节的 S-谷胱甘肽化控制霍乱弧菌毒力

• 批准号: 10648127
• 负责人: Jun Zhu
• 金额: $ 25.61万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-15 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10648127
• 关键词: Affect; Bacteria; Binding; Biochemical; Biological Assay; Catalysis; Cholera; Country; Cues; Cysteine; Cytometry; Disease; Enzymes; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Glutathione; Goals; Host Defense; Image; In Vitro; Infant; Infection; Ligation; Link; Mediating; Messenger RNA; Molecular; Molecular Chaperones; Oxidation-Reduction; Oxidative Stress; Pathogenesis; Post-Translational Protein Processing; Post-Translational Regulation; Poverty; Production; Protein S; Proteins; Proteomics; RNA; RNA Binding; Reactive Oxygen Species; Regulation; Repression; Signal Transduction; Small Intestines; Stimulus; Stress; Sulfhydryl Compounds; System; Testing; Transcriptional Regulation; Untranslated RNA; Vibrio cholerae; Vibrio cholerae infection; Virulence; antioxidant enzyme; biological adaptation to stress; coping; diarrheal disease; disulfide bond reduction; genetic approach; glutaredoxin; gut colonization; human pathogen; in vivo; mass spectrometric imaging; mouse model; novel; pathogenic bacteria; posttranscriptional; programs; protein function; response; spatiotemporal; tool; transcriptome; virulence gene

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.

项目总结 霍乱弧菌是一种人类病原体，它定植于小肠，导致严重腹泻。 被称为霍乱的疾病。霍乱弧菌一旦到达小肠，就会通过 毒力基因由AraC型毒力主调控子ToxT激活。此外，霍乱弧菌必须 应对感染期间的许多宿主防御攻击，包括来自活性氧物种的氧化应激 (ROS)。小调节RNA(SRNAs)是关键的转录后调节因子，帮助细菌快速 对刺激的反应。在我们的初步研究中，我们发现两个sRNA可以抑制grxA的表达， 编码一种谷氧还蛋白，通过催化二硫键还原和蛋白质去氧化来响应ROS胁迫。 谷胱甘肽基化。S-谷胱甘肽基化，一种可逆的硫醇基团的翻译后修饰 半胱氨酸和谷胱甘肽可以改变蛋白质的功能和/或稳定性。我们还发现，母体的毒力 调节剂ToxT是谷胱甘肽基化的(ToxT-SSG)，GrxA的过量生产显著降低了ToxT 谷胱甘肽基化。此外，grxA的失调降低了霍乱弧菌毒力基因的体外表达和 在幼鼠模型中的肠道定植，提示sRNA介导的信号级联可能有助于 霍乱弧菌通过蛋白质S-谷胱甘肽基化致病。因此我们假设宿主信号，如ROS， 诱导sRNA的产生，而sRNAs转录后抑制GrxA的产生，GrxA是一种去蛋白 谷胱甘肽基化酶，保护S谷胱甘肽基化毒力激活剂ToxT的活性池，从而 有助于霍乱弧菌致病的时空调控。为了检验这一假设，我们首先 在目标1中确定sRNA如何调控grxA。我们将使用以下方法识别和表征grxA的sRNA调控因子 依赖于体内邻近连接的候选方法和无偏系统方法rGRIL-Seq SRNA与其靶标结合，选择性浓缩和测序。我们还将调查主机信号 激活grxA调控的sRNA。在目标2中，我们将研究GrxA调节的S谷胱甘肽基化的作用 霍乱弧菌的致病力。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利用谷胱甘肽作为还原型，合成谷胱甘肽修饰的半胱氨酸。因此，我们 假设谷胱甘肽基化促进ToxT功能，霍乱弧菌以腔内GSH为空间调节因子. 引导其殖民的时间线索。我们将研究谷胱甘肽如何影响ToxT的功能/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基化的影响。最后，我们会 用图像质谱仪检测腔内GSH对毒力的影响。拟议的研究将 揭示霍乱弧菌氧化还原感知和调控中新的转录后sRNA信号，并揭示 翻译后蛋白S-谷胱甘肽在控制霍乱弧菌毒力、扩大霍乱弧菌毒力中的作用 控制超出了广泛研究的基因转录。