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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要 Vibrio霍乱是一种人类病原体，可在小肠道上定位，导致严重的腹泻发作疾病称为霍乱。一旦到达小肠，V。Cholerae通过 ARAC型主要毒力调节剂TOXT激活毒力基因。此外，V。Cholerae必须应对感染过程中许多宿主防御攻击，包括活性氧的氧化应激（ROS）。小调节RNA（SRNA）是转录后调节剂，可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，编码通过二硫键还原的催化和蛋白质的催化来应对ROS应激的谷氨酸毒素谷胱甘肽化。 S-谷胱甘肽，一种可逆的翻译后蛋白质修饰与谷胱甘肽的半胱氨酸可以改变蛋白质功能和/或稳定性。我们还发现主要的毒力调节剂TOXT是谷胱甘肽（TOXT-SSG）和GRXA过度生产显着降低TOXT 谷胱甘肽化。此外，GRXA的失调降低了V.霍乱的毒力基因在体外和婴儿小鼠模型中的肠道定植，表明sRNA介导的信号传导级联反应可能有助于 V.霍乱毒力通过蛋白S-谷胱甘肽化。因此，我们假设宿主信号（例如ROS）诱导SRNA的产生，并在转录后抑制GRXA的产生，蛋白质的产生谷胱甘肽酶，保护s-谷胱甘肽的毒力激活剂的活性库，因此促进霍乱发病机理的时空调节。为了检验这一假设，我们将首先确定SRNA如何调节AIM 1中的GRXA。我们将使用GRXA的SRNA调节剂来识别和表征GRXA的SRNA调节剂一种候选方法和公正的系统方法RGRIL-seq，依赖于体内接近的结扎 SRNA结合到目标，选择性富集和测序。我们还将调查主机信号激活用于GRXA调控的SRNA。在AIM 2中，我们将检查GRXA调节的S-谷胱甘肽的影响在V.霍乱毒力上。我们的初步研究还发现，外源性谷胱甘肽（GSH）降低了毒力基因表达。 GRXA使用GSH用作脱脂酸酯修饰的半胱氨酸的降低功率。我们这样假设谷胱甘肽化促进了TOXT功能，而V.霍乱将Lumenal GSH用作时空时间提示指导其殖民化。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检查GRXA对其他蛋白质谷胱甘肽的影响。最后，我们会的使用成像质量细胞仪检查腔GSH对毒力的影响。拟议的研究将在V.霍乱氧化杆菌感应和调节中揭示新的转录后SRNA信号传导，并发现翻译后蛋白S-谷胱甘肽在V.霍乱毒力控制中，扩大霍乱毒素的膨胀超越广泛研究的基因转录。