Redox-regulation in Gram-negative Bacteria

革兰氏阴性细菌中的氧化还原调节

• 批准号: 10292137
• 负责人: Jan-Ulrik Dahl
• 金额: $ 44.38万
• 依托单位: ILLINOIS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10292137
• 关键词: Affect; Amino Acids; Antibiotics; Antimicrobial Resistance; Antioxidants; Bacteria; Bacterial Physiology; Biochemical; Biology; Bladder; Catheters; Cell physiology; Cells; Chlorine; Cysteine; Data; Disease Progression; Enzymes; Epithelial; Escherichia coli; Exposure to; Gene Cluster; Gene Expression; Genes; Genetic Transcription; Goals; Gram-Negative Bacteria; Growth; Growth and Development function; Health; Host Defense; Household; Human; Hypochlorous Acid; Immune; In Vitro; Infection; Inflammation; Intestines; Life Style; Mediating; Microbial Biofilms; Mucous Membrane; Neutrophil Infiltration; Operon; Outcome; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxygen; Phagocytosis; Phenotype; Physiological; Play; Post-Translational Protein Processing; Process; Production; Proteins; Regulation; Regulon; Repression; Resistance; Role; Side; Signal Transduction; Stress; Surface; System; Testing; Time; Toxic effect; Transcription Repressor; Up-Regulation; Urinary tract; Urinary tract infection; Uropathogenic E. coli; acid stress; antimicrobial; biological adaptation to stress; cell growth; commensal bacteria; diguanylate cyclase; enteropathogenic Escherichia coli; experience; fighting; human disease; improved; in vivo; insight; mutant; neutrophil; novel; novel therapeutics; oxidation; pathogen; pathogenic bacteria; repaired; resistance gene; response; stressor; surface coating; transcriptomics; transposon sequencing

PROJECT SUMMARY Bacteria frequently encounter a variety of harmful stressors including reactive oxygen and chlorine species (RO/CS). One particularly potent antimicrobial oxidant is hypochlorous acid (HOCl), which is generated during neutrophil-mediated phagocytosis and by enzymes of the mucosal epithelia to control bacterial colonization. Bacteria have evolved strategies to counteract and reduce the harmful effects of RO/CS, e.g. by upregulating the expression of stress-specific genes. RO/CS-induced changes in gene expression are typically mediated by posttranslational modifications of redox-sensitive amino acid side chains in transcriptional regulators, which affect their ability to activate/repress the expression of the corresponding stress-protective target genes. Our preliminary data show that uropathogenic E. coli (UPEC) respond to sublethal HOCl-stress with the upregulation of an operon harboring three uncharacterized UPEC-specific genes. We identified one of them as a HOCl- sensitive transcriptional repressor that reversibly loses its repressor activity during HOCl-stress. Its inactivation results in the de-repression of the two downstream targets contributing to the increased HOCl resistance of UPEC strains compared to commensal and enteropathogenic E. coli. Moreover, our preliminary data show that sublethal HOCl concentrations cause the induction of numerous biofilm genes and stimulate biofilm formation. The overall goal of this project is to comprehensively understand the cause and effects of RO/CS stress and to characterize specific bacterial defense strategies to RO/CS used to mitigate their damage. Our working hypothesis is that RO/CS specifically and reversibly modify surface-exposed cysteine residues in proteins, which become the key factor in redox signaling by affecting gene expression and/or bacterial physiology. In Aim 1, we will use phenotypic and biochemical strategies to assess the mechanism by which the transcriptional repressor is inactivated in vitro and in vivo. Moreover, we will elucidate the function of one of its downstream targets, which appears to contribute substantially to UPEC’s increased HOCl resistance and identify additional HOCl resistance genes by TnSeq. To characterize the benefits of HOCl-mediated biofilm stimulation, we will analyze the composition of CFT073 biofilms before and after exposure to sublethal HOCl and test whether biofilm cells become more resistant to subsequent HOCl stress or common antibiotics used to treat UTIs (Aim 2). In addition, we will investigate how HOCl stress acts to trigger the increase in biofilm formation in CFT073 by determining the role that the diguanylate cyclase YdeH plays in this process. In Aim 3, we will combine transcriptomic and phenotypic analyses to identify novel regulons contributing to UPEC’s resistance to the surface antimicrobial AgXX, which has previously been shown to generate RO/CS. These studies will provide us with fundamentally new insights into the role that RO/CS play in UPEC. Identifying, characterizing and targeting UPEC-specific defense systems has the potential to increase the body’s own capacity to fight UTIs.

项目摘要 细菌经常遇到各种有害的应激源，包括活性氧和氯 (RO/CS）。一种特别有效的抗微生物氧化剂是次氯酸（HOCl），其在生产过程中产生。 嗜酸性粒细胞介导的吞噬作用和粘膜上皮的酶来控制细菌定植。 细菌已经进化出抵消和减少RO/CS的有害影响的策略，例如通过上调 胁迫特异性基因的表达。RO/CS诱导的基因表达变化通常由 转录调节因子中氧化还原敏感性氨基酸侧链的翻译后修饰， 影响它们激活/抑制相应应激保护靶基因表达的能力。我们 初步数据显示，尿路致病性E.大肠杆菌（UPEC）对亚致死HOCl-胁迫的反应是上调 一个操纵子携带三个未知的UPEC特异性基因。我们确认了其中一个是- 敏感的转录阻遏物，在HOCl-胁迫期间可逆地失去其阻遏物活性。其失活 导致两个下游靶点的去抑制，这两个靶点有助于增加HOCl抗性， UPEC菌株与大肠杆菌和肠致病性E.杆菌此外，我们的初步数据显示， 亚致死HOCl浓度引起许多生物膜基因的诱导并刺激生物膜形成。 本项目的总体目标是全面了解RO/CS应力的原因和影响， 表征用于减轻其损害的RO/CS的特定细菌防御策略。我们的工作 假设RO/CS特异性地可逆地修饰蛋白质中暴露于表面半胱氨酸残基， 通过影响基因表达和/或细菌生理学而成为氧化还原信号传导中的关键因子。目标1： 将使用表型和生化策略来评估转录抑制因子 在体外和体内都是失活的。此外，我们将阐明其下游靶点之一的功能， 似乎对UPEC增加的HOCl抗性有实质性贡献，并确定了额外的HOCl抗性 基因TnSeq为了表征HOCl介导的生物膜刺激的益处，我们将分析 在暴露于亚致死HOCl之前和之后的CFT 073生物膜的组成，并测试生物膜细胞是否 变得对随后的HOCl应激或用于治疗UTI的常用抗生素更具抗性（目的2）。此外，本发明还提供了一种方法， 我们将研究HOCl应激如何触发CFT 073中生物膜形成的增加， 二鸟苷酸环化酶YdeH在这一过程中所起的作用。在目标3中，我们将联合收割机转录组学和 表型分析，以确定有助于UPEC对表面抗菌剂耐药性的新型调节子 AgXX，其先前已显示产生RO/CS。这些研究将为我们提供 对RO/CS在UPEC中发挥的作用的新见解。鉴定、表征和靶向UPEC特异性 防御系统有可能增加身体自身对抗UTI的能力。

项目成果

Redox-Mediated Inactivation of the Transcriptional Repressor RcrR is Responsible for Uropathogenic Escherichia coli's Increased Resistance to Reactive Chlorine Species.

• DOI: 10.1128/mbio.01926-22
• 发表时间: 2022-10-26
• 期刊: mBio
• 影响因子: 6.4

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor.

• DOI: 10.3791/62628
• 发表时间: 2021-06-29
• 期刊: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
• 影响因子: 1.2
• 作者: Sultana, Sadia;Anderson, Greg M.;Hoffmann, Kevin Pierre;Dahl, Jan-Ulrik
• 通讯作者: Dahl, Jan-Ulrik

A novel ruthenium-silver based antimicrobial potentiates aminoglycoside activity against Pseudomonas aeruginosa.

• DOI: 10.1128/msphere.00190-23
• 发表时间: 2023-10-24
• 期刊: MSPHERE
• 影响因子: 4.8
• 作者: Donkor, Gracious Yoofi;Anderson, Greg M.;Stadler, Michael;Tawiah, Patrick Ofori;Orellano, Carl D.;Edwards, Kevin A.;Dahl, Jan-Ulrik
• 通讯作者: Dahl, Jan-Ulrik

A Year at the Forefront of Bacterial Defense Systems Against Neutrophilic Oxidants.

• DOI: 10.1242/bio.059809
• 发表时间: 2023-04-15
• 期刊: Biology open
• 影响因子: 2.4