Soft Metal, Disulfide, and Cysteine Stresses in Escherichia coli

大肠杆菌中的软金属、二硫化物和半胱氨酸应力

• 批准号: 8271819
• 负责人: JAMES A. IMLAY
• 金额: $ 24.76万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8271819
• 关键词: Address; Aerobic; Affinity; Antibiotics; Back; Bacteria; Binding; Biochemistry; Biological; Biota; Cadmium; Catheters; Cells; Chemicals; Chemistry; Complex; Coupled; Coupling; Cysteine; Cystine; Cytoplasm; Cytoplasmic Protein; DNA; Data; Disulfides; Drug Metabolic Detoxication; Ensure; Environment; Enzymes; Escherichia coli; Event; Face; Glutathione Disulfide; Goals; Grant; Habitats; Human Pathology; Hydro-Lyases; Hydroxyl Radical; Immune; In Vitro; Investigation; Iron; Knowledge; Link; Mercury; Metal exposure; Metals; Modeling; Mononuclear; Nature; Organism; Outcome; Oxidation-Reduction; Oxidative Stress; Physiological; Plant Roots; Production; Proteins; Publishing; Pump; Reactive Oxygen Species; Recording of previous events; Risk; Silver; Solvents; Stress; Sulfhydryl Compounds; Sulfides; Sulfur; System; Testing; Toxic effect; Work; Zinc; cell injury; cofactor; coping; copper poisoning; disulfide bond; disulfide compound; fitness; in vivo; interest; metal poisoning; research study; respiratory enzyme; stem

DESCRIPTION (provided by applicant): The goals of this study are to investigate the mechanisms by which soft metals, cystine import, and cysteine accumulation can perturb the fitness of Escherichia coli. These three distinct stresses are connected by the central involvement of cysteine chemistry. Soft metals are present in many habitats, where they comprise a threat to bacteria and higher organisms alike. Their toxicity is underscored by the distribution throughout the biota of dedicated detoxification systems that bind and export them. Some of these metals- notably silver and mercury-have a long history of being used as antibiotics. Nevertheless, we have little knowledge of how they actually damage cells. In recent published work we demonstrated that copper poisons E. coli primarily by inactivating Fe/S-dependent dehydratases. It does so by binding the cysteine residues that coordinate the catalytic iron-sulfur clusters of these enzymes, thereby displacing the iron atoms. Preliminary data demonstrate that silver, mercury, cadmium, and zinc have this effect in vitro, too. This study (Aim 1) will test whether these soft metals exert their toxicity through this mechanism in vivo. It will also determine whether soft metals similarly displace iron from mononuclear enzymes, which employ coordinating groups-often including cysteine-that prefer metals other than iron. Aim 2 focuses upon a separate sulfur problem: the risk of disulfide stress when cystine is imported from aerobic environments. Disulfide stress has conventionally been thought to arise from reactive oxygen species, but recent data do not support this idea. However, disulfide stress is a real risk when cells rapidly import disulfide compounds, such as cystine. In principle such an event would seem likely to propagate disulfide bonds to cytoplasmic proteins. Our study of the high-flux transporter suggests that cystine import is linked to reduction, a tacti that would avoid the release of this disulfide into the cytoplasm. In Aim 2 this model will be rigorously tested. Aim 3 addresses the consequence of rapid cystine import: the excessive accumulation of cysteine, which is toxic in its own right. Our data reveals that E. coli deals with this problem by pumping the cysteine back out of the cell. This investigation will test three plausible mechanisms of cysteine toxicity, and it will identify the exporter(s) that averts it. Collectively this work will illuminate chemical problems that arise from the redox activity and metal affinity of cysteine, as well as the strategies that cells have acquired to protect themselve from it. All three of these sulfur-focused stresses-soft-metal exposure, disulfide import, and cysteine accumulation-occur under conditions that are likely to exist in natural habitats. PUBLIC HEALTH RELEVANCE: The bacteriocidal actions of soft metals have long been exploited, from the administration of mercury as an antibiotic to the coating of catheters with silver. This study seeks to illuminate how these metals exert their effects-which may enable workers to refine how they are employed. We also will examine how E. coli strives to minimize the intracellular concentrations of both disulfide and free-cysteine species. The loss of this control comprises a type of oxidative stress that is believed to contribute to a wide variety of human pathologies.

描述（由申请人提供）：本研究的目的是研究软金属、胱氨酸输入和半胱氨酸积累干扰大肠杆菌适应性的机制。这三种不同的压力是由半胱氨酸化学的中心参与连接。软金属存在于许多栖息地，它们对细菌和高等生物构成威胁。它们的毒性突出表现在整个生物群中分布着专门的解毒系统，这些系统将它们结合并输出。其中一些金属--特别是银和汞--被用作抗生素已有很长的历史。然而，我们对它们实际上如何损害细胞知之甚少。在最近发表的工作中，我们证明了铜毒害E。大肠杆菌中主要通过灭活Fe/S依赖的脱氢酶。它通过结合半胱氨酸残基来实现这一点，半胱氨酸残基与这些酶的催化性铁硫簇合物相协调，从而取代铁原子。初步数据表明，银、汞、镉和锌在体外也有这种作用。本研究（目的1）将测试这些软金属是否通过这种机制在体内发挥其毒性。它还将确定软金属是否同样从单核酶中取代铁，单核酶使用配位基团（通常包括半胱氨酸），这些配位基团更喜欢铁以外的金属。目标2集中在一个单独的硫问题：当胱氨酸从有氧环境中输入时，二硫化物应激的风险。传统上认为二硫化物应力是由活性氧引起的，但最近的数据并不支持这一观点。然而，当细胞快速输入二硫化合物（如胱氨酸）时，二硫应激是一种真实的风险。原则上，这样的事件似乎可能将二硫键传播到细胞质蛋白质。我们对高通量转运蛋白的研究表明，胱氨酸的输入与还原有关，这是一种避免二硫化物释放到细胞质中的策略。在目标2中，将对这一模式进行严格检验。目标3解决了胱氨酸快速输入的后果：半胱氨酸的过度积累，这本身就是有毒的。我们的数据表明，E.大肠杆菌处理 通过将半胱氨酸泵出细胞来解决这个问题。这项研究将测试三种可能的半胱氨酸毒性机制，并将确定避免它的出口商。总的来说，这项工作将阐明半胱氨酸的氧化还原活性和金属亲和力所引起的化学问题，以及细胞已经获得的保护自己免受其害的策略。所有这三种以硫为重点的应激-软金属暴露，二硫化物输入，和半胱氨酸积累-发生在可能存在于自然栖息地的条件下。 公共卫生关系：软金属的杀菌作用早已被开发，从汞作为抗生素的管理到导管的银涂层。这项研究旨在阐明这些金属如何发挥其作用，这可能使工人完善他们如何就业。我们还将研究如何E。大肠杆菌努力使二硫化物和游离半胱氨酸种类的细胞内浓度最小化。这种控制的丧失包括一种类型的氧化应激，其被认为有助于多种人类病理学。