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扰乱大肠杆菌健康的机制。这三种不同的应激通过半胱氨酸化学的核心参与联系起来。软金属存在于许多栖息地，对细菌和高等生物都构成威胁。它们的毒性通过在整个生物群中分布的结合和输出它们的专用解毒系统来强调。其中一些金属，尤其是银和汞，有着悠久的用作抗生素的历史。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，我们证明铜主要通过灭活 Fe/S 依赖性脱水酶来毒害大肠杆菌。它通过结合协调这些酶的催化铁硫簇的半胱氨酸残基，从而取代铁原子来实现这一点。初步数据表明，银、汞、镉和锌在体外也有这种作用。本研究（目标 1）将测试这些软金属是否通过这种机制在体内发挥毒性。它还将确定软金属是否类似地从单核酶中取代铁，单核酶采用的配位基团（通常包括半胱氨酸）更喜欢金属而不是铁。目标 2 关注一个单独的硫问题：从有氧环境输入胱氨酸时产生二硫键应激的风险。传统上认为二硫化物应激是由活性氧引起的，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（例如胱氨酸）时，二硫键应激是一个真正的风险。原则上，这样的事件似乎可能会将二硫键传播到细胞质蛋白上。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，这种策略可以避免这种二硫键释放到细胞质中。在目标 2 中，该模型将接受严格的测试。目标 3 解决了胱氨酸快速输入的后果：半胱氨酸过度积累，其本身是有毒的。我们的数据显示，大肠杆菌处理 通过将半胱氨酸泵回细胞外来解决这个问题。这项调查将测试半胱氨酸毒性的三种可能机制，并将确定避免这种毒性的出口商。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引起的化学问题，以及细胞为保护自己免受半胱氨酸影响而获得的策略。所有这三种以硫为中心的压力——软金属暴露、二硫化物输入和半胱氨酸积累——都发生在自然栖息地可能存在的条件下。 公共健康相关性：软金属的杀菌作用长期以来一直被开发，从使用汞作为抗生素到用银涂覆导管。这项研究旨在阐明这些金属如何发挥其作用，这可能使工人能够改进他们的就业方式。我们还将研究大肠杆菌如何努力降低细胞内二硫键和游离半胱氨酸的浓度。这种控制的丧失包括一种氧化应激，据信这种氧化应激会导致多种人类病理。