Soft Metal, Disulfide, and Cysteine Stresses in Escherichia coli

描述（由申请人提供）：本研究的目的是探讨软金属、半胱氨酸进口和半胱氨酸积累干扰大肠杆菌适合度的机制。这三种不同的压力是由半胱氨酸化学的核心参与联系在一起的。软金属存在于许多栖息地，它们对细菌和高等生物都构成威胁。它们的毒性在整个生物群中分布着专门的解毒系统，这些系统结合并输出它们。其中一些金属——尤其是银和汞——被用作抗生素已有很长的历史。然而，我们对它们是如何损害细胞的知之甚少。在最近发表的工作中，我们证明了铜主要通过灭活铁/硫依赖性脱水酶来毒害大肠杆菌。它是通过结合协调这些酶的催化铁硫簇的半胱氨酸残基来实现的，从而取代了铁原子。初步数据表明，银、汞、镉和锌在体外也有这种效果。本研究（Aim 1）将测试这些软金属是否通过这一机制在体内发挥其毒性。它还将确定软金属是否会以类似的方式取代单核酶中的铁，单核酶使用的配位基团——通常包括半胱氨酸——更喜欢金属而不是铁。目标2侧重于一个单独的硫问题：当胱氨酸从有氧环境进口时，二硫化物应激的风险。二硫化物胁迫通常被认为是由活性氧引起的，但最近的数据不支持这一观点。然而，当细胞快速输入二硫化合物，如胱氨酸时，二硫应激是一个真正的风险。原则上，这样的事件似乎很可能将二硫键传播到细胞质蛋白质上。我们对高通量转运体的研究表明，胱氨酸的输入与还原有关，这种还原可以避免这种二硫化物释放到细胞质中。在目标2中，这个模型将被严格测试。目标3解决了快速输入胱氨酸的后果：半胱氨酸的过度积累，这本身就是有毒的。我们的数据显示大肠杆菌处理

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.