Elucidating the Orchestrated Bacterial Response to Copper Toxicity

项目摘要 尽管有一个专门的铜出口系统，但自古以来铜就对细菌具有广泛的毒性。 铜毒性在实践中被观察到，其中铜表面和工具显著减少医院感染。 此外，在宿主介导的营养免疫期间（在轰击的同时螯合必需金属）， 细菌与有毒金属），先天免疫细胞杀死吞噬目标使用铜，这是严格管制 在主机内。然而，目前的研究是有限的，关于毒性的总体机制和 所使用的克服铜压力的途径与铜输出系统一起工作。通过这个 我们试图了解细菌是如何进化到与有毒金属相互作用并克服随之而来的 应力此外，我们寻求收集一组新的细菌治疗靶点。对于模型系统，我们 使用的是肺炎链球菌，这是一种全球性负担，疫苗仅覆盖了已知菌株的约25%。 到目前为止，使用微阵列数据，我们获得了比较野生型S。pneumoniae菌株TIGR4和铜 铜胁迫下的输出蛋白缺陷型菌株，以及各种技术，我们已经验证了几个 途径及其相应的机制。这些途径包括铜中毒核苷酸合成， 锌与铜对细菌具有协同毒性，糖的输入对铜的介导至关重要 毒性我们将把我们的研究重点放在铜阻遏蛋白CopY上，因为我们有数据表明它不仅 控制铜出口系统，但其他几个系统必须克服铜的压力。我们将 表征这些CopY控制的途径以及在结构上表征CopY，筛选CopY的小分子， 分子疗法，使其与DNA结合，并检查CopY与DNA和蛋白质的相互作用。我们 将阐述我们目前的发现，探索铜如何影响S的新途径。肺炎，并应用 我们的研究结果与其他有毒金属和细菌，希望阐明协调细菌对金属的反应， 应力

PROJECT SUMMARY Despite having a dedicated copper export system, copper has been broadly toxic to bacteria since antiquity. Copper toxicity is seen in practice where copper surfaces and tools significantly reduce nosocomial infections. Furthermore, during host mediated nutritional immunity (sequestering essential metals while bombarding bacterial with toxic metals), innate immune cells kill engulfed targets using copper, which is tightly regulated within the host. However, current research is limited regarding the overarching mechanisms of toxicity and the pathways used overcome copper stress that work in conjunction with the copper export system. Through this proposal, we seek to understand how bacteria evolved to interact with toxic metals and overcome the ensuing stress. Furthermore, we seek to gather a new cohort of bacterial therapeutic targets. For our model system, we are using Streptococcus pneumoniae, a global burden, for which the vaccine only covers ~25% of known strains. Thus far, using microarray data we obtained comparing the wild type S. pneumoniae strain TIGR4 and the copper export protein deficient strain under copper stress, and an assortment of techniques, we have validated several pathways and their corresponding mechanisms. These pathways include copper poisoning nucleotide synthesis, zinc being synergistically toxic to bacteria with copper, and sugar import being vital to mediation of copper toxicity. We will focus our studies around the copper repressor protein CopY as we have data showing it not only controls the copper export system but several other systems necessary to overcome copper stress. We will characterize these CopY-controlled pathways as well as structurally characterize CopY, screen CopY for small molecule therapeutics that keep it bound to DNA, and examine CopY’s interactions with DNA and proteins. We will expound upon our current findings, explore new avenues of how copper affects S. pneumoniae, and apply our findings to other toxic metals and bacteria in hopes to elucidate the orchestrated bacterial response to metal stress.