Leveraging host-imposed metal starvation to elucidate the molecular and environmental factors that dictate metal utilization by the iron/manganese superoxide dismutase superfamily

项目概要/摘要： 超氧化物是一种有毒分子，所有接触氧气的生物都必须科普。尤其如此 病原微生物，作为宿主，利用超氧化物的毒性，通过 氧化爆发为了解毒超氧化物，几乎所有形式的生命，包括严格厌氧菌，产生超氧化物 歧化酶（SOD）。趋同进化导致了三个独立的SOD家族的发展， 其功能依赖于金属。分布最广泛的SOD家族是那些依赖于 铁（Fe）或锰（Mn）的功能。Fe/Mn超家族的成员存在于真核生物中， 古细菌和细菌。尽管经过四十多年的研究，仍无法准确预测所使用的金属 Fe/Mn超家族的成员。预测金属蛋白金属利用率的困难并不在于 仅限于Fe/Mn SOD超家族，但也存在于其他种类的金属酶中。这一缺陷是 由来自不同生物体的SOD之间相对低水平的蛋白质序列同一性驱动， 相同的金属辅因子。此外，环境和分子因素决定了金属的使用， 该蛋白质超家族的成员也是未知的。Fe/Mn SOD超家族的成员是典型的 被认为使用Fe或Mn，但不是两者，作为辅因子。尽管早期的调查表明， 鉴定了Fe/Mn SOD家族成员对Fe和Mn都有活性。这些“变形”的能力 SOD（能够使用Fe或Mn作为催化辅因子）被认为是化学的怪癖。当时它 认为细胞内金属浓度的变化不足以改变SOD结合的金属。 S.金黄色葡萄球菌具有两种超氧化物歧化酶，SodA和SodM，它们约75%相同。起初，双方 据报道，SOD具有Mn依赖性。在感染过程中，宿主限制Mn的可用性， 锰依赖性SOD通过锰结合免疫蛋白钙卫蛋白。最近的研究发现， 关键是有助于S的能力。金黄色葡萄球菌在Mn饥饿时维持对氧化应激的防御， 无论是在文化和感染过程中，而苏打是重要的锰是免费的。生物化学分析 揭示了SodM不是严格的Mn依赖性，而是形成性的，并且利用Fe的能力使其 促进抗氧化应激时，S.金黄色葡萄球菌是Mn限制的主机。这些观察结果支持 形成论的生理作用和金属可用性塑造SOD库的假设 被一个有机体所拥有。本提案中的实验将评估这一假设，并阐明 分子特征决定了Fe/Mn SOD超家族中的金属利用率。目的一：阐明 铁/锰超氧化物歧化酶超家族成员的金属利用的特点。目标二：确定是否环境 金属有效性促进了金属特异性和形成性SOD的保留。金黄色。目标三：阐明 形成层SOD对维持对超氧化物的防御的更广泛的贡献。

Project Summary/Abstract: Superoxide is a toxic molecule that all organisms exposed to oxygen must cope with. This is particularly true for pathogenic microbes, as the host harnesses the toxic properties of superoxide to combat invaders via the oxidative burst. To detoxify superoxide, nearly all forms of life, including strict anaerobes, produce superoxide dismutase (SOD). Convergent evolution has led to the development of three independent SOD families, all of which are dependent on metals for function. The most widely distributed family of SODs are those which depend on iron (Fe) or manganese (Mn) for function. Members of the Fe/Mn superfamily are present in eukaryotes, archaea, and bacteria. Despite over forty years of study, it is not possible to predict accurately the metal utilized by members of the Fe/Mn superfamily of SODs. Difficulties in predicting metalloprotein metal utilization are not confined to the Fe/Mn SOD superfamily but also occur with other classes of metalloenzymes. This deficiency is driven by relatively low levels of protein sequence identity amongst SODs from different organisms that utilize the same metal cofactor. Additionally, the environmental and molecular factors that dictate the metal used by members of this protein superfamily are also unknown. Members of the Fe/Mn SOD superfamily are canonically thought to use either Fe or Mn, but not both, as a cofactor. This idea arose despite early investigations that identified Fe/Mn SOD family members that are active with both Fe and Mn. The ability of these “cambialistic” SODs (able to use either Fe or Mn as a catalytic cofactor) was dismissed as a quirk of chemistry. At the time, it was thought that intracellular metal concentrations did not change enough to alter the metal bound by a SOD. S. aureus possesses two superoxide dismutases, SodA and SodM, which are ~75% identical. Initially, both SODs were reported to be Mn-dependent. During infection, the host restricts the availability of Mn and inactivates Mn-dependent SODs via the Mn-binding immune protein calprotectin. Recent work discovered that SodM critically contributes to the ability of S. aureus to maintain a defense against oxidative stress when Mn-starved, both in culture and during infection, while SodA is important when Mn is freely available. Biochemical analyses revealed that SodM is not strictly Mn-dependent but is instead cambialistic, and the ability to use Fe enables it to promote resistance to oxidative stress when S. aureus is Mn-limited by the host. These observations support a physiological role for cambialism and the hypothesis that metal availability shapes the repertoire of SODs possessed by an organism. The experiments in this proposal will evaluate this hypothesis and elucidate the molecular features that dictate metal utilization in the Fe/Mn SOD superfamily. Aim I: Elucidate the molecular features that dictate metal utilization of Fe/Mn SOD superfamily members. Aim II: Determine if environmental metal availability promotes retention of a metal-specific and cambialistic SOD by S. aureus. Aim III: Elucidate the broader contribution of cambialistic SODs to maintaining a defense against superoxide.