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

项目摘要/摘要： 超氧化物是一种有毒分子，所有暴露在氧气中的有机体都必须应对。这一点对于 病原微生物，作为宿主，利用超氧化物的毒性特性，通过 氧化爆发。为了使超氧化物解毒，几乎所有形式的生命，包括严格的厌氧菌，都会产生超氧化物。 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，它们都是 它们的功能依赖于金属。分布最广的sod家族是那些依赖于 对铁(Fe)或锰(Mn)的作用。铁/锰超家族成员存在于真核生物中， 古生菌和细菌。尽管进行了40多年的研究，但仍不可能准确地预测所使用的金属。 由铁/锰超家族的超家族成员。预测金属蛋白金属利用率的困难并不是 不仅限于铁/锰超家族，而且也存在于其他类别的金属酶中。这个不足之处是 由来自不同生物体的超氧化物歧化酶之间相对较低的蛋白质序列同一性推动的 同样的金属辅因子。此外，决定所用金属的环境和分子因素 这个蛋白质超家族的成员也是未知的。铁/锰超家族的成员是典型的 被认为使用铁或锰中的一种，但不能同时使用两者作为辅因子。这个想法是在早期的调查中出现的 确定了铁和锰都具有活性的Fe/Mn超氧化物歧化酶家族成员。这些“共生体”的能力 超氧化物歧化酶(能够利用铁或锰作为催化辅因子)被认为是化学上的怪癖。当时，它 有人认为，细胞内金属浓度的变化不足以改变被超氧化物歧化酶结合的金属。 金黄色葡萄球菌有两种超氧化物歧化酶，苏打和苏打，它们有75%的同源性。最初，两者都 据报道，超氧化物歧化酶是依赖于锰的。在感染期间，宿主限制了锰的可利用性并使其失活。 锰依赖超氧化物歧化酶通过锰结合免疫蛋白钙保护素。最近的研究发现，SodM 关键有助于金黄色葡萄球菌在缺锰时保持对氧化应激的防御能力， 无论是在培养中还是在感染期间，碳酸饮料都很重要，因为当锰是自由可得的时候。生化分析 揭示了SodM并不是严格依赖于锰，而是共生的，而利用铁的能力使其成为可能 当金黄色葡萄球菌受到宿主的锰限制时，提高对氧化应激的抵抗力。这些观察结果支持 裸子体的生理作用和金属可获得性塑造SOD曲目的假设 被有机体附体的。本提案中的实验将评估这一假设，并阐明 决定铁/锰超家族金属利用的分子特征。目的一：阐明分子 决定铁/锰超家族成员金属利用率的特征。目标二：确定环境是否 金属的可获得性促进了金黄色葡萄球菌对金属特异性和共生的超氧化物歧化酶的保留。目标三：澄清 共生生物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.