Critical Roles for Iron and Copper Detoxification in Borrelia burgdorferi

描述（由申请人提供）：莱姆病是美国最常见的媒介传播疾病。了解病原体伯氏疏螺旋体在自然界中生存的分子机制将有助于推动疾病预防创新策略的发展。我们已经证明，由染色体基因bb 0690编码的疏螺旋体铁和铜结合蛋白A（BicA）的功能对于B是至关重要的。伯氏螺旋体在蜱和哺乳动物的感染性生命周期中存活。我们发现铁和铜存在于B中。在BicA缺陷型突变体和金属转运蛋白缺陷型突变体中，burgdorferi和它们的体内平衡被改变。到目前为止，所有支持或反对B中存在铁的证据。burgdorferi是基于体外实验，其中螺旋体在人工培养基中生长。因此，需要解决的一个关键问题是铁和铜是否在这种蜱传病原体的自然生命周期中发挥任何作用。这就是我们试图通过本提案中概述的实验来解决的问题。铁和铜是许多生物反应中重要的金属辅因子，但铁和铜由于其催化芬顿或类芬顿反应的能力而对细胞有毒。因此，利用铁或铜的生物体已经进化出许多策略来控制游离铁和铜的细胞内水平。金属结合蛋白如铁蛋白和金属硫蛋白代表了这样一种策略。我们的初步数据表明，（i）BicA是一种新的铁结合铁蛋白样分子和铜结合金属硫蛋白之间的融合;（ii）B。 缺乏BicA的burgdorferi具有降低的铁和铜水平，并且对铁和铜毒性更敏感;和（iii）B。Burgdorferi需要BicA才能在小鼠中完全感染，以及在蜱中持续存在并随后传播到新宿主。因此，我们假设BicA对铁和铜的解毒作用对B至关重要。burgdorferi在蜱和哺乳动物中的存活率。为了检验这一假设，我们提出证明（i）铁和铜是由天然BicA从B结合的金属。burgdorferi;（ii）BicA的铁和铜结合保护B。（iii）BicA的铁和铜结合活性对于B是重要的。伯氏螺旋体通过蜱和哺乳动物在其传染性生活周期中存活。这些研究的成功实施不仅将阐明B的分子机制。伯氏菌在自然界中持续存在，但也有助于促进我们对这种重要人类病原体的金属生物学的了解。

DESCRIPTION (provided by applicant): Lyme disease is the most common vector-borne disease in the United States. Understanding the molecular mechanisms by which Borrelia burgdorferi, the etiologic agent, survives in nature will help drive development of innovative strategies for disease prevention. We have demonstrated that the function(s) of Borrelia iron- and copper-binding protein A (BicA), which is encoded by chromosomal gene bb0690, is critically important for B. burgdorferi survival in an infectious life cycle through the tick and te mammal. We have discovered that iron and copper are present in B. burgdorferi and their homeostasis are altered in a mutant deficient in BicA and in a mutant deficient in a metal transporter. To date, all evidence either for or against the presence of iron in B. burgdorferi is based on in vitro experiments where the spirochetes were grown in artificial media. Therefore, a critical question to be addressed is whether iron and copper play any role in the natural life cycl of this tick-borne pathogen. This is the question we are seeking to address with the experiments outlined in this proposal. Iron and copper are important metal cofactors in many biological reactions, but iron and copper can be toxic to cells due to their abilities to catalyze the Fenton r Fenton-like reaction. Hence, organisms utilizing iron or copper have evolved many strategies to control intracellular levels of free iron and copper. Metal-binding proteins such as ferritins and metallothioneins represent one such strategy. Our preliminary data show that (i) BicA is a novel fusion between an iron-binding ferritin-like molecule and a copper-binding metallothionein; (ii) B. burgdorferi lacking BicA has reduced levels of iron and copper, and is more sensitive to iron and copper toxicity; and (iii) B. burgdorferi requires BicA for full infectivity in mice as well as persistence in the tick and subsequent transmission to a new host. Thus, we hypothesize that iron and copper detoxification by BicA is critical to B. burgdorferi survival in the tick and the mammal. To test this hypothesis, we propose to demonstrate that (i) iron and copper are the metals bound by native BicA from B. burgdorferi; (ii) iron- and copper-binding by BicA protects B. burgdorferi from iron and copper toxicity, respectively; (iii) the iron- and copper-binding activities of BicA are important for B. burgdorferi survival in its infectious life cycle through te tick and the mammal. Successful execution of these lines of investigation will not only elucidate molecular mechanisms underlying B. burgdorferi persistence in nature but also help advance our understanding of metal biology in this important human pathogen.