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创新预防策略的发展。我们已经证明，由bb0690编码的伯氏疏螺旋体铁铜结合蛋白A （BicA）的功能对伯氏疏螺旋体通过蜱虫和哺乳动物在感染生命周期中的生存至关重要。我们已经发现，铁和铜是伯氏疏螺旋体的主要过渡金属，并且在BicA突变体缺乏和已知金属转运体突变体缺乏时，这些金属的动态平衡发生了改变。这些数据挑战了该领域目前的教条——铁在莱姆病病原体中缺乏作用。迄今为止，支持或反对伯氏疏螺旋体中存在铁的所有证据都是基于在人工培养基中培养螺旋体的体外实验。因此，一个需要解决的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建议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助因子，但由于游离铁和铜具有催化芬顿反应的能力，因此对细胞具有毒性。因此，利用铁或铜的生物体已经进化出许多策略来控制细胞内游离铁和铜的水平。铁蛋白和金属硫蛋白等金属结合蛋白代表了一种这样的策略。我们的初步数据表明：(i) BicA是铁结合铁蛋白类分子和铜结合金属硫蛋白之间的新型融合；（ii）缺乏BicA的伯氏疏螺旋体铁和铜含量降低，在含铁或铜的培养基中表现出生长缺陷；（iii）伯氏疏螺旋体需要BicA

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 prevention. We have demonstrated that the function(s) of Borrelia iron- and copper-binding protein A (BicA), encoded by bb0690, is critically important for B. burgdorferi survival in an infectious life cycle through the tick and the mammal. We have discovered that iron and copper are among major transition metals in B. burgdorferi and the homeostasis of these metals is altered in a mutant deficient in BicA and in a mutant deficient in a known metal transporter. These data challenge the current dogma in the field - lack of a role for iron in the Lyme disease pathogen. 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 cycle 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 free iron and copper are toxic to cells due to their abilities to catalyze the Fenton 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 exhibits a growth defect in media containing iron or copper; and (iii) B. burgdorferi requires BicA for 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 he mammal. To test this hypothesis, we propose to demonstrate that (i) iron and copper are the metals bound by native BicA in B. burgdorferi; (ii) iron- and copper-binding by BicA protects B. burgdorferi against iron and copper detoxification, respectively; (iii) the iron- and copper-bindin activities of BicA are important for B. burgdorferi survival during its infectious life cycle throuh the tick and the mammal. Successful execution of these lines of investigation will not only elucidate the molecular mechanisms underlying B. burgdorferi persistence in nature but also bring about a paradigm change in our understanding of the Lyme disease spirochete.