Copper as a nutrient for Candida albicans at the host-pathogen interface

 说明(申请人提供)：铜是一种高活性元素，基于其潜在的毒性，动物在传染病期间将铜的有害特性用于抗微生物武器。在动物宿主体内，细菌和真菌病原体都会受到有毒剂量的铜的攻击。然而，铜也是微生物必须从宿主获得的一种必需营养物质。目前，对宿主-病原菌竞争铜作为一种营养物质的了解很少。为了阐明铜在传染病中的生物学作用，这项研究建议将重点放在白色念珠菌上，这是人类最常见的真菌病原体。白念珠菌需要铜来维持细胞外超氧化物歧化酶(SOD)家族的活性，这些酶保护酵母免受宿主氧化猝发的影响，对毒力是重要的。我们的发现表明，这些铜超氧化物歧化酶在结构和功能上都是史无前例的，因为它们缺乏锌辅助因子，并且含有一个高度不规则的开放铜位，可以很容易地从宿主中捕获铜。我们发现白念珠菌胞内的铜-超氧化物歧化酶也是值得注意的，因为当酵母缺乏铜时，这种酶被非铜的替代品(Mn-SOD3)所取代。这种SOD酶的交换是对铜饥饿的大规模适应的一部分，令人惊讶的是，在体内白念珠菌入侵肾脏期间，这种铜应激反应变得明显。据我们所知，这是感染期间宿主限制铜的第一个证据。我们假设，宿主不仅可以攻击铜含量升高的病原体，还可以阻止入侵的微生物摄入这种营养物质，而白色念珠菌可以同时适应这两种情况。在这里，我们将使用白色念珠菌中的铜超氧化物歧化酶家族作为两个极端的宿主铜利用率的读数，即巨噬细胞的高铜和肾脏感染的低铜。在真菌与巨噬细胞氧化爆发的过程中，胞外铜超氧化物歧化酶变得尤为重要，我们认为白色念珠菌有效地利用巨噬细胞铜充电其超氧化物歧化酶来进行抗氧化防御。在目标1(定义白色念珠菌中不同的SOD5样蛋白的金属结合特性和功能)中，我们将胞外超氧化物歧化酶的铜结合能力作为这些酶在宿主-病原体界面保持活性的预测指标。此外，我们还将探索细胞外超氧化物歧化酶家族中一个未知成员，即SOD6的功能，作为一个潜在的真菌发病机制中需要铜的新实体。在目标2(了解白色念珠菌在巨噬细胞感染过程中利用宿主铜作为营养物质)中，我们检测了巨噬细胞感染模型中细胞外真菌超氧化物歧化酶的激活，并确定了巨噬细胞铜最终被输送到真菌超氧化物歧化酶活性部位的途径。最后，目标3(了解宿主-病原体界面的真菌铜饥饿反应)将探索感染的肾脏模型，并确定宿主阻止白色念珠菌产生铜的机制，这是第一个有文献记载的涉及铜的营养免疫反应。总而言之，这些研究有望为铜作为一种营养物质在真菌发病中的意义提供新的见解。

 DESCRIPTION (provided by applicant): Copper is a highly reactive element and based on its potential toxicity, animals use the deleterious properties of Cu in anti-microbial weaponry during infectious disease. Both bacterial and fungal pathogens are attacked with toxic doses of Cu inside the animal host. However, Cu is also an essential nutrient that microbes must acquire from the host. Currently, little is understood regarding host-pathogen competitions for Cu as a nutrient. To shed light into the biology of Cu during infectious disease, this research proposal focuses on Candida albicans, the most prevalent of human fungal pathogens. C. albicans requires Cu to maintain activity of a family of extracellular superoxide dismutase (SOD) enzymes that protect the yeast from the host oxidative burst and are important for virulence. Our findings show these Cu-SODs are unprecedented in structure and function in that they lack a Zn co-factor and contain a highly irregular open Cu site that may easily capture Cu from the host. We find the intracellular Cu-SOD of C. albicans is also remarkable in that this enzyme is replaced with a non-Cu alternative (Mn-SOD3) when the yeast is starved for Cu. This swapping of SOD enzymes is part of a large adaptation to Cu starvation, and surprisingly this Cu stress response becomes evident during C. albicans invasion of the kidney in vivo. To our knowledge this is the first documented evidence for host limitation of Cu during infection. We hypothesize that the host not only attacks pathogens with elevated Cu, but can also withhold this nutrient from invading microbes and C. albicans can adapt to both. Here we shall use the family of Cu SODs in C. albicans as a read-out for fungal Cu utilization during two extremes of host Cu availability, namely the high Cu of macrophages and the low Cu of kidney infection. The extracellular Cu-SODs become particularly important during fungal encounters with the oxidative burst of macrophages and we propose that C. albicans efficiently utilizes macrophage Cu to charge its SODs for an anti-oxidant defense. In Aim 1 (To define the metal binding properties and function of diverse SOD5-like proteins in C. albicans) we address the Cu binding capacities of the extracellular SODs as predictive indicators of how well these enzymes can maintain activity at the host-pathogen interface. Additionally we will explore the function of an unknown member of the extracellular SOD family, namely SOD6, as a potentially new Cu- requiring entity for fungal pathogenesis. In Aim 2 (To understand C. albicans utilization of host copper as a nutrient during macrophage infection), we examine activation of the extracellular fungal SODs in a macrophage infection model and define the pathway by which macrophage Cu is ultimately delivered to the active site of the fungal SOD enzymes. Lastly, Aim 3 (To understand the fungal Cu starvation response at the host-pathogen interface) will explore the kidney model of infection and define the mechanism whereby the host withholds Cu from C. albicans as the first documented nutritional immunity response involving Cu. Collectively, these studies promise to provide new insight into the implications for Cu as a nutrient for fungal pathogenesis.