The novel family of superoxide dismutase enzymes in Candida albicans

描述(由申请人提供)：具有传染性的白色念珠菌仍然是公共卫生中的一个重要问题，特别是对免疫受损的人。一组对白色念珠菌的毒力因子包括一种含铜的超氧化物歧化酶(SOD)酶家族，该酶能清除有毒的超氧自由基。尽管它们在毒力方面具有公认的重要性，但几乎对白色念珠菌Sod的生物化学或这些酶是如何通过插入铜辅助因子在细胞中被激活的知之甚少。在这项研究应用中，我们计划开发新的系统来监测宿主对白念珠菌胞内铜和含铜超氧化物歧化酶的影响。白念珠菌中含铜超氧化物歧化酶的两种类型将被检测：一种是胞浆中的铜/锌超氧化物歧化酶(SOD1)，另一种是通过GPI锚连接到细胞壁的铜/锌超氧化物歧化酶家族的胞外成员(SOD5)。两者对毒力都很重要，在我们的初步分析中，两者都显示出一些其他铜/锌SOD中没有的独特特征。例如，在正常(非传染性)实验室生长条件下，细胞内的SOD1蛋白是不活跃的，酵母转而使用一种不寻常的胞质锰-SOD3来清除超氧化物。胞外SOD5也是独一无二的，因为它似乎只与铜结合，并且缺乏不变的锌位点 其他真核生物铜锌超氧化物歧化。我们认为，这些不寻常的铜氧化物是为了利用金属上的宿主效应而进化出来的。在感染过程中，巨噬细胞吞噬酶体使病原体缺乏锰、锌和铁，同时试图杀死含有高水平铜和活性氧的病原体。主机的这种特殊环境似乎是激活需要铜的SOD的理想环境。我们推测，在感染期间，细胞内和细胞外的SOD5都立即被铜充电，代表着对抗宿主氧化爆发的第一道防线。我们将开始通过开发系统来检验这一假设，在该系统中，我们可以监测白念珠菌在感染巨噬细胞期间细胞内铜和铜/锌超氧化物歧化酶活性的变化。我们还将开发一种系统，用于从细胞壁或酵母分泌的酶活性SOD5中回收SOD5，这将被证明是监测巨噬细胞感染期间细胞外SOD活性的宝贵工具。通过对重组SOD5的金属分析，我们将确定该酶是否真的是只含铜的超氧化物歧化酶。这些研究还处于探索、分析和开发阶段。这个两年计划的发现和新系统将促进我们更长期的机制研究，以了解宿主对病原体金属和SOD酶的影响。这些基础研究有可能催生针对铜的新疗法的开发，并防止对毒力至关重要的白色念珠菌SOD的激活。

DESCRIPTION (provided by applicant): The infectious yeast Candida albicans remains an important concern in public health, particularly with immune-compromised individuals. One set of virulence factors for C. albicans includes a family of copper containing superoxide dismutase (SOD) enzymes that scavenge toxic superoxide free radicals. In spite of their established importance in virulence, virtually nothing is known about the biochemistry of C. albicans SODs or how these enzymes are activated in cells through insertion of the copper co-factor. In this research application, we plan to develop new systems for monitoring the effects of the host on intracellular copper and copper containing SODs of C. albicans . Two types of Cu-containing SODs in C. albicans will be examined: a Cu/Zn SOD in the cytosol (SOD1), and an extracellular member of the Cu/Zn SOD family attached to the cell wall through GPI anchors (SOD5). Both are important for virulence and in our preliminary analyses, both exhibit some distinctive features not seen in other Cu/Zn SODs. For example, under normal (non-infectious) laboratory growth conditions, the intracellular SOD1 protein is inactive and the yeast instead uses an unusual cytosolic manganese-SOD3 to remove superoxide. The extracellular SOD5 is also unique in that it appears to bind only copper, and is lacking the zinc site that is invariant among other eukaryotic Cu/Zn SODs. We propose that these unusual Cu-SODs have evolved to exploit the host effects on metals. During infection, the macrophage phagolysosome starves pathogens of manganese, zinc and iron, while attempting to kill pathogens with high levels of copper and reactive oxygen. This particular environment of the host seems ideal for activating copper requiring SODs. We hypothesize that during infection, both the intracellular SOD1 and extracellular SOD5 are immediately charged with copper, representing the first line of defense against the oxidative burst of the host. We will begin to test this hypothesis by developing systems in which we can monitor changes in intracellular copper and Cu/Zn SOD activity that occur in C. albicans during infection of macrophages. We will also develop a system for recovering enzymatically active SOD5 from the cell wall or secreted from yeast, a tool that will prove invaluable for monitoring extracellular SOD activation during macrophage infection. Through metal analysis of recombinant SOD5, we will address whether this enzyme is truly a copper-only SOD. These studies are in the exploratory, assay-development phase. The findings and new systems that stem from this 2 year program will foster our more long-term mechanistic studies geared towards understanding host effects on pathogen metals and SOD enzymes. These basic research studies have the potential to seed development of new therapies that target copper and prevent activation of the C. albicans SODs essential for virulence.