The novel family of superoxide dismutase enzymes in Candida albicans

白色念珠菌中超氧化物歧化酶的新家族

基本信息

批准号：
8502621
负责人：
Valeria C Culotta
金额：
$ 19.04万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-02 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8502621
关键词：
Address Basic Science Binding Biochemistry Biological Assay Biological Availability Biology Candida albicans Cell Wall Cells Charge Copper Cuprozinc Superoxide Dismutase Cytosol Development Disulfides Environment Enzymes Eukaryota Exhibits Exploratory/Developmental Grant Family Fluorescent Probes Fostering Free Radicals Growth Immune Immune response Immunocompromised Host Individual Infection Iron Laboratories Lead Manganese Metals Methods Mission Monitor Names Oxygen Pathogenicity Phagolysosome Phase Population Proteins Public Health Reactive Oxygen Species Recombinants Research Respiratory Burst Seeds Site Staging Superoxide Dismutase Superoxides System Testing Virulence Virulence Factors Yeasts Zinc assay development extracellular factor C killings macrophage member novel novel therapeutics oxidation pathogen prevent programs research study stem tool

项目摘要

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.

描述（由申请人提供）：传染性酵母白色念珠菌仍然是公共卫生中的一个重要问题，特别是对于免疫受损的个体。白色念珠菌的一组毒力因子包括一系列含铜超氧化物歧化酶 (SOD) 酶，可清除有毒的超氧化物自由基。尽管它们在毒力方面具有重要意义，但实际上人们对白色念珠菌 SOD 的生物化学或这些酶如何通过插入铜辅助因子在细胞中被激活一无所知。在这项研究应用中，我们计划开发新系统来监测宿主对白色念珠菌细胞内铜和含铜 SOD 的影响。将检查白色念珠菌中两种类型的含铜 SOD：细胞质中的铜/锌 SOD (SOD1)，以及通过 GPI 锚定附着在细胞壁上的铜/锌 SOD 家族的细胞外成员 (SOD5)。两者对于毒力都很重要，在我们的初步分析中，两者都表现出一些其他 Cu/Zn SOD 中未见的独特特征。例如，在正常（非感染性）实验室生长条件下，细胞内 SOD1 蛋白不活跃，酵母反而使用不寻常的胞质锰 SOD3 来去除超氧化物。细胞外 SOD5 的独特之处还在于它似乎仅结合铜，并且缺乏在不同条件下不变的锌位点。其他真核铜/锌 SOD。我们认为这些不寻常的 Cu-SOD 已经进化到利用对金属的宿主效应。在感染过程中，巨噬细胞吞噬溶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。宿主的这种特殊环境似乎非常适合激活需要 SOD 的铜。我们假设在感染过程中，细胞内 SOD1 和细胞外 SOD5 立即带有铜，代表了宿主对抗氧化爆发的第一道防线。我们将开始通过开发系统来测试这一假设，在该系统中我们可以监测巨噬细胞感染期间白色念珠菌中发生的细胞内铜和铜/锌 SOD 活性的变化。我们还将开发一种从细胞壁或酵母分泌的酶活性 SOD5 中回收的系统，该工具对于监测巨噬细胞感染期间细胞外 SOD 的激活将具有不可估量的价值。通过重组 SOD5 的金属分析，我们将确定这种酶是否真的是纯铜 SOD。这些研究正处于探索性测定开发阶段。这个为期 2 年的计划的发现和新系统将促进我们更长期的机制研究，旨在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推动新疗法的开发，这些疗法以铜为靶点，并防止白色念珠菌毒力所必需的 SOD 激活。