Comparative and functional genomics of C. neoformans

描述（由申请人提供）：致病性真菌新型隐球菌对艾滋病患者造成威胁生命的感染，因此对全球1.34亿艾滋病毒感染者构成重大威胁。近缘种加蒂隐球菌最近成为免疫正常人群的主要病原体。该项目的长期目标是获取知识，这将导致对抗真菌感染的新策略。特别是，我们希望对哺乳动物宿主中病原体生长所需的因素有一个详细的了解，并确定有用的治疗靶点。在这方面，铁的有效性是宿主环境的关键指标，也是病原体增殖所必需的营养物质。此外，哺乳动物在一种称为营养免疫的过程中主动阻止病原体吸收铁，因此病原体必须积极争夺铁。铁对新生梭状菌的发病机制尤为重要，因为这种金属的可用性不仅影响生长，而且影响多糖胶囊的大小，而多糖胶囊是主要的毒力因素。为了填补我们对真菌病原体争夺铁的机制的理解空白，第一个具体目标是确定支持从血红素中获取铁的细胞表面蛋白的分子功能。这些蛋白质都有助于血红素的强劲生长，包括一种分泌的甘露蛋白，它是一种候选的血红素结合蛋白，以及三种铁还原酶。第二个具体目标将描述血红素运输和加工的细胞内机制。插入突变筛选鉴定出25个突变导致血红素生长缺陷的基因，其中一些基因编码细胞内运输机制。这一结果导致假设血红素是通过内吞作用获得并运输到液泡中进行铁提取和再循环。在已知和候选转运功能的特定步骤中存在缺陷的突变体将被构建并测试其处理血红素的能力。最后一个具体的目的是评估血红素和铁获取功能在新生芽孢杆菌毒力中的作用。缺乏铁获取功能组合的突变体将在隐球菌病的小鼠吸入模型中进行测试，并且将在不同铁来源的背景下检查巨噬细胞中的突变体增殖。这些研究将为隐球菌病期间特定宿主铁源和真菌摄取策略的相对重要性提供一个全面的观点。

DESCRIPTION (provided by applicant): The pathogenic fungus Cryptococcus neoformans causes life-threatening infections in AIDS patients and therefore poses a major threat to the >34 million people worldwide who are infected with HIV. The related species Cryptococcus gattii has recently emerged as a primary pathogen of immunocompetent people. The long-term goal of this project is to acquire knowledge that will lead to new strategies to combat fungal infections. In particular, we want to establish a detailed understanding of the factors required for pathogen growth in mammalian hosts and identify useful targets for therapy. In this regard, iron availabilit is a key indicator of the host environment as well as an essential nutrient for pathogen proliferation. In addition, mammals actively withhold iron from pathogens in a process termed nutritional immunity, and pathogens must therefore aggressively compete for iron. Iron is particularly important for the pathogenesis of C. neoformans because the availability of this metal not only influences growth but also the size of the polysaccharide capsule that is the major virulence factor. To fill gaps in our understanding of the mechanisms by which fungal pathogens compete for iron, the first specific aim is to determine the molecular functions of cell surface proteins that support iron acquisition from heme. These proteins each contribute to robust growth on heme and include a secreted mannoprotein that is a candidate heme-binding protein as well as three ferric reductases. A second specific aim will characterize the intracellular machinery for heme trafficking and processing. An insertional mutagenesis screen identified 25 genes in which mutations caused growth defects on heme, and some of these genes encode intracellular trafficking machinery. This result led to the hypothesis that heme is acquired by endocytosis and transported to the vacuole for iron extraction and recycling. Mutants with defects at specific steps in known and candidate transport functions will be constructed and tested for their ability to process heme. A final specific aim will evaluate the roe of heme and iron acquisition functions in the virulence of C. neoformans. Mutants lacking combinations of iron acquisition functions will be tested in mouse inhalation models of cryptococcosis and the proliferation of the mutants in macrophages will be examined in the context of different iron sources. These studies will provide a comprehensive view of the relative importance of specific host iron sources and fungal uptake strategies during cryptococcosis.