Comparative and functional genomics of C. neoformans

描述(由申请人提供)：真菌病原体新生隐球菌在艾滋病患者中导致危及生命的感染的高发病率，因此这种真菌对全球4000万艾滋病毒感染者构成重大威胁。此外，近缘物种C.gattii最近已成为免疫功能正常人群的主要病原体。我们的长期目标是获得将导致抗击真菌感染的新战略的知识。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，以便确定阻止感染的关键靶点。在这方面，铁的有效性是寄主环境的重要指标，也是病原体的基本营养。因此，我们计划研究新生弧菌中铁的调节和获取，作为了解铁在真菌发病中的作用的模型。铁水平对新生隐孢子菌的发病机制特别重要，因为可获得性影响多糖壳的大小，而多糖壳是真菌的主要毒力因素。我们的假设是，铁对毒力和生长的影响是通过一个由主要的铁反应调节因子Cir1(隐球菌铁调节因子)控制的网络来实现的。我们相信，Cir1和这个网络的特征将确定新生葡萄球菌致病所必需的铁获取机制。我们的第一个具体目标是描述Cir1的关键功能。我们将专门确定Cir1是否直接与铁结合，以及该蛋白是否与编码铁摄取功能的靶基因的启动子区域结合。这项工作将包括识别与Cir1相互作用的调控因子的蛋白质组学方法。这些因子可能参与了铁获取的靶基因的调节以及与毒力相关的其他功能。我们的第二个特定目标将表征Hap转录因子复合体在从血红素中获取铁的作用。HAP基因的表达受Cir1调控，我们有初步数据表明，这些基因是血红素使用所必需的。我们的第三个具体目标将使用转录图谱来研究Cir1在使用宿主中丰富的铁源(血红素和转铁蛋白)中的作用。最后，我们将构建编码候选功能基因缺陷的突变体，以便在感染期间使用血红素和转铁蛋白。可能的靶标(例如，血红素加氧酶)已经被确定，我们预计我们在Aim 3的微阵列工作将确定其他功能。在这项工作结束时，我们将对连接铁与毒力的调控网络有更深入的了解，我们也将知道新生葡萄球菌在感染过程中用来从宿主那里窃取铁的功能。这些信息将突出与铁相关的关键功能，这些功能可以作为治疗隐球菌病的靶点。公共卫生相关性：该项目的相关性来自于控制免疫系统受损的人类的真菌感染的迫切需要。特别是，感染艾滋病毒的4000万或更多人死于真菌病的几率很高。这项研究将专门研究通过靶向真菌病原体在感染期间获得营养铁的能力来控制感染的可能性。

DESCRIPTION (provided by applicant): The fungal pathogen Cryptococcus neoformans causes a high incidence of life-threatening infections in AIDS patients and this fungus therefore poses a major threat to the > 40 million people worldwide who are infected with HIV. In addition, the related species C. gattii has recently emerged as a primary pathogen of immunocompetent people. Our long-term goal is to acquire knowledge that will lead to new strategies to combat fungal infections. In particular, we want to develop a detailed understanding of the factors required for growth of pathogens in mammalian hosts in order to identify key targets to block infection. In this regard, iron availability is an important indicator of the host environment as well as an essential nutrient for pathogens. Therefore, we plan to investigate iron regulation and acquisition in C. neoformans, as a model for understanding the role of iron in fungal pathogenesis. Iron levels are particularly important for the pathogenesis of C. neoformans because availability influences the size of the polysaccharide capsule that is the major virulence factor of the fungus. Our hypothesis is that the influence of iron on virulence and growth occurs through a network controlled by the master iron-responsive regulator Cir1 (Cryptococcus iron regulator). We believe that the characterization of Cir1 and this network will identify mechanisms of iron acquisition that are essential for C. neoformans to cause disease. Our first specific aim is to characterize the key functions of Cir1. We will specifically determine whether Cir1 directly binds iron and whether the protein binds the promoter regions of target genes encoding iron uptake functions. This work will include proteomic approaches to identify regulatory factors that interact with Cir1. These factors may participate in the regulation of target genes for iron acquisition and other functions relevant to virulence. Our second specific aim will characterize the role of the Hap transcription factor complex in iron acquisition from heme. The expression of the HAP genes is regulated by Cir1 and we have preliminary data that these genes are required for heme use. Our third specific aim will employ transcriptional profiling to examine the role of Cir1 in the use of iron sources (heme and transferrin) that are abundant in the host. Finally, we will construct mutants that are defective in genes encoding candidate functions for the use of heme and transferrin during infection. Likely targets (e.g., heme oxygenase) have been identified and we anticipate that our microarray work in aim 3 will identify additional functions. At the conclusion of this work, we will have a deeper understanding of the regulatory network that links iron to virulence, and we will know the functions that C. neoformans uses to steal iron from the host during infection. This information will highlight the key iron-related functions that can be targeted to treat Cryptococcal disease. PUBLIC HEALTH RELEVANCE: The relevance of this project comes from the pressing need to control fungal infections in humans with impaired immune systems. In particular, the 40 million or more people infected with HIV have a high chance of succumbing to fungal disease. The research will specifically examine the potential to control infections by targeting the ability of fungal pathogens to acquire the nutrient iron during infection.