Targeting copper homeostasis in the fungal pathogen, Cryptococcus neoformans

描述(申请人提供)：新生隐球菌是一种人类真菌病原体，在免疫功能受损和健康的人中都会发展为系统性感染，导致致命的隐球菌性脑膜炎。目前针对新生弧菌感染的治疗方法不是很有效，因此需要更有效的治疗来预防感染，特别是在脆弱的患者群体中，如器官移植接受者、艾滋病毒/艾滋病患者和接受化疗的癌症患者。铜是真菌和人类多种酶的重要辅助因子，但过量的铜是有毒的，需要生物严格控制细胞内铜的水平。研究表明，新生芽孢杆菌铜感应转录因子Cuf1是小鼠感染模型致病所必需的。虽然Cuf1激活了编码铜输入蛋白的基因的表达，但我的博士后导师丹尼斯·J·蒂勒(Dennis J.Thiele)最近的实验室工作表明，Cuf1激活了许多对铜的获得和铜的解毒至关重要的基因，以及编码未知功能的蛋白质的基因。此外，其他人的研究表明，被激活的巨噬细胞作为一种抗菌剂，在吞噬小体的管腔内积累铜。由于肺泡巨噬细胞是抵御新城疫杆菌感染的第一道防线，了解Cuf1铜感应转录因子及其靶基因编码的蛋白在新城疫霉菌毒力中起着至关重要的作用。在这个应用程序中，我概述了通过三个具体目标来实现这一总体目标的实验。在第一个目的中，我将进行遗传和生化实验，以确定依赖Cuf1的基因是否直接或间接受到该转录因子的调控，并确定这些基因在铜获得或解毒中的作用。在第二个目标中，我概述了评估Cuf1依赖基因突变的实验，以确定它们对体外肺泡巨噬细胞吞噬存活的贡献。在第三个目标中，使用两个 野生型小鼠和我的博士后导师实验室创造的小鼠在巨噬细胞铜积累方面存在特定缺陷，我概述了实验，以破译依赖Cuf1的基因和宿主铜稳态机制在宿主-病原体相互作用中扮演的角色。我通过在真菌发病机制、铜金属调控和哺乳动物宿主铜代谢遗传学的交叉点上进行这个项目将获得的新专业知识将大大有助于理解铜在宿主-病原体轴中的作用，并将提供 我接受了尖端的培训，开始了作为一名有竞争力的独立教员的职业生涯。

DESCRIPTION (provided by applicant): Cryptococcus neoformans is a human fungal pathogen that can progress to systemic infection in both immunocompromised and healthy individuals, leading to lethal cryptococcal meningitis. Current therapies to address C. neoformans infection are not highly effective, therefore more efficacious treatments are needed to prevent infection, particularly in vulnerable patient population such as organ transplant recipients, HIV-AIDS patients and cancer patients undergoing chemotherapy. Copper (Cu) serves as an essential co-factor for a wide variety of enzymes in fungi and humans, but excess Cu is toxic requiring organisms to maintain tight control of intracellular Cu levels. Studies suggest that the C. neoformans Cu-sensing transcription factor, Cuf1, is required for virulence in mouse infection models. While Cuf1 activates the expression of genes encoding proteins that carry out Cu import, recent work in the laboratory of my postdoctoral sponsor, Dennis J. Thiele, has demonstrated that Cuf1 activates many genes that are essential for both Cu acquisition and Cu detoxification, as well as genes encoding proteins of unknown function. Moreover, work by others suggests that activated macrophages accumulate Cu within the lumen of the phagosome as an anti-microbial agent. Since alveolar macrophages are the first line of defense against C. neoformans infection, it is important to understand how the Cuf1 Cu-sensing transcription factor, and the proteins encoded by Cuf1 target genes, play a critical role in C. neoformans virulence. In this application I outline experiments to accomplish this overall goal through three Specific Aims. In the first aim I will carry out genetic and biochemical experiments to establish whether the genes that are dependent on Cuf1 are regulated directly or indirectly by this transcription factor and to ascertain the role of these genes in Cu acquisition or detoxification. In the second aim I outline experiments to evaluate mutants in Cuf1-dependent genes to ascertain their contribution to survival to alveolar macrophage phagocytosis in vitro. In the third aim, using both wild type mice and mice created in my postdoctoral sponsor's laboratory that are specifically defective in macrophage Cu accumulation, I outline experiments to decipher what roles Cuf1-dependent genes and the host Cu homeostasis machinery play in host-pathogen interactions. The new expertise I will gain by carrying out this project at the intersection of fungal pathogenesis, Cu metalloregulation and the genetics of mammalian host Cu metabolism will contribute significantly to understanding the role of Cu in the host-pathogen axis and will provide me with cutting-edge training to launch a career as a competitive independent faculty member.