Targeting copper homeostasis in the fungal pathogen, Cryptococcus neoformans

描述（由申请人提供）：新型隐球菌是一种人类真菌病原体，可在免疫功能低下和健康个体中发展为全身性感染，导致致命的隐球菌脑膜炎。目前治疗新生芽胞杆菌感染的方法并不十分有效，因此需要更有效的治疗方法来预防感染，特别是在器官移植受者、hiv -艾滋病患者和接受化疗的癌症患者等易感患者群体中。铜（Cu）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，需要生物体严格控制细胞内的铜水平。研究表明，新生C. us -sensing转录因子Cuf1在小鼠感染模型的毒力中是必需的。虽然Cuf1激活了编码铜输入蛋白的基因的表达，但我的博士后资助人Dennis J. Thiele的实验室最近的工作表明，Cuf1激活了许多对铜获取和铜解毒至关重要的基因，以及编码功能未知的蛋白质的基因。此外，其他人的研究表明，活化的巨噬细胞在吞噬体的管腔内积累Cu作为抗微生物剂。由于肺泡巨噬细胞是抵御新生C.感染的第一道防线，因此了解Cuf1 cu感应转录因子和Cuf1靶基因编码的蛋白如何在新生C.毒力中发挥关键作用是很重要的。在本应用程序中，我通过三个具体目标概述了实现这一总体目标的实验。在第一个目标中，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节，并确定这些基因在Cu获取或解毒中的作用。在第二个目标中，我概述了评估cuf1依赖基因突变的实验，以确定它们对肺泡巨噬细胞体外吞噬存活的贡献。在第三个目标中，两者都使用

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. PUBLIC HEALTH RELEVANCE: Cryptococcus neoformans is a fungal pathogen that infects both immunocompromised and healthy individuals and if left untreated, C. neoformans can result in lethal fungal meningitis. Recently, it has become clear that copper plays a critical rolein the outcome of infection, with the host using the toxic properties of copper and pathogens encoding copper defense mechanisms. In this application I outline experiments to characterize the C. neoformans copper metabolism mechanisms in the context of infection with the aim of identifying novel and more efficacious drug targets against this deadly pathogen.