Mechanism of GPCR Signaling-mediated Fungal Cell Gigantism

描述（由申请人提供）：该提案侧重于真菌病原体新型隐球菌中的G蛋白偶联受体（GPCR）信号传导，该病原体感染肺部并经常传播到中枢神经系统，导致危及生命的脑膜炎。开发新的药物靶点以更好地治疗隐球菌感染是一个迫切的未满足的医疗需求。为了满足这一需求，更好地了解真菌感知宿主信号并适应敌对宿主环境的疾病机制至关重要。感染期间的一种适应策略是显著扩大酵母细胞的大小，这是一种新的细胞形态发生，称为细胞巨人症或“泰坦”细胞形成。肺部感染期间的真菌细胞巨人症最近被认为是一种新的毒力因子，是疾病发生和传播所必需的。尽管泰坦细胞在真菌发病机制中很重要，但宿主信号和真菌受体诱导泰坦细胞的作用尚不清楚。申请人的研究表明，Gpa1 （G蛋白¿亚基）G蛋白信号转导途径参与泰坦细胞的产生。一种新的G蛋白偶联受体（GPCR） Gpr5已被确定为促进泰坦细胞形成所必需的。这些新发现引出了一个核心假设，即真菌细胞通过细胞内的细胞感应宿主的特定信号

DESCRIPTION (provided by applicant): The proposal focuses on G protein-coupled receptor (GPCR) signaling in the fungal pathogen Cryptococcus neoformans, which infects the lung and often disseminates to the central nervous system to cause life- threatening meningitis. There is an urgent unmet medical need for developing new drug targets for better treatment of cryptococcal infection. To meet this need, it is critical to better understand the disease mechanisms by which the fungus senses host signals and adapts to the hostile host environment. One such adaptation strategy during infection is to dramatically enlarge yeast cell size, a novel cell morphogenesis called cell gigantism or "titan" cell formation. Fungal cell gigantism during lung infection has been recently recognized as a new virulence factor that is required for disease initiation and dissemination. Despite its importance in fungal pathogenesis, the host signals and fungal receptors for titan cell induction remain unknown. Studies from the applicant demonstrated that the Gpa1 (G protein ¿ subunit) G protein signal transduction pathway is involved in the production of titan cells. A novel G protein-coupled receptor (GPCR), Gpr5, has been identified as being essential for promoting titan cell formation. These new discoveries led to the central hypothesis that fungal cells sense host specific signals through the Gpr5 orphan receptor, which then activates Gpa1-mediated signaling to regulate fungal cell morphogenesis. The long-term goal is to understand the molecular basis of cryptococcal cell gigantism in order to provide the mechanistic details needed to develop new antifungal agents that interfere with GPCR function and inhibit titan cell formation during Cryptococcus infection. Guided by strong preliminary data, the hypothesis will be tested by two specific aims: 1) Define the function of Gpr5 in G-protein signaling activation and fungal titan cell regulation, and 2) Identify and characterize host signals required for Gpr5-mediated fungal cell gigantism. Under the first aim, strains having various levels of Gpr5 activity will be used to determine the role of Gpr5 in fungal titan cell production and pathogenesis in murine models, and to characterize how Gpr5 regulates the activation of Gpa1 as measured by cellular cAMP production. Under the second aim, a spent medium that can stimulate titan cell formation in vitro will be used to identify active compounds that are responsible for Gpr5-dependent cell gigantism. A Saccharomyces-based heterologous expression system will be used to analyze potential ligands for Gpr5 activation. The approach is innovative, because we have developed an in vitro system for titan cell formation that allows us to identify host factors responsible for cell gigantism. The proposed research is significant, because it aims to understand the molecular basis of the host-pathogen interaction during the development of virulence factors, which is critical for fungal pathogenesis. Ultimately, such knowledge will facilitate efforts to design new anti-fungal agents to perturb GPCR signaling in a therapeutically beneficial manner.