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。有一个迫切的未满足的医疗需求，开发新的药物靶标，以更好地治疗隐球菌感染。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。在感染过程中，一种这样的适应策略是显着扩大酵母细胞的大小，一种新的细胞形态发生称为细胞增殖或“泰坦”细胞形成。肺部感染中的真菌细胞增殖是一种新的致病因子，是疾病发生和传播的必要条件。尽管它在真菌发病机制中的重要性，宿主信号和真菌受体的泰坦细胞诱导仍然是未知的。申请人的研究表明，Gpa 1（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），Gpr 5，已被确定为是必不可少的促进泰坦细胞的形成。这些新的发现导致了一个中心假设，即真菌细胞通过细胞膜感受宿主特异性信号。 Gpr 5孤儿受体，然后激活Gpa 1介导的信号传导，以调节真菌细胞形态发生。长期目标是了解隐球菌细胞增殖的分子基础，以便提供开发新的抗真菌药物所需的机制细节，这些药物在隐球菌感染期间干扰GPCR功能并抑制泰坦细胞形成。在强有力的初步数据的指导下，该假设将通过两个具体目标进行测试：1）定义Gpr 5在G蛋白信号传导激活和真菌泰坦细胞调节中的功能，以及2）鉴定和表征Gpr 5介导的真菌细胞增殖所需的宿主信号。在第一个目标下，将使用具有不同水平的Gpr 5活性的菌株来确定Gpr 5的作用。 Gpr 5在真菌泰坦细胞生产和发病机制在小鼠模型，并表征如何Gpr 5调节Gpa 1的激活，通过细胞cAMP的生产。在第二个目标下，可以在体外刺激泰坦细胞形成的废培养基将用于鉴定负责Gpr 5依赖性细胞增殖的活性化合物。将使用一种基于链霉菌的异源表达系统来分析Gpr 5激活的潜在配体。这种方法是创新的，因为我们已经开发了一种体外泰坦细胞形成系统，使我们能够识别负责细胞增殖的宿主因子。这项研究具有重要意义，因为它旨在了解毒力因子发展过程中宿主-病原体相互作用的分子基础，这对真菌致病至关重要。最终，这些知识将有助于设计新的抗真菌剂，以治疗上有益的方式干扰GPCR信号传导。

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.