GPCR signaling cascades in Cryptococcus neoformans

描述（由申请方提供）：新型隐球菌是一种致病性担子菌真菌，感染中枢神经系统，引起脑膜脑炎。本研究的目的是了解控制该模式致病真菌有性生殖和毒力的信号机制。关于G蛋白调控细胞类型和毒力的信号转导通路的研究已经取得了巨大的进展。先前已经表征了涉及cAMP和MAP激酶信号传导的两个非常保守的级联。但真菌病原体如何感知环境和宿主信号，触发这些G蛋白控制的途径，在很大程度上仍然未知。我们已经鉴定了一个G蛋白偶联受体（GPCR）家族，并发现其中一个GPCR，Gpr 4，感测氨基酸并与Ga蛋白Gpa 1相互作用以激活cAMP信号。我们的研究进一步揭示了Gpa 1需要感知多种信号，这可能为理解G蛋白如何通过受体依赖性和受体非依赖性机制激活提供新的范式。这些研究将为理解配体-GPCR-G蛋白相互作用如何控制微生物致病机制提供新的方法。本研究将应用裂解泛素系统研究GPCR家族与相关Ga蛋白的相互作用。将开发酵母异源表达系统以筛选单个GPCR的潜在配体。已经产生了每个GPCR的基因缺失突变体，并将在体外和小鼠感染模型中研究它们在信号传导和发病机制中的作用。本项目的具体目标是：1）识别和表征参与cAMP信号传导的传感器，并验证多传感器系统在C中运作的假设。新型生物激活Ga蛋白Gpa 1; 2）阐明GPCR家族与Ga蛋白Gpa 1、Gpa 2和Gpa 3之间的相互作用; 3）识别潜在的GPCR配体及其在发育和毒力中的作用。本项目的研究结果将填补控制有性生殖的胞外信号感受和信号转导级联与C.新人类了解配体- GPCR-G蛋白相互作用和真菌发育的调控将为开发新的药物靶点提供见解，并可能提供控制C。新生菌和其他病原真菌，具有相当重要的医学意义。新型隐球菌是一种感染中枢神经系统的人类真菌病原体，如果不治疗，通常会导致致命的脑膜炎。了解配体- GPCR-G蛋白的相互作用和真菌发育的调控将为开发新的药物靶点提供见解，并可能提供控制C neoformans和其他病原真菌的手段，这些病原真菌具有相当重要的医学意义。

DESCRIPTION (provided by applicant): Cryptococcus neoformans is a pathogenic basidiomycetous fungus that infects the central nervous system causing meningoencephalitis. This project's objective is to understand signaling mechanisms controlling sexual reproduction and virulence in this model pathogenic fungus. Tremendous advances have been achieved in studies on the role of G protein regulated signal transduction pathways that control cell type and virulence. Two well-conserved cascades involving cAMP and MAP kinase signaling have been previously characterized. But it still remains largely unknown how fungal pathogens sense environmental and host signals that trigger these G protein governed pathways. We have identified a family of G protein-coupled receptors (GPCRs) and discovered that one GPCR, Gpr4, senses amino acids and interacts with the Ga protein Gpa1 to activate cAMP signaling. Our studies further reveal Gpa1 is required to sense multiple signals, which may provide new paradigms to understand how G proteins are activated by both receptor-dependent and receptor-independent mechanisms. The studies proposed here will provide new approaches to understand how ligand-GPCR-G protein interactions control microbial pathogenesis. The split-ubiquitin system will be applied to study the interactions between the family of GPCRs and associated Ga proteins. A yeast heterologous expression system will be developed to screen for potential ligands of individual GPCRs. Gene deletion mutants for each GPCR have been generated and their roles in signaling and pathogenesis will be studied in vitro and in a murine infection model. The specific aims of this project are to: 1) Identify and characterize sensors involved in cAMP signaling, and to test the hypothesis that a multiple sensor system operates in C. neoformans to activate the Ga protein Gpa1; 2) Elucidate interactions between the GPCR family and the Ga proteins Gpa1, Gpa2, and Gpa3; 3) Identify potential GPCR ligands and their roles in development and virulence. Outcomes of this project should fill critical research gaps between extracellular signal sensing and signal transduction cascades controlling sexual reproduction and virulence factor production in C. neoformans. Understanding ligand- GPCR-G protein interactions and regulation of fungal development will provide insights to develop new drug targets, and may provide the means to control C. neoformans and other pathogenic fungi, which have medical significance of considerable importance. Cryptococcus neoformans is a human fungal pathogen that infects the central nervous system and often causes meningitis that is fatal if untreated. Understanding ligand- GPCR-G protein interactions and regulation of fungal development will provide insights to development new drug targets, and may provide the means to control C neoformans and other pathogenic fungi, which have medical significance of considerable importance.