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. ? ? ?
