Molecular analysis of calcineurin in human pathogenic fungi

Abstract

Environmental sensing and the retrieval of essential nutrients from the environment are general metabolic traits that are associated with the growth of P.brasiliensis and A.fumigatus in an inhospitable environment such as its human host. Calcium is a ubiquitous second messenger that functions in signal transduction pathways in eukaryotic organisms. Calcium signaling is important for several cellular processes, including fertilization and development, exocytose, muscle contraction, motility, chemotaxis, cell division, differentiation, programmed cell death and chromatin remodeling in multicellular eukaryotes. Our project proposes to study the system of calcineurin regulation in two human pathogenic fungi, P.brasiliensis and A.fumigatus, and the relationship between this system and two essential aspects of pathogenicity in these organisms: 1) the mycelium-to-yeast transition in P.brasiliensis and 2) the sensing of the human host environment and the nutrient acquisition in A.fumigatus. In the last four years, our group has been actively working with both pathogenic fungi, developing a series of resources and molecular tools aiming to understand the mechanisms of virulence/pathogenicity in these organisms. Thus, the main objectives of this proposal are: 1) to analyze microarray gene expression of the P.brasiliensis mycelium-to-yeast transition blocked by cyclosporine; 2) by using yeast two-hybrid screening, to identify cDNAs that correspond to genes that encode P.brasiliensis proteins that interact with calcineurin during the mycelium-to-yeast transition; 3) construction of a vector for transformation and RNAi inhibition in P.brasiliensis; 3) molecular characterization of an A.fumigatus gene that encodes the homologue of the Crz1 (CrzA) transcription factor that is transported to the nucleus after dephosphorylation by calcineurin; 5) to identify genes that suppress by high copy number the absence of calA e crzA and microarray gene expression analysis of the wild type and calA e crzA mutants in different stressing conditions; 6) characterization of genes involved in the inorganic phosphate utilization and their relationship with calA and crzA mutant; and 7) molecular characterization of A.fumigatus genes that encode proteins with Ca+2-binding domains (EF-hands). (AU)