Aspergillus fumigatus is a major obstacle to the successful treatment of bone marrow and solid organ transplant recipients worldwide. The organism is a potent opportunistic fungal pathogen, causing severe invasive infections that result in mortality rates that approach 90 percent. The continued expansion of organ transplantation programs, and the lack of effective antifungal therapy to treat invasive aspergillosis, is driving the need for a more detailed understanding of the A. fumigatus genes that contribute to pathogenesis. Unfortunately, the genetic tractability of A. fumigatus has lagged behind most other fungal systems, which limits the type of experiments that can be performed on this organism. Inducible promoter systems are one of the most important tools in fungal genetics and have proven to be instrumental for the elucidation of gene function in a number of species. The purpose of this proposal is to develop the technology for a regulatable gene expression system in A. fumigatus, focusing on the prokaryotic tetR/tetO system that has been applied to other eukaryotes. The first aim of the project is to engineer the tetR/tetO system so that a gene can be switched off in A. fumigatus in the presence of tetracycline. This will be accomplished by creating a promoter containing one or more copies of the tet operator sequence linked to a minimal TATA-promoter, and using this hybrid promoter to drive expression of an E. coli lacZ reporter gene. The expression cassette will be transformed into strains of A. fumigatus that constitutively express an artificial transactivator comprised of the tet repressor tetR linked to the Herpes simplex VP16 activator domain, and the activity of the reporter gene will be quantitated in the presence or absence of tetracycline. The second aim seeks to determine whether this system can be used to manipulate A. fumigatus gene expression in vivo. Strains carrying the tet-regulated reporter system will be used for infection in a mouse model of invasive aspergillosis, and the expression of the reporter gene in mouse tissues will be determined in the presence and absence of tetracycline. The ability to manipulate gene expression in vivo would provide a unique opportunity to assess the contribution of a specific A. fumigatus gene product to the pathogenesis of aspergillosis.