Invasive pulmonary aspergillosis (IPA) is a fungal disease with high morbidity and mortality. Immunocompromised patients are at the greatest risk. The genetic program used by Aspergillus spp. during invasive growth is unknown, but its elucidation is critical to developing novel antifungal therapies. Work with A. fumigatus, the primary cause of aspergillosis, is hampered by the absence of critical molecular and genetic tools that would facilitate studies of gene regulation, gene function and a gene's role in pathogenesis. For this reason, part of this proposal is to develop the basic tools needed to advance the study of gene activity and function in A. fumigatus. We are developing molecular tools and auxotrophic mutants in the A. fumigatus strain AF293 to facilitate our investigations. In conjunction with the development of auxotrophic mutants, we will develop a set of transformation and expression vectors based on the different nutritional markers. We have performed preliminary suppression subtractive hybridization (SSH) experiments to identify genes that have increased expression during invasive growth. We will test the hypothesis that genes whose expression increases during invasive growth contribute to the progression of IPA. Using SSH and a murine model of IPA, we will identify those fungal genes that are highly expressed during invasive growth. We will use the A. fumigatus genome data being generated from the public sequencing project to establish the function of genes identified through SSH and in time explore the regulation of these genes and determine the signals that control their expression. These studies will provide us with a snapshot of the genetic program used by the fungus to grow in this unusual physiological state, leading to novel therapeutic approaches to managing IPA.