The fungal pathogen Cryptococcus neoformans causes a high incidence of life-threatening infections in AIDS patients and this fungus therefore poses a major threat to the > 40 million people worldwide who are infected with HIV. In addition, the related species C. gattii has recently emerged as a primary pathogen of immunocompetent people. Our long-term goal is to acquire knowledge that will lead to new strategies to combat fungal infections. In particular, we want to develop a detailed understanding of the factors required for growth of pathogens in mammalian hosts in order to identify key targets to block infection. In this regard, iron availability is an important indicator of the host environment as well as an essential nutrient for pathogens. Therefore, we plan to investigate iron regulation and acquisition in C. neoformans, as a model for understanding the role of iron in fungal pathogenesis. Iron levels are particularly important for the pathogenesis of C. neoformans because availability influences the size of the polysaccharide capsule that is the major virulence factor of the fungus. Our hypothesis is that the influence of iron on virulence and growth occurs through a network controlled by the master iron-responsive regulator Cir1 (Cryptococcus iron regulator). We believe that the characterization of Cir1 and this network will identify mechanisms of iron acquisition that are essential for C. neoformans to cause disease. Our first specific aim is to characterize the key functions of Cir1. We will specifically determine whether Cir1 directly binds iron and whether the protein binds the promoter regions of target genes encoding iron uptake functions. This work will include proteomic approaches to identify regulatory factors that interact with Cir1. These factors may participate in the regulation of target genes for iron acquisition and other functions relevant to virulence. Our second specific aim will characterize the role of the Hap transcription factor complex in iron acquisition from heme. The expression of the HAP genes is regulated by Cir1 and we have preliminary data that these genes are required for heme use. Our third specific aim will employ transcriptional profiling to examine the role of Cir1 in the use of iron sources (heme and transferrin) that are abundant in the host. Finally, we will construct mutants that are defective in genes encoding candidate functions for the use of heme and transferrin during infection. Likely targets (e.g., heme oxygenase) have been identified and we anticipate that our microarray work in aim 3 will identify additional functions. At the conclusion of this work, we will have a deeper understanding of the regulatory network that links iron to virulence, and we will know the functions that C. neoformans uses to steal iron from the host during infection. This information will highlight the key iron-related functions that can be targeted to treat Cryptococcal disease.