Project Summary/Abstract. Recent estimates indicate that meningoencephalitis caused by the pathogenic fungus Cryptococcus neoformans is responsible 15% of deaths in AIDS patients. Along with other fungal pathogens, C. neoformans is therefore a major threat to the 37 million people worldwide living with HIV. A complex of related species originally designated Cryptococcus gattii has recently emerged as a primary pathogen of immunocompetent people. The long-term goal of our research program is to acquire knowledge that will lead to new strategies to combat cryptococcal infections. In particular, we are working to acquire a detailed understanding of the factors required for fungi to proliferate in vertebrate hosts. In particular, we seek to identify new targets for therapy. Our focus is on iron as an essential nutrient for pathogen proliferation and an important indicator of the host environment. Iron is especially important because mammals actively withhold iron from pathogens through a process called nutritional immunity. Pathogens must therefore be able to successfully compete for iron in order to cause disease. We have shown that iron influences the growth of C. neoformans and also the size of the polysaccharide capsule that is the major virulence factor. Our efforts have focused on characterizing the mechanisms of iron sensing and exploiting the regulatory information to identify targets required for iron acquisition. The first specific aim is to characterize the monothiol glutaredoxin Grx4 as a key sensor of iron availability. Grx4 interacts with the iron regulator Cir1 and the proteins regulate iron homeostasis and the expression of virulence factors. We seek to understand the mechanisms of iron sensing and how the iron signal influences gene expression. A second specific aim will investigate the interaction of Grx4 with a network of transcription factors. Two strong candidates have been identified (HapX and Gat201) and the interactions and regulatory influences of these proteins with Grx4 will be characterized. A final specific aim is based on highly productive genetic screens that identified components of the intracellular machinery for heme trafficking. Mutants lacking trafficking functions for heme acquisition will be constructed and tested in mouse inhalation models of cryptococcosis. Additionally, a heme sensor has been developed to detect heme availability in trafficking mutants in culture and in cryptococcal cells during proliferation in different host tissue locations. Overall, these studies will provide a comprehensive view the integration of iron sensing with the regulation of uptake strategies that are critical during cryptococcosis. !