Project Summary. Fungal mediated disease progression is highlighted by populations of fungal cells that form a community referred to as a biofilm. For therapeutic success, contemporary antifungal therapies must be effective at the site of infection in the context of an established fungal biofilm. Critically, it is now clear that emergent properties arise from fungal biofilms that directly alter virulence, disease progression, and antifungal drug susceptibility. However, the mechanisms through which filamentous fungal biofilm emergent properties impact virulence, disease progression, and antifungal susceptibility remain a significant knowledge gap. The long-term goal of this project is focused on defining the molecular mechanisms of Aspergillus fumigatus biofilm mediated disease progression mechanisms to inform contemporary and novel therapeutic approaches. In the prior funding period, we made important progress that surprisingly revealed heterogeneity in A. fumigatus biofilm morphology across clinical isolates. Differences in biofilm morphology altered virulence and disease progression in vivo in murine models of aspergillosis. We discovered that long term growth in a low oxygen environment gives rise to a biofilm morphology we termed H-MORPH and that a novel fungal specific gene cluster that contains a protein with unknown function was sufficient for H-MOPRH formation. Significantly, we identified H-MORPH clinical isolates from both acute invasive aspergillosis patients and patients with chronic aspergillosis, suggesting H-MORPH can arise in human disease. We observed that H-MOPRH occurs in vivo in a murine model of invasive pulmonary aspergillosis and contributes to worse disease outcomes compared to the contrasting N-MORPH isolates. In aim 1, we will define the genetic pathway(s) that regulate A. fumigatus the development of this unique population level morphotype utilizing the newly discovered biofilm architecture factor (baf) gene family as a tool to dissect the underlying mechanisms. In aim 2, we will define the differences in fungal metabolism that underly N-MORPH and H-MORPH morphotypes and test the hypothesis that H- MORPH biofilms are carbon catabolite de-repressed which leads to increased fitness in vivo. In aim 3, we test the hypothesis that H-MORPH strain metabolism is immune modulatory through alterations in fungal pathogen associated molecular pattern exposure. Taken together, our proposed studies will fill significant knowledge gaps related to the discovery of distinct A. fumigatus morphotypes that directly impact virulence. Advancing our understanding of this knowledge gap is expected to lay the foundation for new diagnostic and therapeutic strategies to combat highly virulent and drug resistant strains of this important human fungal pathogen.