A significant challenge faced by obligate aerobic eukaryotic pathogens during infection is low oxygen microenvironments. The ability to acquire sufficient oxygen in the face of oxygen depletion has now been shown to be critical for Aspergillus fumigatus and other eukaryotic pathogen's virulence. However, a major gap in knowledge is how obligate aerobic fungi acquire oxygen in the face of oxygen depletion. An in vitro experimental evolution experiment conducted under low oxygen conditions to identify mechanisms of A. fumigatus hypoxia fitness revealed an unexpected change in the fungal mycelium, or biofilm, morphology. A substantial increase in fungal colony furrowing, a so called rugose colony morphology, was observed in the evolved strain with a concomitant increase in hypoxia fitness compared to the parental strain. Importantly, this morphological change and increased hypoxia fitness strongly correlates with virulence. Examination of a large collection of A. fumigatus strains with increased virulence and hypoxia fitness reveals similar colony morphological changes. Preliminary whole genome sequencing of the evolved strain identified a mutation in a novel unstudied fungal specific gene we currently call eefA. Over-expression or loss of eefA dramatically affects fungal colony morphology, hypoxia fitness, and virulence. In this proposal, we will test the hypothesis that increased colony furrowing represents a novel mechanism for fungal oxygen acquisition that is critical for virulence. Using molecular genetics, biochemical, and host-pathogen interaction approaches, we will define the novel function of eefA in mediating fungal oxygen acquisition and virulence. Preliminary data strongly link eefA with the ability of hyphae to adhere and form furrows that promote oxygen access to fungal cells deep within the mycelium. How fungal colony morphology and structure affects A. fumigatus virulence is unstudied and represents a new paradigm for a mechanism of in vivo fitness in the face of low oxygen stress. Consequently, the proposed studies will reveal new insights into A. fumigatus virulence mechanisms and are expected to identify novel therapeutic approaches to thwart fungal oxygen acquisition in vivo to improve disease outcomes.