PROJECT SUMMARY/ABSTRACT Mucormycosis, caused by Mucorales fungi, is a life-threatening infection that occurs in patients immunocom- promised by diabetic ketoacidosis, neutropenia, corticosteroid use, and/or increased serum iron. Rhizopus species are the most common cause of mucormycosis. Because of the rising prevalence of mucormycosis risk factors, the incidence of the infection has risen. Despite disfiguring surgery and aggressive antifungal therapy, the mortality of mucormycosis remains >40%, and approaches 100% in patients with disseminated disease. Clearly new strategies to prevent and treat mucormycosis are needed. Commonly initiated via inhalation of spores, clinical hallmarks of mucormycosis is the virtually uniform pres- ence of extensive angioinvasion with resultant vessel thrombosis and tissue necrosis. These features highlight the importance of the ability of the fungus to invade alveolar epithelial cells (AECs) to initiate the infection. They also emphasize the significance of penetrating the endothelium during the progression and dissemination of the disease. Finally, the extensive tissue necrosis points to the presence of fungal toxins. During the last funding cycle, we determined that Mucorales initiate infection when the CotH invasins bind to integrins on AECs. This binding triggers the activation of AEC epidermal growth factor receptor (EGFR) to in- duce invasion of germinated spores with an unidentified mechanism. We also discovered that germinated spores disseminate through the pericytes and extracellular matrix to invade endothelial cells via CotH interact- ing with Glucose Regulated Protein 78. We also made the seminal discovery that Mucorales damage host cells by producing a toxin called mucoricin, so named because of its similarities to the plant toxin, ricin. Both CotH invasins and mucoricin are required for pathogenesis and antibodies targeting either protein are protective in mice. Specifically, our preliminary data show that the combination of binding of CotH invasins and subsequent production of mucoricin cause disintegration of AECs (potentially by activating the ADP-ribosylation factor 6 (Arf6)), excessive vascular leak, tissue edema, and organ failure; all of which are hallmarks of mucormycosis and are associated with antifungal therapy failure. Thus, we propose to build on these exciting data and deter- mine the molecular mechanisms by which Mucorales induce disintegration of AECs and vascular leak and de- fine the intracellular events of mucoricin-induced host cell entry and death. We will also evaluate the efficacy of novel therapeutic strategies targeting Arf6, host receptors, mucoricin and mucoricin-induced host cell death pathways in mice. We use a mechanism-based approach to examine two hallmarks of mucormycosis: angioinvasion and tissue necrosis. These studies will lead to novel therapies that target mucoricin, host receptors and host cell- activation pathways. Some of these therapies are FDA-approved drug...