Project 1

Lundquist Institute For Biomedical Innovation At Harbor-Ucla Medical Center, Torrance CA

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT Mucormycosis, caused by Mucorales fungi, is a life-threatening infection that occurs in patients immunocompromised by diabetic ketoacidosis, neutropenia, corticosteroid use, and/or increased serum iron. Commonly initiated via inhalation of spores, clinical hallmarks of mucormycosis are the virtually uniform presence 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. We determined that Mucorales initiate infection when the spore coating protein (CotH) invasins bind to integrins α3β1 on AECs. This binding triggers the activation of AEC epidermal growth factor receptor (EGFR) to induce invasion of germinated spores. We also discovered that germinated spores disseminate through the pericytes and extracellular matrix to invade endothelial cells via CotH interacting with Glucose Regulated Protein 78 (GRP- 78). We also made the seminal discovery that Mucorales damage host cells by a cell-associated ricin-like toxin, named mucoricin. Both CotH invasins and mucoricin are required for pathogenesis and antibodies targeting either protein are protective in mice. More recently, we discovered that Mucorales also secrete another toxin that belongs to the sphingolipid family, and is most operative during hypoxia and hematogenous dissemination. Our goal is to gain insights into the mechanisms by which toxins dampens the host immune response and to develop multiple immunotherapeutic strategies that will collectively nullify critical virulence factors of Mucorales and enhance disease diagnostics. Thus, in Aim 1 we will determine the mechanism by which mucoricin prevents neutrophil coordinated clustering (swarming) and cause death to this first-line innate host defense. We will also assess the benefit of combining a protective anti-mucoricin monoclonal antibody (MAb) with antifungals and other non-cellular FDA-approved immunomodulators (cytokines, immune checkpoint inhibitors, and hematopoietic growth factors). In Aim 2 we will characterize the role and the mechanism of action of the sphingolipid toxin (sphingotoxin) in mucormycosis pathogenesis by using genetic and biochemical blocking strategies and conduct comparative in vitro and in vivo transcriptomic studies using isogenic strain pairs lacking the ability to secrete the toxin. In Aim 3, we will examine the feasibility of developing a loop-mediated isotheral amplification (LAMP) assay for rapid diagnostics of mucormycosis using unique targets of Mucorales including rDNA, CotH, mucoricin, and other genes identified by the Genetic/Transcriptomic Core. Overall, our well-integrated proposed aims will mechanistically define the contribution of toxins to mucormycosis pathogenesis, and identify novel immunotherapeutic strategies and diagnostics that will be candidates for clinical testing.