PROJECT SUMMARY/ABSTRACT This proposal describes a mentored three-year training plan that will provide the applicant with broad training in fungal pathogenesis, cellular immunology, and cell biology. The sponsor’s clinical and laboratory expertise in modeling and analyzing immune responses to microbial pathogens, the dedicated training plan, and institutional strengths in immunology, microbial pathogenesis, and cell biology all contribute to an outstanding training environment and a high likelihood of success. Aspergillus fumigatus is an opportunistic fungal pathogen and is the most common etiological agent of invasive aspergillosis (IA). Despite being constantly exposed to A. fumigatus conidia, immunocompetent individuals rarely get IA, because cells of the innate immune system are rapidly recruited to the lung where they phagocytose and clear conidia asymptomatically. However, IA is major complication for immunosuppressed individuals, such as those receiving solid organ or hematopoietic stem cell transplant and patients with hematological malignancies. Because these patient groups are expanding and there is currently a lack of effective anti-fungal drugs (fungus attributable mortality in these risk groups is upwards of 20%) there is a significant unmet need to understand the host immune response to IA. Preliminary data from a murine model of IA suggests that in the lung, neutrophils upregulate lysosome associated genes in response to fungal engagement. Regulation of lysosome biogenesis is controlled by the master transcription factors TFEB and TFE3 (TFEB/3). Additionally, TFEB/3 are activated in macrophages (MΦ) in response to stimulation with swollen heat-killed conidia. My training and research objective are to integrate the role of TFEB/3 and lysosome biogenesis in the context of the pulmonary anti-fungal immune responses and to acquire training in the technical disciplines needed for these studies. I hypothesize that A. fumigatus activates TFEB/3 in leukocytes through C-type lectin receptor signaling, and that TFEB/3 contribute to sterilizing immunity in the lung, by promoting intracellular killing of A. fumigatus conidia. Specific Aim 1 of the proposed research will determine the molecular basis for TFEB/3 activation in response to A. fumigatus. Using a ΜΦ cell line as a model leukocyte and both genetic and pharmacological strategies to inhibit key signaling molecules, I will uncover the relative contributions of various signaling pathways to the activation of TFEB/3 in ΜΦs. Specific Aim 2 of the proposed research I will define the contribution of TFEB/3 to anti-Aspergillus immunity. I will use a genetically engineered mouse model to deplete TFEB/3 from myeloid cells combined with our model of IA to uncover the contribution of TFEB/3 to murine survival, to the maintenance of lung tissue integrity, and to the ability of leukocytes to effectively clear conidia from the lung. These studies will reveal novel mechanisms of phagolysosomal ki...