Project Abstract Cryptococcus species are environmental fungi that cause disease primarily in immunocompromised populations, including a deadly cryptococcal meningitis that contributes to 15% of HIV/AIDS-related deaths. When inhaled into the lungs, these fungi must adapt rapidly to survive a variety of stresses encountered in the human host, including high temperature stress, changes in pH and oxidative stress. In cases of persistent disease, Cryptococcus must evade host immune defenses and resist antifungal drug treatment. Adaptive genomic changes in Cryptococcus known to enhance virulence or cause drug resistance during infection include base substitutions, small insertions/deletions and aneuploidy. Transposable elements (TEs) are small, mobile DNA elements present in the genomes of most eukaryotic organisms that are capable of causing significant genomic changes and phenotypic variation. The potential role of TEs in Cryptococcus and other pathogenic fungal species (Candida and Aspergillus) in contributing to fungal pathogenesis or drug resistance is largely unexplored. We recently identified TE mobilization in Cryptococcus deneoformans as a significant cause of mutation in a murine model of infection. Mutations by TEs in reporter genes for drug resistance were dramatically elevated at high temperature (37° host-relevant temperature) in vitro, suggesting that heat stress stimulates TE mobility in the cryptococcal genome. Additionally, we demonstrated TE insertion as a cause of drug resistance to clinical antifungal agents rapamycin/FK506 and 5-fluorocytosine in vitro. Our study was the first to identify TE mobilization as a cause of mutation during infection in a pathogenic fungus. In addition, TE mutagenesis in response to heat stress had not been described previously in any model yeast species. Remarkably, the heat- responsive TEs identified in C. deneoformans include both DNA transposons and retrotransposons, each with distinct modes of mobilization and preferred sites of genomic integration. In the proposed research, we seek to 1) determine whether heat stress is the primary cause of increased TE mobilization during C. deneoformans infection, 2) identify regulators of heat stress-induced TE mutagenesis in C. deneoformans, and 3) determine whether TEs mobilize in other cryptococcal species (C. neoformans or C. gattii) in vitro or during host infection. Elucidating the mechanisms of adaptive change that enhance fungal pathogenesis or enable drug resistance is critical in developing and maintaining effective antifungal treatments. This study will further our understanding of the types of stress-induced mutations that arise during cryptococcal infection that may contribute to disease persistence and variations in clinical outcomes for patients. In addition, our study will highlight a set of experimental approaches, infection models and sequencing tools that can be used to identify and quantitate genetic mutations (TE and non-TE) in a broad rang...