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 range of pathogenic and non-pathogenic species.