Project Summary Environmental microbes are affected by and evolve to succeed in the changing environments they inhabit. As a result, changes in the environment can select for microbes that are more or less capable of causing human infections. Cryptococcus is a genus of opportunistic fungal pathogens that cause approximately 200,000 deaths in HIV/AIDS individuals. Cryptococcus species grow in an environmental niche before causing “accidental” infections of mammals with no known return to the environment after infection or spread between hosts. Notably, the pathogens within the genus split from the nonpathogens approximately 100 million years ago, concurrent with the development of the ability to grow at human body temperature. In the proposed work, I will use the Cryptococcus genus to explore the genetic basis of two pathogenesis relevant traits that are changing in the environment because of human activities, with an eye towards using Cryptococcus as a model to understand how these changes in environment may alter other environmental fungi. Project 1 will focus on the transition to thermotolerance that occurred in the Cryptococcus genus. Elevated global temperatures as a result of climate change will likely select for environmental microbes that can grow at higher temperatures. I will employ high-throughput genetics approaches (TN-seq) to characterize genome-wide contributions to growth at various temperatures across the entire Cryptococcus genus, including both pathogens and nonpathogens. I will then use allele swap experiments to explore effects of sequence variation, lineage-specific gene content, and transcriptional reprogramming. Project 2 will focus on resistance to azole class drugs. Modern agricultural practices employ large amounts of azole drugs to control fungal plant pathogens. Environmental fungi are also commonly exposed to these drugs and thus are being selected for drug resistance. I will use a diploid complex trait genetics approach (RH-seq) that is based on TN-seq to explore the basis of azole drug resistance in a large (n=387) strain collection of environmental and clinical isolates from Africa. I will again use allele swap experiments to validate drug resistance mutations and test the contribution of epistasis to antifungal drug resistance. This work will reveal fundamental mechanisms underlying adaptation to high temperatures and antifungal drugs. It will also generate mutant libraries across an entire genus and mapping populations for a large number of C. neoformans isolates that can be used in future projects across the community. An expanded understanding of heat tolerance and of drug resistance may aid in the development of novel antifungal agents or more efficient use of the currently available drugs, leading to improved outcomes for treatment of invasive fungal diseases. Finally, a better understanding of how human activities lead to changes in environmental microbes can provide both predictions and potential guidance for intervention.

Narrative Many pathogens live in the environment prior to encountering a mammalian host. As a result, human interactions with the environment can alter the population of pathogens we are likely to encounter and thus with which we are infected. This proposal aims to understand how heat tolerance and drug resistance are controlled by genetic variation at two different time scales (across a genus and within a species) of opportunistic environmental fungal pathogens in order to better predict how pathogens are likely to be altered by human activities.