Utilizing Natural Variation and De Novo Mutation to Understand Cryptococcus Evolution

The evolution of pathogenesis, in many cases, is a story of competition between host and microbe; however, many opportunistic pathogens are primarily found in niches other than the host environment. Such pathogens frequently lack host-to-host transmission, and there may be limited opportunities for an infectious population to be re-dispersed back into the environment. Observations such as these motivate the hypothesis that the evolution of virulence traits in opportunistic pathogens may be primarily driven by environmental selective pressures, rather than the host-environment per se.

For Cryptococcus the ability to survive interactions with macrophages and the ability to grow at host body temperatures are indispensable to its pathogenic capabilities. The work presented here aims to dissect the genetic underpinnings of these virulence traits using the abundant natural variation of Cryptococcus and using the accumulation de novo mutations associated with growth under relevant stressors.

An important aspect of the hypotheses surrounding Cryptococcus evolution is the predator-prey interactions it has with free-living amoeba. Amoebae are able to consume Cryptococcus cells in a manner similar to how macrophages phagocytose and digest infectious cells. This similarity is the basis of the “Amoeboid Predator-Fungal Animal Virulence Hypothesis” which posits that amoeba act as training grounds for environmental fungal pathogens and thus inadvertently select for resistance to immune phagocytes. I tested this hypothesis by using QTL mapping to identify genes and alleles that are involved in amoebae resistance in both C. neoformans and C. deneoformans. I identified QTL that contribute to amoeba resistance, and discovered that the largest effect QTL in both species localize to homologous regions of the genome, suggesting a shared mechanism of amoeba resistance. In C. neoformans, this QTL also contributes to variation in melanization. I identified a causal variant for this QTL, a non-coding deletion upstream of a transcription factor, BZP4. Contrary to the predictions of the Amoeboid Predator-Fungal Animal Virulence Hypothesis, I did not find an association between the ability to survive amoeba predation and virulence in either in vitro or in vivo models of infection. These findings suggest a re-evaluation of the amoeba predation model for the evolution of pathogenesis, suggesting that factors other than amoeba may provide the significant selective pressures that underlie virulence ability.

I extended my quantitative analyses of Cryptococcus to two important factors involved in both environmental and disease contexts: thermal and low pH tolerance. In doing so, I discovered multiple pleiotropic QTL involved in general growth that also dictate stress tolerance in both high temperature and low pH environments. By fitting growth data to a Gompertz growth model and QTL mapping based on the parameters of this model, I discovered a novel QTL that effects lag, the time it takes for a population of cells to begin growing at an exponential rate. This lag QTL is pleiotropic across growth conditions. I identified a candidate allele for the lag QTL, a 9-bp deletion in CNAG_01111, a gene that has been found to impact growth initiation in other species of fungi.

Finally, taking a complimentary approach to understanding the role of genes in environmental survival, I experimentally evolved a C. neoformans strain in conditions of thermal stress and fludioxonil stress. I discovered that strains evolved at high temperatures lose tolerance to fludioxonil and strains evolved in fludioxonil lose temperature tolerance. Furthermore, the loss of fludioxonil tolerance in the high temperature evolved strains can be partially rescued by growing them on media containing fludioxonil. This rescue results in a proportional loss of thermal tolerance. Studying the genomic changes behind the evolved phenotypes I discovered multiple large scale deletions and one multi-gene duplication associated with fludioxonil resistance and a single multi-gene deletion associated with thermal tolerance. There are also a variety of small scale mutations associated with each evolved condition, including mutations of genes in the HOG and ergosterol pathway that are responsible for fludioxonil resistance. Mutations in uncharacterized multidrug transporters are frequently associated with fludioxonil resistance, suggesting that the evolved strains might also have altered resistance to other antifungals. These findings highlight the polygenic and pleiotropic genetic architecture of adaptation in C. neoformans on an ever warming planet with increased use of agricultural antifungals. The trade-offs found may represent a good sign for the use of phenylpyrroles as an agricultural antifungal.

Collectively, my work sheds light on genes and alleles involved in environmental survival while also making important connections back to human disease. It also exhibits the importance of utilizing the natural variation of fungal pathogens to study the evolutionary hypothesis surrounding virulence traits. The studies reported here also provide significant groundwork for many new insights into virulence genes and the origins of Cryptococcus pathogenicity.