Amoeba Predation of Cryptococcus: A Quantitative and Population Genomic Evaluation of the Accidental Pathogen Hypothesis

The “Amoeboid Predator-Fungal Animal Virulence Hypothesis” posits that interactions with environmental phagocytes shape the evolution of virulence traits in fungal pathogens. In this hypothesis, selection to avoid predation by amoeba inadvertently selects for traits that contribute to fungal escape from phagocytic immune cells. Here, we investigate this hypothesis in the human fungalpathogens Cryptococcus neoformans and Cryptococcus deneoformans. Applying quantitative trait locus (QTL) mapping and comparative genomics, we discovered a cross-species QTL region that is responsible for variation in resistance to amoeba predation. In C. neoformans, this same QTL was found to have pleiotropic effects on melanization, an established virulence factor. Through fine mapping and population genomic comparisons, we identified the gene encoding the transcription factor BZP4 that underlies this pleiotropic QTL and we show that decreased expression of this gene reduces melanization and increases susceptibility to amoeba predation. Despite the joint effects of BZP4 on amoeba resistance and melanin production, we find no relationship between BZP4 genotype and escape from macrophages or virulence in murine models of disease. Our findings provide new perspectives on how microbial ecology shapes the genetic architecture of fungal virulence, and suggests the need for more nuanced models for the evolution of pathogenesis that account for the complexities of both microbe-microbe and microbe-host interactions. The goal of the RNAseq experiment was to determine the change in expression when Cryptococcus neoformans cells are in coculture with Acanthamoeba castellanii or alone. This was accomplished by growing C. neoformans cells on V8 media and either adding amoeba or a control to the plate they are on. Cells were then left to interact for 48 hours. Cells were then collected for sequencing. 14 strains in total were used. With two of those strains having a single duplicate. The two strains with the duplicates are the parent strains a cross that generated the other 12 samples.