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 fungal pathogens 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. Author summary A prominent hypothesis for the evolution of many environmental pathogens proposes that opportunistic pathogenesis is an “accidental” by-product of selection to survive encounters with microbial predators. Chief among the predators that have been suggested as relevant to the evolution of virulence are phagocytic amoebae. Amoebae share many characteristics with macrophages and other primary immune cells that microbial pathogens encounter during infection of animal hosts. This has led to the suggestion that amoebae may act as “training grounds” for both bacterial and fungal pathogens. In this study we test key tenets of the accidental pathogen hypothesis by examining two related questions: “Do alleles important for survival in the face of amoeba predation correspond to known virulence genes? And does genetic variation that increases resistance to amoeba predation increase virulence potential?” We carried out quantitative trait locus (QTL) mapping in two species of the human fungal pathogen Cryptococcus and identified an orthologous QTL, shared by the two species, where allelic variation is a key predictor of resistance to amoeba predation. In C. neoformans we show that this QTL corresponds to a deletion upstream of a transcription factor gene, BZP4 . Variation at BZP4 also predicts melanin synthesis, another trait implicated in Cryptococcus virulence. Although BZP4 genotype is a strong predictor of resistance to amoeba predation, we find no correlation between genetic variation at this locus and the ability to proliferate in macrophages or to kill animal hosts. Our findings suggest that the evolutionary landscape of fungal virulence is complex, and highlights the importance of accounting for natural genetic variation when evaluating evolutionary hypotheses. ### Competing Interest Statement The authors have declared no competing interest.