Amoeba Predation of Cryptococcus neoformans Results in Pleiotropic Changes to Traits Associated with Virulence

Amoeboid predators, such as amoebae, are proposed to select for survival traits in soil microbes such as Cryptococcus neoformans; these traits can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Consistent with this notion, incubation of various fungal species with amoebae enhanced their virulence, but the mechanisms involved are unknown. In this study, we exposed three strains of C. neoformans (1 clinical and 2 environmental) to predation by Acanthamoeba castellanii for prolonged times and then analyzed surviving colonies phenotypically and genetically. Surviving colonies comprised cells that expressed either pseudohyphal or yeast phenotypes, which demonstrated variable expression of traits associated with virulence, such as capsule size, urease production, and melanization. Phenotypic changes were associated with aneuploidy and DNA sequence mutations in some amoeba-passaged isolates, but not in others. Mutations in the gene encoding the oligopeptide transporter (CNAG_03013; OPT1) were observed among amoeba-passaged isolates from each of the three strains. Isolates derived from environmental strains gained the capacity for enhanced macrophage toxicity after amoeba selection and carried mutations on the CNAG_00570 gene encoding Pkr1 (AMP-dependent protein kinase regulator) but manifested reduced virulence in mice because they elicited more effective fungal-clearing immune responses. Our results indicate that C. neoformans survival under constant amoeba predation involves the generation of strains expressing pleiotropic phenotypic and genetic changes. Given the myriad potential predators in soils, the diversity observed among amoeba-selected strains suggests a bet-hedging strategy whereby variant diversity increases the likelihood that some will survive predation.