TY - JOUR
T1 - Amoeba predation of Cryptococcus neoformans results in pleiotropic changes to traits associated with virulence
AU - Fu, Man Shun
AU - Liporagi-Lopes, Livia C.
AU - dos Santos Júnior, Samuel R.
AU - Tenor, Jennifer L.
AU - Perfect, John R.
AU - Cuomo, Christina A.
AU - Casadevall, Arturo
N1 - Funding Information:
A.C. is supported by National Institutes of Health grants AI052733, AI15207, and HL059842. C.A.C is supported by National Institute of Allergy and Infectious Diseases, National Institutes of Health, award U19AI110818. J.R.P is supported by Public Health Services grants AI73896 and AI93257.
Funding Information:
We thank the Broad Institute Microbial Omics Core for generating the DNA libraries and the Genomics Platform for the sequencing for this study. We thank H. J. Tsai for advice on figure design. A.C. is supported by National Institutes of Health grants AI052733, AI15207, and HL059842. C.A.C is supported by National Institute of Allergy and Infectious Diseases, National Institutes of Health, award U19AI110818. J.R.P is supported by Public Health Services grants AI73896 and AI93257.
Publisher Copyright:
© 2021 Fu et al.
PY - 2021
Y1 - 2021
N2 - 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 macro-phages. 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. IMPORTANCE Cryptococcus neoformans is a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immuno-compromised patients. A major question in the field is how an environmental yeast such as C. neoformans becomes a human pathogen when it has no need for an animal host in its life cycle. Previous studies showed that C. neoformans increases its pathoge-nicity after interacting with its environmental predator amoebae. Amoebae, like macro-phages, are phagocytic cells that are considered an environmental training ground for pathogens to resist macrophages, but the mechanism by which C. neoformans changes its virulence through interactions with protozoa is unknown. Our study indicates that fungal survival in the face of amoeba predation is associated with the emergence of pleiotropic phenotypic and genomic changes that increase the chance of fungal survival, with this diversity suggesting a bet-hedging strategy to ensure that some forms survive.
AB - 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 macro-phages. 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. IMPORTANCE Cryptococcus neoformans is a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immuno-compromised patients. A major question in the field is how an environmental yeast such as C. neoformans becomes a human pathogen when it has no need for an animal host in its life cycle. Previous studies showed that C. neoformans increases its pathoge-nicity after interacting with its environmental predator amoebae. Amoebae, like macro-phages, are phagocytic cells that are considered an environmental training ground for pathogens to resist macrophages, but the mechanism by which C. neoformans changes its virulence through interactions with protozoa is unknown. Our study indicates that fungal survival in the face of amoeba predation is associated with the emergence of pleiotropic phenotypic and genomic changes that increase the chance of fungal survival, with this diversity suggesting a bet-hedging strategy to ensure that some forms survive.
KW - Acanthamoeba castellanii
KW - Amoeba
KW - Cryptococcus neoformans
KW - Evolution
KW - Host-pathogen interactions
KW - Macrophages
KW - Opportunistic fungi
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U2 - 10.1128/mBio.00567-21
DO - 10.1128/mBio.00567-21
M3 - Article
C2 - 33906924
AN - SCOPUS:85104734140
SN - 2161-2129
VL - 12
JO - mBio
JF - mBio
IS - 2
M1 - e00567-21
ER -