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Amoeba predation of Cryptococcus neoformans results in pleiotropic changes to traits associated with virulence

View ORCID ProfileMan Shun Fu, Livia C. Liporagi-Lopes, View ORCID ProfileSamuel R. dos Santos Júnior, Jennifer L. Tenor, View ORCID ProfileJohn R. Perfect, View ORCID ProfileChristina A. Cuomo, Arturo Casadevall
doi: https://doi.org/10.1101/2020.08.07.241190
Man Shun Fu
1Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Maryland, USA
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Livia C. Liporagi-Lopes
1Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Maryland, USA
2Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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Samuel R. dos Santos Júnior
1Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Maryland, USA
3Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil
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Jennifer L. Tenor
4Division of Infectious Diseases, Department of Medicine and Department of Molecular Genetics and Microbiology, Duke University, North Carolina, USA
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John R. Perfect
4Division of Infectious Diseases, Department of Medicine and Department of Molecular Genetics and Microbiology, Duke University, North Carolina, USA
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Christina A. Cuomo
5Infectious Disease and Microbiome Program, Broad Institute, Cambridge, Massachusetts, USA
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Arturo Casadevall
1Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Maryland, USA
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  • For correspondence: acasade1@jhu.edu
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Abstract

Phagocytic amoeboid predators such as amoeba have been proposed to select for survival traits in soil microbes such as Cryptococcus neoformans that can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Several prior studies have shown that incubation of various fungal species with amoeba can enhance their virulence. However, the mechanisms by which fungi adapt to amoeba and thus change their virulence are unknown. In this study we exposed three strains of C. neoformans (1 clinical and 2 environmental) to predation by Acanthamoeba castellanii for prolonged periods of time and then analyzed surviving colonies phenotypically and genetically. Surviving colonies were comprised of cells that expressed either pseudohyphal or yeast phenotypes, which demonstrated variable expression of such 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 for the oligopeptide transporter (CNAG_03013; OPT1) were observed among amoeba-passaged isolates from each of the three strains. In addition, isolates derived from environmental strains gained the capacity for enhanced macrophage toxicity after amoeba selection and carried mutations on the CNAG_00570 gene, which encodes Pkr1 (AMP-dependent protein kinase regulator) but were less 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, which confer increase resistance against protozoal predation. Given the myriad of 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.

Author summary Cryptococcus neoformans is a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immunocompromised patients. Cryptococcosis is a worldwide concern due to its high mortality rate. 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 animal host in its life cycle. Previous studies showed evidence that C. neoformans increases its pathogenicity after interacting with its environmental predator amoebae. Amoebae behave like macrophages, an important immune cell in human body, so it is considered as a training ground for pathogens to resist macrophages. However, how C. neoformans changes its virulence through interacting with amoebae is unknown. Here, we exposed C. neoformans to amoebae for a long period of time. We found that C. neoformans cells recovered from amoebae manifested numerous changes to phenotypes related to its virulence and one of the amoeba-passaged C. neoformans cells had enhanced ability to kill macrophages. We further analyzed their genome sequences and found various mutations in different cells of amoeba-passaged C. neoformans, showing that DNA mutations may be the major cause of the phenotypic changes after interacting with amoebae. 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.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted August 07, 2020.
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Amoeba predation of Cryptococcus neoformans results in pleiotropic changes to traits associated with virulence
Man Shun Fu, Livia C. Liporagi-Lopes, Samuel R. dos Santos Júnior, Jennifer L. Tenor, John R. Perfect, Christina A. Cuomo, Arturo Casadevall
bioRxiv 2020.08.07.241190; doi: https://doi.org/10.1101/2020.08.07.241190
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Amoeba predation of Cryptococcus neoformans results in pleiotropic changes to traits associated with virulence
Man Shun Fu, Livia C. Liporagi-Lopes, Samuel R. dos Santos Júnior, Jennifer L. Tenor, John R. Perfect, Christina A. Cuomo, Arturo Casadevall
bioRxiv 2020.08.07.241190; doi: https://doi.org/10.1101/2020.08.07.241190

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