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Updated: Jul 1, 2025
Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions
Published on: August 26, 2021
Corrected and republished from: "Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal
Rafael F Castelli1,2, Alana Pereira3, Leandro Honorato4
1Instituto Carlos Chagas, Fundação Oswaldo Cruz (Fiocruz), Curitiba, Brazil.
Abstract:
Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants expected to lack NOP16 expression, we observed a reduced EV production. Whole-genome sequencing, RNA-Seq, and cellular proteomics revealed that, contrary to our initial findings, these mutants expressed Nop16 but exhibited altered expression of 14 genes potentially involved in sugar transport. Based on this observation, we designated these mutant strains as Past1 and Past2, representing potentially altered sugar transport. Analysis of the small molecule composition of EVs produced by wild-type cells and the Past1 and Past2 mutant strains revealed not only a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the Past1 and Past2 mutant strains were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the mutant cells in G. mellonella. These results connect EV biogenesis, cargo, and cryptococcal virulence.
Insights
Mutant fungal strains showed reduced extracellular vesicle (EV) production and altered small molecule cargo, leading to decreased virulence in infection models. Wild-type EVs restored virulence, linking EV production, cargo, and fungal pathogenicity.
Area of Science:
- Mycology
- Cell Biology
- Infectious Diseases
Background:
- Fungal extracellular vesicles (EVs) contain small molecules with poorly understood biological roles.
- The gene NOP16 is implicated in the efficacy of benzimidazoles against Cryptococcus deuterogattii.
Purpose of the Study:
- To investigate the role of NOP16 in Cryptococcus deuterogattii.
- To characterize the impact of altered NOP16 expression on EV production and cargo.
- To determine the contribution of EVs to fungal virulence.
Main Methods:
- Phenotypic characterization of C. deuterogattii mutants.
- Whole-genome sequencing, RNA-Seq, and cellular proteomics.
- Analysis of small molecule composition in EVs.
- Virulence assessment using a Galleria mellonella infection model.
Main Results:
- Mutants (Past1, Past2) exhibited reduced EV production and altered EV small molecule composition.
- These mutants showed altered expression of genes involved in sugar transport.
- Past1 and Past2 strains were hypovirulent in the G. mellonella model.
- Co-injection with wild-type EVs, but not mutant EVs, restored virulence.
Conclusions:
- NOP16 expression is linked to EV biogenesis and cargo.
- EVs play a significant role in Cryptococcus deuterogattii virulence.
- Altered EV production and cargo impact fungal pathogenicity.
