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Updated: Jun 25, 2026
Setting a Successful Sorting for Extracellular Vesicle Isolation
Published on: October 11, 2024
Sec6-dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus neoformans
John Panepinto1, Kazimierz Komperda, Susana Frases
1Section of Infectious Diseases, Department of Medicine, University of Illinois at Chicago College of Medicine, Chicago, IL, USA.
Abstract:
The cell wall of pathogenic fungi such as Cryptococcus neoformans, provides a formidable barrier to secrete virulence factors that produce host cell damage. To study secretion of virulence factors to the cell periphery, sec6 RNAi mutant strains of C. neoformans were tested for virulence factor expression. The studies reported here show that SEC6 RNAi mutant strains were defective in a number of virulence factors including laccase, urease as well as soluble polysaccharide and demonstrated attenuated virulence in mice. Further analysis by transmission electron microscopy detected the production of abundant extracellular exosomes in wild-type strains containing empty plasmid, but a complete absence in the iSEC6 strain. In addition, a green fluorescent protein-laccase fusion protein demonstrated aberrant localization within cytoplasmic vesicles in iSEC6 strains. In contrast, iSEC6 strains retained normal growth at 37 degrees C, as well as substantially normal capsule formation, phospholipase activity and total secreted protein. These results provide the first molecular evidence for the existence of fungal exosomes and associate these vesicles with the virulence of C. neoformans.
Insights
Cryptococcus neoformans SEC6 RNAi mutant strains show defects in virulence factors and attenuated virulence in mice. This study provides the first evidence for fungal exosomes and links them to C. neoformans virulence.
Area of Science:
- Mycology
- Cell Biology
- Infectious Diseases
Background:
- Pathogenic fungi like Cryptococcus neoformans possess a cell wall that facilitates the secretion of virulence factors, contributing to host cell damage.
- Understanding the mechanisms of virulence factor secretion is crucial for developing targeted antifungal therapies.
Purpose of the Study:
- To investigate the role of SEC6 in the secretion of virulence factors in Cryptococcus neoformans.
- To explore the potential involvement of fungal exosomes in C. neoformans pathogenesis.
Main Methods:
- Utilized SEC6 RNAi mutant strains of C. neoformans for virulence factor expression analysis.
- Employed transmission electron microscopy to examine extracellular vesicle production.
- Assessed the localization of a green fluorescent protein-laccase fusion protein.
Main Results:
- SEC6 RNAi mutant strains exhibited defects in laccase, urease, and soluble polysaccharide secretion, along with attenuated virulence in a mouse model.
- Transmission electron microscopy revealed abundant extracellular exosomes in wild-type strains but their absence in SEC6 RNAi strains.
- Aberrant cytoplasmic localization of a laccase fusion protein was observed in SEC6 RNAi strains, while other functions remained largely unaffected.
Conclusions:
- This study presents the first molecular evidence for the existence of fungal exosomes.
- Fungal exosomes are associated with the virulence of Cryptococcus neoformans.
- SEC6 plays a critical role in the secretion pathway of C. neoformans, potentially involving exosome biogenesis.
