Related Experiment Video
Updated: May 26, 2026
Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins
Published on: December 24, 2017
Use of fungal derived polysaccharide-conjugated particles to probe Dectin-1 responses in innate immunity
Jenny M Tam1, Michael K Mansour, Nida S Khan
1Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, GRJ-5-504, Boston, MA 02114, USA.
Abstract:
The number of life-threatening fungal infections has risen in immunocompromised patients, and identification of the rules that govern an appropriate immune response is essential to develop better diagnostics and targeted therapeutics. The outer cell wall component on pathogenic fungi consists of β-1,3-glucan, and Dectin-1, a pattern recognition receptor present on the cell surface of innate immune cells, binds specifically to this carbohydrate. A barrier in understanding the exact immunological response to pathogen-derived carbohydrate epitopes is the presence of multiple types of carbohydrate moieties on fungal cell walls. To dissect the immunological mechanisms used to recognize pathogens, a system of "fungal like particles" was developed that consisted of polystyrene beads, which mimicked the three dimensional shape of the fungus, coated covalently with purified β-1,3-glucan derived from Saccharomyces cerevisiae. The morphology of the β-1,3-glucan layer was examined by immunofluorescence, flow cytometery, and immuno-transmission electron microscopy. The covalent linkages of the β-1,3-glucan to the polystyrene surface were stable after subjecting the beads to detergents. By pre-treating β-1,3-glucan beads with laminarinase, a specific β-1,3-gluconase, the reactivity of the anti-β-1,3-glucan antibody was abrogated in comparison to treatment with proteinase K indicating that the coating of these beads was predominantly β-1,3-glucan. TNF-α was also measured by stimulating bone-marrow derived macrophages with the β-1,3-glucan beads, and showed a dose dependent response compared to soluble β-glucan, insoluble β-1,3-glucan, uncoated beads, and soluble β-1,3-glucan mixed with uncoated beads. Finally, β-1,3-glucan beads were incubated with GFP-Dectin-1 expressing macrophages and imaged using confocal microscopy. β-1,3-beads were taken up within minutes and retained Dectin-1 recruitment to the phagosome as compared to uncoated beads. These data describe a unique fungal-like particle system that will permit immunologists to probe the critical steps in early recognition of pathogen-derived fungal carbohydrate antigens by innate immune cells.
Insights
Researchers developed fungal-like particles to study immune responses to fungal infections. These particles mimic fungal cell walls, enabling better understanding of how innate immune cells recognize and respond to fungal pathogens.
Area of Science:
- Immunology
- Mycology
- Biotechnology
Background:
- Rising incidence of life-threatening fungal infections in immunocompromised individuals necessitates improved diagnostics and therapeutics.
- Understanding the immune response to fungal cell wall components, particularly β-1,3-glucan, is crucial.
- Challenges exist in dissecting immune recognition due to complex fungal cell wall carbohydrate structures.
Purpose of the Study:
- To develop a novel system for studying the immunological recognition of fungal carbohydrate antigens.
- To create "fungal-like particles" (FLPs) mimicking fungal morphology and cell wall composition.
- To investigate the interaction of FLPs with innate immune cells, specifically Dectin-1.
Main Methods:
- Synthesized polystyrene beads coated with purified β-1,3-glucan from Saccharomyces cerevisiae.
- Characterized FLP morphology and β-1,3-glucan integrity using immunofluorescence, flow cytometry, and immuno-transmission electron microscopy.
- Assessed immune cell activation (TNF-α production) and Dectin-1 engagement using bone-marrow derived macrophages stimulated with FLPs.
Main Results:
- Developed stable, β-1,3-glucan-coated FLPs confirmed by enzymatic and antibody-based assays.
- Demonstrated dose-dependent TNF-α production by macrophages stimulated with FLPs, indicating effective immune activation.
- Observed rapid uptake of FLPs by Dectin-1 expressing macrophages and sustained Dectin-1 recruitment to phagosomes.
Conclusions:
- The developed fungal-like particle system provides a robust tool for dissecting early innate immune recognition of fungal carbohydrate antigens.
- This system facilitates detailed investigation into the mechanisms of Dectin-1-mediated fungal pathogen recognition.
- The findings pave the way for improved diagnostics and targeted immunotherapies for fungal infections.
