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Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans
Published on: March 19, 2015
Biotinylation and characterization of Cryptococcus neoformans cell surface proteins
A J Foster1, R A Bird, S N Smith
1Life and Health Sciences, Aston University, Birmingham, UK.
Journal of Applied Microbiology
|July 26, 2007
Summary
Researchers developed a novel method to identify cryptococcal cell-surface proteins using biotinylation and flow cytometry. This technique revealed dynamic protein distribution, particularly around cell division sites in Cryptococcus neoformans.
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- Cryptococcus neoformans is a significant fungal pathogen.
- Understanding cryptococcal cell-surface proteins is crucial for developing targeted therapies.
- Current methods for analyzing cell-surface proteins are limited.
Purpose of the Study:
- To develop and validate a novel procedure for isolating and characterizing cryptococcal cell-surface proteins.
- To investigate the distribution and exposure of these proteins in situ.
- To identify specific protein entities on the cryptococcal cell wall.
Main Methods:
- Biotinylation of cell-surface proteins using sulfo-NHS-biotin.
- Labeling with fluorescein isothiocyanate (FITC)-streptavidin.
- Analysis by flow cytometry for protein exposure and ligand-receptor interactions (EC50, Fmax, Hn).
- Visualization of protein distribution using confocal microscopy.
- Isolation and characterization by affinity chromatography and SDS/PAGE.
Main Results:
- Successful labeling and visualization of cell-surface proteins in both acapsulate and encapsulated C. neoformans.
- Flow cytometry provided quantitative data on protein exposure and binding affinity.
- Confocal microscopy revealed localized protein distribution, especially at cell division sites.
- SDS/PAGE identified two major protein bands (43 and 57 kDa) on acapsulate cell walls.
- Stationary phase and acapsulate cells showed higher affinity for biotin.
Conclusions:
- A novel, versatile method for in situ identification and characterization of cryptococcal cell-surface proteins was established.
- The technique is applicable to both acapsulate and encapsulated C. neoformans.
- Protein distribution is dynamic and localized, particularly around sites of cell division and cell wall trauma.
