Abstract
Detailed analysis of dynamic fungal cell wall components is crucial to our understanding of fungal systematics and the biology and
physiology of fungal growth. In fungal pathogens this is of particular importance in examining their response to stress. However,
current methodologies do not permit fast and accurate or quantitative analysis of cell wall carbohydrate components. Here, we provide
a novel method permitting simultaneous quantitative analysis of the major cell wall components of Candida species relying on triplestaining
with fluorescent labeling of chitin, β-glucans and mannans. Quantification is based on flow cytometry whereas qualitative
analysis can be performed by direct imaging using fluorescence microscopy. We validated the method by determining the in vitro
responses of different Candida species to the challenge of antifungal treatment with caspofungin. The assay facilitated rapid analysis
of adaptive changes in cell wall composition upon caspofungin-induced damage. The method can be exploited for comprehensive
quantitative analysis of Candida cell wall components, with relevant implications for clinical diagnosis of fungal infections in general.
physiology of fungal growth. In fungal pathogens this is of particular importance in examining their response to stress. However,
current methodologies do not permit fast and accurate or quantitative analysis of cell wall carbohydrate components. Here, we provide
a novel method permitting simultaneous quantitative analysis of the major cell wall components of Candida species relying on triplestaining
with fluorescent labeling of chitin, β-glucans and mannans. Quantification is based on flow cytometry whereas qualitative
analysis can be performed by direct imaging using fluorescence microscopy. We validated the method by determining the in vitro
responses of different Candida species to the challenge of antifungal treatment with caspofungin. The assay facilitated rapid analysis
of adaptive changes in cell wall composition upon caspofungin-induced damage. The method can be exploited for comprehensive
quantitative analysis of Candida cell wall components, with relevant implications for clinical diagnosis of fungal infections in general.
| Original language | English |
|---|---|
| Article number | 101 |
| Number of pages | 9 |
| Journal | Journal of Microbiology and Modern Techniques |
| Volume | 2 |
| Issue number | 1 |
| Early online date | 20 Oct 2017 |
| DOIs | |
| Publication status | Published - 31 Dec 2017 |
Bibliographical note
This work was supported by the European Commission within the FP7 Framework Programme [Fungitect-Grant No 602125]. We also thank Thomas Sauer, Vienna Biocenter Campus (VBC), Austria, for technical support at the FACS facility of the MFPL, Karl Kuchler, MFPL-Department of Medical Biochemistry, Medical University of Vienna, Max F. Perutz Laboratories, Campus Vienna Biocenter, Vienna, Austria and Ernst Thuer, Centre for Genomic Regulation, Barcelona, Spain, for advice on statistical approaches. Neil Gow acknowledges the support of the Wellcome Trust and the MRC Centre for Medical MycologyKeywords
- Candida spp
- cell wall
- triple staining
- chitin
- glucans
- mannans
- flow cytometry