Determination of chitin content in fungal cell wall: An alternative flow cytometric method

Abstract

The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3D/chs3D) and genes encoding predicted GlycosylPhosphatidylInositol (GPI)Àanchored proteins (pga31D/D and pga62D/D), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3D/chs3D and pga31D/D especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher cell wall chitin content. Although no relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi. ' 2013 International Society for Advancement of Cytometry Key terms chitin; flow cytometry; paradoxical effect; echinocandins CHITIN is a b-1,4-homopolymer of N-acetylglucosamine that is synthesized by chitin synthase enzymes (1). This polysaccharide is present in most fungi and together with b-1,3-glucan, plays a fundamental role in maintaining fungal cell integrity and conferring structural rigidity during growth and morphogenesis (1-3). Mutations in glucan synthase genes reduce glucan levels in the cell wall while stimulating salvage pathways, leading to increased chitin synthesis. This pathway restores the strength of the cell wall matrix and prevents antifungal action (4). This compensatory increase in cell wall chitin synthesis enables some Candida species to grow at high caspofungin concentrations, a phenomenon termed paradoxical growth effect. As chitin is not present in human cells, inhibition of chitin synthesis has been proposed as a potential, selective antifungal target.The assessment of cell wall chitin content based on glucosamine release through acid hydrolysis has been used extensively; however, this method is very laborious and time consuming (5-7). Epifluorescence microscopy has also been widely used to quantify chitin levels in fungi stained by Calcofluor White (CFW), a specific chitin dye (7-12). However, with this approach, only a limited number of yeast cells can be analyzed and the quantification of the fluorescence emitted cannot be performed ...

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