Abstract
Cryptococcus neoformans var. grubii is considered to be the major cause of cryptococcosis in immunosuppressed patients. Understanding cell wall glycoproteins using lectins is of medical interest and can contribute to specific therapy. The aim of this study was to evaluate the carbohydrates on the cell wall of Cryptococcus neoformans var. grubii clinical isolates, using a fluorescein isothiocyanate-lectin binding protocol. Thirty yeast strains stocked in the culture collection were cultivated for 2 days at 30 °C with shaking. Cells were obtained by centrifugation, washed in phosphate-buffered saline, and a suspension of 107 cells/mL was obtained. To determine the binding profile of lectins, concanavalin A (Con A), wheat germ agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), and peanut agglutinin (PNA) conjugated to fluorescein were used. All the tested clinical isolates of Cryptococcus neoformans var. grubii were intensely stained by WGA, moderately stained by Con A, and weakly stained by PNA and UEA-I. Thus, Cryptococcus can be detected in clinical specimens such as blood and cerebrospinal fluid using the fluorescent lectin WGA, which may be considered as an option for detection in cases of suspected cryptococcosis with low laboratory sensitivity. Future applications may be developed using this basic tool.
Highlights
In developed countries, Cryptococcus neoformans var. grubii is considered to be the major cause of cryptococcosis, a serious human infection that results in death in some cases [1,2,3]
All of the tested strains of C. neoformans var. grubii had intense staining with wheat germ agglutinin (WGA), moderate staining with concanavalin A (Con A), and weak staining with peanut agglutinin (PNA) and Ulex europaeus agglutinin I (UEA-I) when compared with positive and negative controls
The cryptococcal cells were collected by centrifugation, washed in phosphate-buffered saline (PBS), and counted in a Neubauer chamber to obtain a standard suspension of 107 cells/mL following the method of Rodrigues et al [9]
Summary
Cryptococcus neoformans var. grubii is considered to be the major cause of cryptococcosis, a serious human infection that results in death in some cases [1,2,3]. The mechanisms that are involved in its pathogenesis can best be studied by starting with an understanding of cell wall glycoproteins, which are important characteristics of fungi of medical interest [4] In this context, different lectins have been used to determine a pattern of cellular glycoconjugates in Cryptococcus species, including C. neoformans var. The expression of N-acetyl-D-glucosamine may be analysed using wheat germ agglutinin (WGA), that of methyl-α-D-mannoside using concanavalin A (Con A), the L-fucose using Ulex europaeus agglutinin I (UEA-I), and for detection of D-galactose, and glucose/mannose on the cell wall surface, peanut agglutinin (PNA) is considered the most specific lectin [5]. These methods are laborious and may eventually provide inconclusive identifications [9] In this context, the aim of this study was to evaluate the expression of N-acetyl-D-glucosamine, methyl-α-D-mannoside, L-fucose, D-galactose, and glucose/mannose on the cell wall surface of. C. neoformans var. grubii through staining protocols using wheat germ agglutinin (WGA), concanavalin A (Con A), Ulex europaeus agglutinin I (UEA-I), and peanut agglutinin (PNA) lectins
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