RESUMO
Glycoside hydrolases (GHs) are enzymes that participate in many biological processes of fungi and other organisms by hydrolyzing glycosidic linkages in glycosides. They play fundamental roles in the degradation of carbohydrates and the assembly of glycoproteins and are important subjects of studies in molecular biology and biochemistry. Based on amino acid sequence similarities and 3-dimensional structures in the carbohydrate-active enzyme (CAZy), they have been classified in 171 families. Members of some of these families also exhibit the activity of trans-glycosydase or glycosyl transferase (GT), i.e., they create a new glycosidic bond in a substrate instead of breaking it. Fungal glycosidases are important for virulence by aiding tissue adhesion and colonization, nutrition, immune evasion, biofilm formation, toxin release, and antibiotic resistance. Here, we review fungal glycosidases with a particular emphasis on Sporothrix species and C. albicans, two well-recognized human pathogens. Covered issues include a brief account of Sporothrix, sporotrichosis, the different types of glycosidases, their substrates, and mechanism of action, recent advances in their identification and characterization, their potential biotechnological applications, and the limitations and challenges of their study given the rather poor available information.
RESUMO
The cell wall (CW) of fungi exhibits a complex structure and a characteristic chemical composition consisting almost entirely of interacting crystalline and amorphous polysaccharides. These are synthesized by a number of sugar polymerases and depolymerases encoded by a high proportion of the fungal genome (for instance, 20% in Saccharomyces cerevisiae). These enzymes act in an exquisitely coordinated process to assemble the tridimensional and the functional structure of the wall. Apart from playing a critical role in morphogenesis, cell protection, viability and pathogenesis, the CW represents a potential target for antifungals as most of its constituents do not exist in humans. Chitin, ß-glucans and cellulose are the most frequent crystalline polymers found in the fungal CW. The hexosamine biosynthesis pathway (HBP) is critical for CW elaboration. Also known as the Leloir pathway, this pathway ends with the formation of UDP-N-GlcNAc after four enzymatic steps that start with fructose-6-phosphate and L-glutamine in a short deviation of glycolysis. This activated aminosugar is used for the synthesis of a large variety of biomacromolecules in a vast number of organisms including bacteria, fungi, insects, crustaceans and mammalian cells. The first reaction of the HBP is catalyzed by GlcN-6-P synthase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16), a critical enzyme that has been considered as a potential target for antifungals. The enzyme regulates the amount of cell UDP-N-GlcNAc and in eukaryotes is feedback inhibited by the activated aminosugar and other factors. The native and recombinant forms of GlcN-6-P synthase has been purified and characterized from both prokaryotic and eukaryotic organisms and demonstrated its critical role in CW remodeling and morphogenesis after exposure of some fungi to agents that stress the cell surface by interacting with wall polymers. This review deals with some of the cell compensatory responses of fungi to wall damage induced by Congo Red and Calcofluor White.
Assuntos
Sporothrix , beta-Glucanas , Animais , Antifúngicos , Benzenossulfonatos , Parede Celular/metabolismo , Celulose , Quitina , Vermelho Congo , Glutamina , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Hexosaminas/análise , Hexosaminas/metabolismo , Humanos , Mamíferos/metabolismo , Polímeros/análise , Sporothrix/metabolismo , Açúcares , Difosfato de Uridina , beta-Glucanas/análiseRESUMO
The most frequently isolated human fungal pathogen is Candida albicans which is responsible for about 50% of all Candida infections. In healthy individuals, this organism resides as a part of the normal microbiota in equilibrium with the host. However, under certain conditions, particularly in immunocompromised patients, this opportunistic pathogen adheres to host cells causing serious systemic infections. Thus, much effort has been dedicated to the study of its physiology with emphasis on factors associated to pathogenicity. A representative analysis deals with the mechanisms of glycoprotein assembly as many cell surface antigens and other macromolecules that modulate the immune system fall within this chemical category. In this regard, studies of the terminal protein glycosylation stage which occurs in Golgi vesicles has led to the identification of nucleotidases that convert glycosyltransferase-generated dinucleotides into the corresponding mononucleotides, thus playing a double function: their activity prevent inhibition of further glycosyl transfer by the accumulation of dinucleotides and the resulting mononucleotides are exchanged by specific membrane transporters for equimolecular amounts of sugar donors from the cytosol. Here, using a simple protocol for protein separation we isolated a bifunctional nucleotidase from C. albicans active on GDP and UDP that was characterized in terms of its molecular mass, response to bivalent ions and other factors, substrate specificity and affinity. Results are discussed in terms of the similarities and differences of this nucleotidase with similar counterparts from other organisms thus contributing to the knowledge of a bifunctional diphosphatase not described before in C. albicans.
Assuntos
Candida albicans , Candidíase , Humanos , Pirofosfatases/metabolismoRESUMO
It is well documented that disturbance of cell surface by some agents triggers compensatory responses aimed to maintain the cell wall integrity in fungi and other organisms. Here, the thermodimorphic fungus Sporothrix globosa, a member of the pathogenic clade of the Sporothrix complex, was propagated in yeast-peptone-dextrose medium under conditions to obtain the mycelium (pH 4.5, 27-28 °C) or the yeast (pH 7.8, 32-34 °C) morphotypes in the absence and presence of the wall-interacting dyes Congo Red (CR) and Calcofluor White (CFW) either alone or in combination. After different periods of time, growth, cell morphology and activity of glucosamine-6-phosphate synthase (GlcN-6-P synthase), an ubiquitous enzyme that plays a crucial role in cell wall biogenesis, were determined. CR and to a lower extent CFW affected growth and morphology of both fungal morphotypes and significantly increased enzyme activity. Notoriously, CR or CR in combination with CFW induced the transient conversion of yeasts into conidia-forming filamentous cells even under culture conditions adjusted for yeast development, most likely as a strategy to evade the noxious effect of the dye. After sometime, hypha returned to yeast cells. An hypothetical model to explain the effect of CR on morphology and enzyme activity based on the possible role of membrane-spanning proteins known as mechanosensors is proposed. Results are discussed in terms of the fungal responses to cell wall damage.