RESUMO
In the cell walls of the pathogenic yeast phases of Paracoccidioides brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum, the outer α-(1,3)-glucan layer behaves as a virulence factor. In H. capsulatum, an α-(1,4)-amylase gene (AMY1) is essential for the synthesis of this polysaccharide, hence related to virulence. An orthologous gene to H. capsulatum AMY1 was identified in P. brasiliensis and also labeled AMY1. P. brasiliensis AMY1 transcriptional levels were increased during the yeast phase, which correlates with the presence of α-(1,3)-glucan as the major yeast cell wall polysaccharide. Complementation of a H. capsulatum amy1 mutant strain with P. brasiliensis AMY1, suggests that P. brasiliensis Amy1p may play a role in the synthesis of cell wall α-(1,3)-glucan. To study some biochemical properties of P. brasiliensis Amy1p, the enzyme was overexpressed, purified and studied its activity profile with starch and amylopeptin. It showed a relatively higher hydrolyzing activity on amylopeptin than starch, producing oligosaccharides from 4 to 5 glucose residues. Our findings show that P. brasiliensis Amy1p produces maltooligosaccharides which may act as a primer molecule for the fungal cell wall α-(1,3)-glucan biosynthesis by Ags1p.
Assuntos
Parede Celular/genética , Proteínas Fúngicas/genética , Glucanos/biossíntese , Histoplasma/genética , Paracoccidioides/genética , alfa-Amilases/genética , Sequência de Aminoácidos , Amilopectina/metabolismo , Parede Celular/enzimologia , Proteínas Fúngicas/metabolismo , Expressão Gênica , Teste de Complementação Genética , Histoplasma/enzimologia , Histoplasma/patogenicidade , Dados de Sequência Molecular , Mutação , Paracoccidioides/enzimologia , Paracoccidioides/patogenicidade , Filogenia , Alinhamento de Sequência , Amido/metabolismo , Especificidade por Substrato , Virulência , alfa-Amilases/metabolismoRESUMO
Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, an important human systemic mycosis in Latin America. Recently, the existence of three different phylogenetic species (S1, PS2, and PS3) of P. brasiliensis was demonstrated. Despite being genetically isolated, all three species were capable of inducing disease in both humans and animals, although lower virulence has been found with the PS2 species. The available molecular methods developed to characterize and type strains have not been useful for assigning isolates to the described species, creating the need for molecular markers capable of distinguishing genetically isolated groups. Here, we describe a PCR and sequencing-based microsatellite marker system that is stable, easy to assay, adaptable to large series of isolates, and discriminatory enough to be used as a typing system in identifying the three proposed species of P. brasiliensis. In addition, this system provides an unambiguous tool for strain discrimination between two (S1 and PS2) of the three phylogenetic species.