Expression of Paracoccidioides brasiliensis AMY1 in a Histoplasma capsulatum amy1 mutant, relates an α-(1,4)-amylase to cell wall α-(1,3)-glucan synthesis.

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.

Effects of histoplasmin M antigen chemical and enzymatic deglycosylation on cross-reactivity in the enzyme-linked immunoelectrotransfer blot method.

The enzyme-linked immunoelectrotransfer blot (EITB) method was evaluated as a suitable method for detecting antibodies against M antigen of Histoplasma capsulatum by use of both glycosylated and deglycosylated M protein of histoplasmin (HMIN). Sera from patients with histoplasmosis, paracoccidioidomycosis, blastomycosis, coccidioidomycosis, and aspergillosis were tested by the EITB with glycosylated M protein of HMIN. This assay demonstrated 100% sensitivity with histoplasmosis serum samples, all of which reacted with the 94-kDa glycoprotein (M antigen). Although the EITB is highly sensitive, it is not specific for histoplasmosis when glycosylated M protein is used as an antigen. A total of 81% of paracoccidioidomycosis, 25% of blastomycosis, 33% of coccidioidomycosis, 73% of aspergillosis, and 16% of tuberculosis serum samples cross-reacted with M protein of HMIN and yielded patterns indistinguishable from those obtained with histoplasmosis serum samples. The EITB reactions with both untreated M antigen and M antigen altered by periodate oxidation or by deglycosylation with endoglycosidases were compared. Cross-reactions with heterologous sera in the EITB could be attributed to periodate-sensitive carbohydrate epitopes, as reflected by the increase in the test specificity from 46.1 to 91.2% after periodate treatment of M protein. The EITB for the detection of antibodies to M antigen is a potential diagnostic test for histoplasmosis, provided that periodate-treated M protein is used as an antigen.

Regulaciones bioquímicas en el dimorfismo y la virulencia de hongos patogenos para humanos / Biochemical regulations in the dimorphism and virulence of pathogenic fungi for humans

Se revisan diversos mecanismos bioquímicos involucrados en el control de la virulencia y el dimorfismo de hongos patógenos para humanos. Entre ellos, la participación de grupos sulfidricos y disulfuros, los receptores hormonales y las proteinas intra- y extracelulares en histoplasma capsulatum, paracoccidioides brasiliensis y Coccidioides immitis

Actividades enzimáticas en levaduras de Histoplasma capsulatum / Enzymatic activities from Histoplasma capsulatum yeasts

Se determinaron actividades enzimáticas en 5 cepas de Histoplasma capsulatum en fase levaduriforme (EH46, EH 50, EH51, EH52, EH53) por el micrométodo del APIZYM. De las 19 enzimas probadas se encontraron actividades de fosfatasa alcalina, esterasa lipasa C8, fosfatasa ácida y fosfoamidasa en todas las cepas estudiadas, mientras que la lipasa C14 y la leucina arilamidasa, sólo se determinaron en 4 cepas. Estas últimas enzimas no aparecieron en la cepa EH53 de alta virulencia

Amino acid synthetic media for fungal pathogens based on aminopeptidase specificities: Histoplasma capsulatum, Blastomyces dermititidis, Paracoccidioides brasiliensis and Cryptococcus neoformans.

The development of simple and chemically defined liquid media for Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Crypto-occus neoformans according to their aminopeptidases profiles as amino acid requirement was described. When 1.5% purified agar was added, these media also supported excellent mycelial growth and sporulation of the deep mycoses. H. capsulatum was converted to and maintained in yeast phase when 0.1% L-cystine was added to the solid medium incubated at 37 degrees C.