Characterization of a secreted aspartyl protease of the fungal pathogen Paracoccidioides brasiliensis

Abstract

Paracoccidioides brasiliensis is a thermally dimorphic fungus that causes para coccidioidomycosis, a human systemic disease prevalent in Latin America. Proteases have been described as playing an important role in the host invasion process in many pathogenic microorganisms. Here we describe the identifi cation and characterization of a secreted aspartyl protease ( Pb SAP), isolated from a cDNA library constructed with RNAs of mycelia transitioning to yeast cells. Recombinant Pb SAP was produced in Escherichia coli , and the purifi ed protein was used to develop a polyclonal antibody that was able to detect a 66 kDa protein in the P. brasiliensis proteome. Pb SAP was detected in culture supernatants of P. brasiliensis and this data strongly suggest that it is a secreted molecule. The protein was located in the yeast cell wall, as determined by immunoelectron microscopy. In vitro deglycosylation assays with endoglycosidase H, and in vivo inhibition of the glycosylation by tunicamycin demonstrated N -glycosylation of the Pb SAP molecule. Zymogram assays indicated the presence of aspartyl protease gelatinolytic activity in yeast cells and culture supernatant.