Abstract
Paracoccidioides brasiliensis is a thermally dimorphic fungus that causes paracoccidioidomycosis, 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 identification and characterization of a secreted aspartyl protease (PbSAP), isolated from a cDNA library constructed with RNAs of mycelia transitioning to yeast cells. Recombinant PbSAP was produced in Escherichia coli, and the purified protein was used to develop a polyclonal antibody that was able to detect a 66 kDa protein in the P. brasiliensis proteome. PbSAP 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 PbSAP molecule. Zymogram assays indicated the presence of aspartyl protease gelatinolytic activity in yeast cells and culture supernatant.
Acknowledgements
This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Ensino Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG), Secretaria de Ciência e Tecnologia do Estado de Goiás (SECTEC-GO) and Financiadora de Estudos e Projetos (FINEP, grants 0106121200 and 010477500). The authors wish to thank Dr George S. Deepe Jr, Division of Infectious Diseases, College of Medicine, University of Cincinnati for critical review of the manuscript. We also thank Dr Maria José S. Mendes-Giannini, Faculdade de Ciências Farmaceuticas, UNESP, Araraquara, Brazil, for helpful suggestions.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.