Paracoccidioides brasiliensis malate synthase is a cell surface protein and is related to the fungus adhesion to the extracellular matrix protein and cell interaction

Abstract

Paracoccidioides brasiliensis, a pathogenic fungus, is the agent of paracoccidioidomycosis (PCM). This is a pulmonary mycosis acquired through inhalation of fungal airborne propagules that can disseminate to several organs and tissues leading to a severe form of the disease. Adhesion to host cells and their invasion are essential steps for pathogen internalization and dissemination. Inside the host, P. brasiliensis may use the glyoxylate cycle for intracellular survival. In the present study, we evidenced that P. brasiliensis malate synthase (PbMLS) is located on the fungal cell surface and is secreted. PbMLS was overexpressed in Escherichia coli, and polyclonal antibody was obtained against this protein. Using confocal laser scanning microscopy, PbMLS was detected in the cytoplasm and in the mother-cell wall, but mainly in P. brasiliensis yeast-phase budding cells. Applying flow cytometry, we observed that PbMLSr and its respective polyclonal antibody inhibited the interaction of P. brasiliensis with cultured epithelial cells A549 in vitro. The results showed the presence of PbMLS in the culture filtrate of yeast cells (parasitic phase), its surface location in P. brasiliensis and its binding to ECM in Far-Western blot and ELISA assays and to A549 cell membranes. Reduction in the adherence of P. brasiliensis to A549 cells by anti-PbMLSr suggests that PbMLS could contribute to active fungal interaction and disease progression in humans due to its ability to act as a probable adhesin. These observations indicate that cell wall-associated PbMLS could mediate binding of fungal cells to the host, thus contributing to the adhesion of fungus to host tissues and to the dissemination of this infection.