THE MALATE SYNTHASE OF PARACOCCIDIOIDES BRASILIENSIS IS A LINKED SURFACE PROTEIN THAT BEHAVES AS AN ANCHORLESS ADHESIN.

Benedito Rodrigues da Silva Neto (UFG); Julhiany de Fátima da Silva (UNESP); Renata Silva do Prado (UFG); Maria José Soares Mendes-giannini (UNESP); Henrique Leonel Lenzi (FIOCRUZ); Maristela Pereira (UFG); Célia Maria de Almeida Soares (UFG)

Paracoccidioidomycosis (PCM), the most important systemic mycosis in Latin America, is a chronic granulomatous disease that affects about 10 million people. Paracoccidioides brasiliensis, a thermally dimorphic fungus pathogen, is the pulmonary infective agent. The pathogenic fungus P. brasiliensis is the agent of PCM. This is a pulmonary mycosis acquired by inhalation of fungal airborne propagules that can disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside the host, P. brasiliensis may use the glyoxylate cycle for intracellular survival. Here, we provide evidence that the malate synthase of P. brasiliensis (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. By using Confocal Laser Scanning Microscopy, PbMLS was detected in the cytoplasm and in the cell wall of the mother, but mainly of budding cells of the P. brasiliensis yeast phase. By flow cytometry PbMLSr and its respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis with in vitro cultured epithelial cells A549. 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 cells membranes. The 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 through its ability to act as a probable adhesin. In addition, the absence of conventional secretion or cell wall anchoring motifs defines PbMLS as a probable anchorless adhesin that could contribute to virulence by promoting P. brasiliensis infection and dissemination. Here it is demonstrated that PbMLS is the first fungal MLS localized on the cell surface which interferes with the infection process.These observations indicated that cell wall-associated PbMLS could be mediating the binding of fungal cells to the host, thus contributing to the adhesion of fungus to host tissues and to the dissemination of infection, behaving as an anchorless adhesin.