Resumo

The thermodimorphic fungus Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), an endemic systemic disease in Latin America. The disease is acquired when mycelium fragments or conidia of the fungus are inhaled by the host. In the lungs the fungus is submitted to a range of stresses including the nutritional starvation. Among other nutrients, the nitrogen metabolism is essential for fungal growth and establishment in host tissues, once nitrogen-dependent pathways are closely related to pathogenicity. Pathogenic organisms presents a regulatory system responsive to nitrogen availability in the milieu, as well as alternative nutrient source acquiring mechanisms. The regulation of nitrogen metabolism comprises a complex system in the infectious process. This work aims to characterize the Paracoccidioides, isolate Pb01, proteome under nitrogen starvation. The fungus was cultivated in Fava-Netto´s medium and subsequently transferred to minimal medium McVeigh & Morton (MMCM) in the presence and absence of nitrogen sources. Growth curve and the viability of the fungal cells were performed. Based on the viability of fungal cells, the protein extracts were obtained by incubation of Paracoccidioides yeast cells during 6 hours on minimal medium with and without nitrogen sources. Following, the cells were centrifuged, washed, lysed and the protein supernatant from control and treated conditions were obtained. The protein extracts were quantified by using the Bradford method. The protein profile was analyzed by SDS-PAGE. The total protein extracts were subjected to tryptic digestion. The digested peptides were submitted to ultra performance liquid chromatography coupled to mass spectrometer (UPLC-MS^E) for protein identification and quantification. The lists of identified proteins are under characterization and functional annotation by using bioinformatics tools. The proteome analysis of Paracoccidioides sp isolate Pb01-like, will expand the knowledge about the biology of this fungus, besides elucidating new virulence factors that may help us to understand the mechanisms used by Paracoccidioides to obtain a successful infection in the host.