Resumo

Fungi of the complex Paracoccidioides are thermo-dimorphic fungus that causes Paracoccidioidomycosis (PCM), a systemic mycosis that affects the rural population, particularly in Latin America. In this work we use two phylogenetic isolates of Pb01 and PbEPM83, aiming the characterization of proteins secreted by both isolates. Extracellular proteins secreted by fungi may be the main mediators of host-parasite interactions. In this sense we intend to characterize and compare the secretomes in order to elucidate some particular features among members of Paracoccidioides phylogenetic species. We utilized the method of two-dimensional gels coupled with mass spectrometry to identify proteins differentially expressed among the isolates. The proteomic analysis revealed an average of 350 and 355 spots in secretomes of PbEPM83 and Pb01, respectively. Were identified 27 and 52 proteins / isoforms preferentially secreted by PbEPM83 and Pb01, respectively. Moreover, we identified 67 proteins constitutively expressed in both isolates. After identification, the protein sequences were classified into functional categories and the vast majority of the proteins were related to metabolism, energy, defense and virulence. The proteins were further subjected to bioinformatic analysis to predict secretory pathways and potential antigenic epitopes. Our next step, which is ongoing, is the choice of some specific target proteins for construction of mutants for gene silencing. Also, these mutants will be used to do in vitro and in vivo analyses to assess their effect on phagocytosis (macrophages) and fungal load recovered from organ in experimentally infected animals. Many of the proteins found in our secretome such as superoxide dismutase, heat shock proteins, cytochrome c, formamidase, triose phosphate isomerase, 14-3-3 were also found in vesicles secreted by Histoplasma capsulatum. Our results of secretome differential between two phylogenetic species include various proteins of interest that add new information on the mechanisms of pathogenesis and virulence of these isolates. Our secretome assists for cytosolic molecules or from other cellular compartments, which perform new functions and are released out of the cell to act somehow on the host, to achieve survival and establish infection.