Vesicle and vesicle-free extracellular proteome of Paracoccidioides brasiliensis: comparative analysis with other pathogenic fungi.

Microorganisms release effector molecules that modulate the host machinery enabling survival, replication, and dissemination of a pathogen. Here we characterized the extracellular proteome of Paracoccidioides brasiliensis at its pathogenic yeast phase. Cell-free culture supernatants from the Pb18 isolate, cultivated in defined medium, were separated into vesicle and vesicle-free fractions, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. In vesicle and vesicle-free preparations we identified, respectively, 205 and 260 proteins with two or more peptides, including 120 overlapping identifications. Almost 70% of the sequences were predicted as secretory, mostly using nonconventional secretory pathways, and many have previously been localized to fungal cell walls. A total of 72 proteins were considered as commonly transported by extracellular vesicles, considering that orthologues have been reported in at least two other fungal species. These sequences were mostly related to translation, carbohydrate and protein metabolism, oxidation/reduction, transport, response to stress, and signaling. This unique proteomic analysis of extracellular vesicles and vesicle-free released proteins in a pathogenic fungus provides full comparison with other fungal extracellular vesicle proteomes and broadens the current view on fungal secretomes.