Virulence and Stress-Related Proteins Are Differentially Enriched and N-Terminally Acetylated in Extracellular Vesicles from Virulent Paracoccidioides brasiliensis

Extracellular vesicles (EVs) are bilayer-membrane cellular components that deliver protected cargo to the extracellular environment and can mediate long-distance signaling. We have previously reported that EVs isolated from the virulent fungal pathogen Paracoccidioides brasiliensis Vpb18 can revert the expression, in the attenuated variant Apb18, of stress-related virulence traits. We presently show that the Vev and Aev, respectively, produced by these variants display distinct proteomes, with prevalent functional enrichment in Vev related to oxidative stress response, signal transduction, transport, and localization, in addition to richer protein–protein interaction. Proteome sequences were obtained by nanoflow liquid chromatography coupled with tandem mass spectrometry (nano LC-ESI-MS/MS). The Vev and corresponding Vpb18 proteomes also differed, suggesting a selective bias in vesicle protein cargo. Moreover, sublethal oxidative (VevOxi) and nitrosative (VevNO) stress modulated the Vev proteome and a positive correlation between VevOxi/VevNO-enriched and Vev-enriched (relative to Aev) proteins was observed. Out of 145 fungal virulence factors detected in Vev, 64% were enriched, strongly suggesting that molecules with virulence roles in Paracoccidioides are selectively concentrated in Vev. Our study significantly advanced the field by exploring protein N-terminal acetylation to a dimension rarely investigated in fungal EV proteomics. The proportion of N-terminally acetylated proteins in Vev was higher than in Vpb18 and the presence of Nt-acetylation in Vev-enriched virulence factors varied across the samples, suggesting that it might interfere with protein sorting into EVs and/or protein functionality. Our findings highlight the relevance of our fungal model to unraveling the significance of fungal EVs in pathogenesis and phenotypic transfer.