Virulence and Stress-Related Proteins Are Differentially Enriched and N-Terminally Acetylated in Extracellular Vesicles from Virulent <em>Paracoccidioides brasiliensis</em>
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Extracellular vesicles (EVs) are by layered-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 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, localization, besides richer protein-protein interaction. Proteome sequences were obtained by nano-flow 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 was observed between VevOxi/VevNO-enriched and Vev-enriched (relative to Aev) proteins. Among 145 fungal virulence regulators detected in Vev, 64% were Vev-enriched, strongly suggesting that they selectively concentrate molecules with virulence roles in Paracoccidioides. Our study significantly advanced the field by exploring the protein N-terminal acetylation to a dimension that has scarcely been 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 regulators varied across 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 unravel the significance and nuances of fungal EVs in pathogenesis and phenotypic transfer.