| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1087-1 | ||||
Resumo:Extracellular vesicles (EVs) are heterogeneous rounded structures exported by cells in all kingdoms. They transport a variety of cell biomolecules protected by a bi-layered membrane and therefore can play significant roles in long-distance signaling between pathogen-hosts and microorganisms. We have previously reported that vEVs, isolated from the fungal pathogen Paracoccidioides brasiliensis vPb18 originally highly virulent, can recover the expression of virulence traits of attenuated Pb18 (aPb18) upon aPb18-vEV co-incubation. The effect was similar to that evoked by recovery of aPb18 from organs of infected mice. We also observed that vEVs and aEVs vary in many aspects that include differential stimulation of murine macrophages in vitro. However, both aEV and vEV preparations exacerbated the experimental infection in our supposedly prophylactic immunization protocol. In the present work, we compared the vEV and aEV proteomes to understand their differences at the protein level. EVs were isolated from washes of aPb18 and vPb18 cultures in defined Ham F-12 agar medium supplemented with 0.5% glucose. Proteins were extracted from the samples, digested, and the resulting peptides were analyzed by nano flow liquid chromatography coupled with tandem mass spectrometry (nano LC-ESI-MS/MS). In this study, 932 vEV and 860 aEV proteins were validated, from which over 700 (including ribosomal proteins) were detected in both samples. Abundance of each protein was estimated by label-free quantification. Protein comparison between samples was possible thorough the validation of the peak areas relatively to the equal peptide amount loaded in the spectrometry analysis. Using highly stringency parameters with minimum difference of 4-fold in peak areas, we found 54 differentially abundant proteins between vEVs (41) and aEVs (13). Proteins that were 13 to 37-fold more abundant in vEVs include chromate and copper transporters (while a zinc transporter was more abundant in aEV), peptidyl-prolyl cis-trans isomerase, and 5-oxoprolinase. Among the others, we can point out stress-related thioredoxin, formamidase, RAS2, high osmolarity signaling protein SHO1, and heat shock protein Sti1; an alpha-1,2-mannosidase was more represented in vEV, while a glucan endo-1 3-beta-glucosidase was more abundant in aEV. We found exclusively represented proteins in both vEVs (163) and aEVs (153), however these proteins were generally not among the most abundant. Among the vEV unique proteins we can mention GTP-binding proteins RHO3 and RHB1, high osmolarity signaling protein SHO1, elongation factor Tu (EF-Tu), chitin biosynthesis proteins, and enolase. When both vEVs and aEVs are considered, the plasma membrane ATPase is the most abundant protein. The top 10 highest abundant proteins among the vEV included glyceraldehyde-3-phosphate dehydrogenase, a cell surface protein involved in fungal adhesion, and the Hsp90-like protein that is involved in regulating the proliferation and adaptation of the Paracoccidioides to different environmental conditions. Our results do not necessarily match those of differentially expressed proteins in Paracoccidioides brasiliensis aPb18 and vPb18 (Castilho et al., 2014), suggesting that the EV content does not reflect the cell content. The role of differentially represented proteins in extracellular vesicles from virulent Pb18 is under investigation.
Financial support: FAPESP (2018/13588-0, 2022/11123-5), CNPq (Universal 408843/2021-7), CAPES Palavras-chave: extracellular vesicles, proteome, Paracoccidioides, virulento, atenuado Agência de fomento:FAPESP, CAPES, CNPq | |||||