Deisiany Gomes Ferreira¹, Bruna Fernandes Gonçalves², Marcel Ivan Ramirez Araya³, Sónia Carina Silva², Melyssa Negri¹

Introduction: Fusarium oxysporum is an opportunistic filamentous fungus associated with a wide spectrum of infections, ranging from superficial to disseminated. One of its main virulence factors is biofilm formation. This microbial community establishes and develops due to a quorum sensing system, which mediates cell-to-cell communication through signaling molecules released via extracellular vesicles (EVs). EVs are nanostructures capable of carrying various components within them and are frequently associated with cell-to-cell communication and pathogen-host interaction. When associated with biofilms, EVs play roles in different stages, from cell adhesion, matrix production, biofilm maturation and regulation, to dispersion and virulence factors. This research aimed to evaluate the protein profiles of EVs obtained from the supernatant and the biofilm matrix of F. oxysporum to analyze the differences between the content carried out of the biofilm (supernatant – biofilm vesicles, BVs) and what remains retained within it (matrix – MVs). Materials and Methods: To form the biofilm, an inoculum of 1.2×10⁷ conidia/mL of F. oxysporum CMRP 2925 was prepared in SDB and incubated for 48 hours. After this period, the supernatant was separated, and the extracellular matrix was extracted by mechanical scraping. Both fractions were then filtered through 0.45μM syringe filters. Following filtration, differential centrifugation (2000 xg, 5 min; 6000 xg, 30 min; 15,000 xg, 1h) was performed to isolate the EVs. After this preliminary preparation, we employed a label-free proteomics approach to identify changes in protein abundance of EVs from the biofilm: BVs and MVs. Results and Discussion: Proteomic analysis resulted in the identification of 419 total proteins. Of these, 49 were exclusively expressed in the supernatant, 15 only in the extracellular matrix, and 355 were expressed in both situations. Among the 355, the supernatant showed a higher abundance of proteins (259) compared to the extracellular matrix (34). Regarding the function of the detected proteins, BVs showed a higher relative frequency (p<0.05) for proteins associated with defense and virulence, cellular transport, and metabolism (biological processes), as well as extracellular region, membrane, plasma membrane, and vacuole (related to cellular content), and hydrolase activity and transporter activity (molecular function). In contrast, proteins from MVs showed a higher relative frequency (p<0.05) for proteins associated with the cell cycle and DNA processing, and protein synthesis (biological processes), cytoplasm, nucleus, ribosome (related to cellular content), DNA and RNA binding, and structural molecule activity (molecular function). Comparing the two situations, it is possible to observe that EVs have altered protein profiles depending on where they are isolated (supernatant or matrix). This characteristic indicates that fungal cells send different proteins outside the biofilm (BVs) with roles more associated with metabolism, defense, and virulence, while MVs, which remain retained within the biofilm, are more associated with regulation, especially gene regulation. Conclusion: From the analysis of the isolated proteins in this study, it was concluded that EVs play an important role both inside and outside the biofilm, being responsible for carrying different proteins to meet the diverse needs of the microbial community.

Agradecimentos: We would like to thank the State University of Maringá (UEM) and the University of Minho (UMINHO) for the provided space. We also extend our gratitude to the Coordination for the Improvement of Higher Education Personnel – Brazil (CAPES) (funding code 001) for their financial support in the development of this work.