Identification and multi-omics analysis of extracellular vesicles from the plerocercoids of Spirometra mansoni (Cestoda: Diphyllobothriidae)

Parasitic extracellular vesicles (EVs) play crucial roles in the growth and development of parasites. However, we remain unaware of the EVs from the plerocercoids of Spirometra mansoni. In this study, firstly, the plerocercoids of S. mansoni were cultured in vitro to enrich EVs, which were then characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. Next, the protein components and miRNAs of EVs were subsequently analysed using data-independent acquisition (DIA) and high-throughput sequencing, respectively. The results showed that EVs derived from the plerocercoids exhibit a typical double-layered membrane structure as spherical or elliptical vesicles ranging from 80 to 150 nm in diameter. The molecular markers HSP70, TSG101, HSP90, CD81, and β-Actin were significantly expressed in these EVs. Micro-proteomic analysis revealed 2,729 proteins, with functional annotation indicating that EV proteins are enriched primarily in pathways such as intracellular transport/secretion, cellular processes, and signal transduction. High-throughput miRNA sequencing revealed that the identified EVs contained 37 known miRNAs and 118 novel miRNAs. Functional enrichment analysis indicated that miRNAs primarily participate in host interactions by regulating DNA integration, apoptosis, cholesterol metabolism, and cAMP signaling pathways. Immunological analysis demonstrated that the antiserum against EVs elicited a mixed Th1/Th2 immune response, which was predominantly Th1-type. In this study, S. mansoni plerocercoid-derived EVs were successfully isolated and characterized. Systematic profiling of EV components via microproteomics and high-throughput miRNA sequencing provides a foundation for further investigations into the functional mechanisms of EVs.