Multi-omics characterization of putative VSP-like surface antigens reveals a dynamic antigenic repertoire in...

Scuticociliatosis, caused by the parasitic ciliate Philasterides dicentrarchi, is one of the most important infectious diseases affecting turbot (Scophthalmus maximus) aquaculture. Despite the critical role of parasite surface antigens in host-parasite interactions, their molecular diversity and expression dynamics remain poorly understood. Here, we applied an integrated multi-omics approach combining genome mining, transcriptomics, structural annotation, phylogenetic analysis, proteomics and immunological assays to identify and characterize a repertoire of putative variant surface protein-like (VSP-like) antigens in P. dicentrarchi. Throughout this study, the term VSP-like is used as a descriptive designation for cysteine-rich surface proteins sharing structural characteristics with both Giardia variant-specific surface proteins (VSPs) and ciliate immobilization antigens (i-antigens), without implying functional or evolutionary equivalence with either protein family. Genome and transcriptome analyses identified a diverse repertoire of cysteine-rich surface proteins displaying predicted N-terminal signal peptides, cysteine-rich extracellular domains and C-terminal membrane-associated regions compatible with glycosylphosphatidylinositol (GPI) anchoring. Phylogenetic reconstruction resolved these proteins into several distinct clades, whereas LC-MS/MS analysis confirmed the expression of multiple VSP-like families in trophozoites. Integration of structural, phylogenetic and proteomic data with B-cell epitope prediction enabled the rational prioritization of putative candidate vaccine antigens for future experimental evaluation. Comparative analyses further revealed dynamic modulation of the VSP-like-associated antigenic profile during successive parasite passages. RT-qPCR demonstrated differential VSP2 transcript abundance between early and late infective passages, whereas ELISA and two-dimensional immunoblot analyses revealed marked differences in anti-rVSP2 antigen recognition among parasite populations. Moreover, sequencing of cloned transcripts and exposure of trophozoites to immune turbot serum revealed shifts in the expressed antigenic repertoire, suggesting that host immune factors may contribute to modulation of VSP-like expression. Together, these findings provide the first comprehensive molecular characterization of a repertoire of putative VSP-like surface antigens in P. dicentrarchi. They reveal a dynamic antigenic repertoire associated with parasite adaptation and host immune interactions, establish a framework for investigating antigenic variation in parasitic scuticociliates, and provide a rational basis for the future experimental evaluation of vaccine antigens against scuticociliatosis.