Uncovering how major extracellular proteins drive virulence and functional compensation in the mollusk pathogen Vibrio europaeus

• The Vibrio europaeus secretome is dominated by three major proteins: VemA, VepA, and GbpA. • VemA is essential for virulence against Manila clam juveniles. • Deletion of vemA , vepA , gbpA triggers secretory reprogramming. • The triple mutant (Δ vemA Δ vepA Δ gbpA) exhibits reduced virulence despite an enriched secretome and increased OMV production. • Compensatory secretion mechanisms reveal novel adaptive strategies in Vibrio europaeus . Bivalve aquaculture is a major component of global seafood production. However, its sustainability is severely threatened by recurrent outbreaks of vibriosis, caused by Vibrio europaeus that has emerged as a significant threat worldwide, affecting key aquaculture shellfish species. This study presents the first comprehensive characterization of the extracellular products (ECPs) and secretome of the mollusk pathogen Vibrio europaeus . A total of 108 different proteins were identified in the wild-type (WT) secretome, with the extracellular proteins VemA, VepA, and GbpA, accounting for nearly 70% of the total secreted protein content. A significant proportion of cytoplasmic proteins were also detected in the WT secretome, consistent with their secretion through outer membrane vesicles (OMVs) observed by electron microscopy. Functional assays confirmed that VemA is essential for virulence toward Manila clam juveniles ( Ruditapes philippinarum ), whereas VepA and GbpA appear to play complementary or modulatory roles. Surprisingly, simultaneous deletion of vemA , vepA , and gbpA genes induced extensive secretory reprogramming, with this mutant secreting 823 proteins, nearly eight times more than the WT, and exhibiting novel enzymatic activities. Approximately half of the secreted proteins in the triple mutant were cytoplasmic in origin, likely associated with a hypervesiculation phenotype observed by electron microscopy. Despite its expanded repertoire of virulence-associated proteins, the triple mutant displayed the lowest virulence in challenge assays, suggesting that the loss of the main ECPs triggers compensatory stress responses that remodel secretion but does not restore the virulence phenotype. Overall, these findings demonstrate that VemA, VepA, and GbpA are key determinants of virulence and secretory homeostasis in V. europaeus , revealing an adaptive mechanism linking OMV-mediated secretion, proteomic plasticity, and pathogenic potential in marine Vibrio species and provide better understanding how pathogens evolve to maintain or expand their virulence capacity.