Identification of outer membrane protein TolC as the major adhesin and potential vaccine candidate for Vibrio harveyi in hybrid grouper, Epinephelus fuscoguttatus (♀) × E. lanceolatus (♂).

Vibrio harveyi is a serious pathogen of scale drop and muscle necrosis disease in marine commercial fishes. Adhesion to and colonization of the host cells surfaces is the first and crucial step for pathogenic bacterial infection, which is usually mediated by outer membrane proteins (Omps). The objectives of this study were to identify the major adhesin in Omps that plays the essential role in adhesion of V. harveyi to the host cells, and to assess the potential of this adhesin as a vaccine candidate for V. harveyi infection. We observed that pathogenic V. harveyi adhered to the surface of grouper embryonic cells (GEM cells) and induced apoptosis of them. Native Omps were extracted from nine different V. harveyi strains, and five common Omp bands were isolated by SDS-PAGE analysis. Western blot analysis and an anti-native Omp antibodies blocking assay indicated that one strong and several weak immunoreactivity Omps bands presence. Next, a total of five Omps, including TolC, Agg (Agglutination protein), Omp47, Fla (Flagellin), and OmpW, were identified and their encoding genes were cloned, characterized, and expressed in E. coli. The purified recombinant TolC could competitively inhibit the invasion of V. harveyi to GEM cells in vitro, and anti-TolC antibody also could significantly block the adhesion of V. harveyi to GEM cells. When used to immunize hybrid groupers, the recombinant TolC could confer significant protection to fish against experimental V. harveyi challenge. These data suggested that outer membrane protein TolC functions as a major adhesin in V. harveyi and could be a potential vaccine candidate for V. harveyi infection.