Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-sen University, Guangzhou, 510275, PR China; State Key Laboratory of Biocontrol / MOE Key Laboratory of Aquatic Product Safety, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, PR China; Institute of Aquatic Economic Animals, and Guangdong Province Key Laboratory for Aquatic Economic Animals, Sun Yat-sen University, Guangzhou, 510275, PR China.
State Key Laboratory of Biocontrol / MOE Key Laboratory of Aquatic Product Safety, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, PR China; Institute of Aquatic Economic Animals, and Guangdong Province Key Laboratory for Aquatic Economic Animals, Sun Yat-sen University, Guangzhou, 510275, PR China.
Fish Shellfish Immunol. 2019 Mar;86:143-151. doi: 10.1016/j.fsi.2018.11.037. Epub 2018 Nov 16.
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.
哈维弧菌是一种严重的海洋商业鱼类鳞片脱落和肌肉坏死疾病的病原体。黏附和定植宿主细胞表面是致病性细菌感染的第一步和关键步骤，通常由外膜蛋白（OMPs）介导。本研究的目的是鉴定在 OMPs 中起主要黏附作用的主要黏附素，该黏附素在哈维弧菌黏附宿主细胞中起着至关重要的作用，并评估该黏附素作为哈维弧菌感染疫苗候选物的潜力。我们观察到致病性哈维弧菌黏附在石斑鱼胚胎细胞（GEM 细胞）表面，并诱导其凋亡。从 9 株不同的哈维弧菌菌株中提取天然 OMPs，通过 SDS-PAGE 分析分离出 5 种共同的 Omp 带。Western blot 分析和抗天然 Omp 抗体阻断试验表明，存在 1 种强和几种弱免疫反应性 Omp 带。接下来，共鉴定出 5 种 OMPs，包括 TolC、Agg（凝集蛋白）、Omp47、Fla（鞭毛蛋白）和 OmpW，并克隆、表征和在大肠杆菌中表达它们的编码基因。纯化的重组 TolC 可在体外竞争性抑制哈维弧菌对 GEM 细胞的侵袭，抗 TolC 抗体也可显著阻断哈维弧菌与 GEM 细胞的黏附。当用于免疫杂交石斑鱼时，重组 TolC 可显著保护鱼类免受实验性哈维弧菌的侵袭。这些数据表明，外膜蛋白 TolC 是哈维弧菌的主要黏附素之一，可作为哈维弧菌感染的潜在疫苗候选物。
