Adhesion Function Of The OmpU Of Vibrio Mimicus And Determination Of The Receptor-binding Domain Of The Adhesin

Vibrio mimicus (V.mimicus) is a wide host pathogen causing ascetic fluid disease in aquatic animals, which results in serious threat to aquaculture. Bacterial adherence, which is interaction between adhesins of pathogenic bacteria and receptors of host cells actually, plays an essential role in causing infection. The infection process includs adhesion, growth, toxin-producing, penetrate deeper tissue or damage tissue cells. It is a new strategy for prevention and treatment of bacterial disease to ascertain major adhesins and its key receptor-binding domain, then to design and develope adhesion antagonists based on key receptor-binding domain. In our previous studies, the outer membrane protein U (OmpU) of V. mimicus has been proven to be a conservative protein with adhesion motif at N-terminal, six linear B-cell epitopes of the protein were identified, and rabbit anti-OmpU antibody can inhibit V.mimicus adhesion to human cells. Here, the adhesion model of Epithelioma Papulosum Cyprinid (EPC) cells was established for detecting adhesive ability of Vibrio mimicus or adhesin protein, then the adhesion function of OmpU and its key receptor-binding domain were confirmed.Firstly, the adhesion model of EPC cells was established, and then the adhesive characteristics of V. mimicus to EPC cells were assayed. The results showed that V.mimicus 04-14 strain could cluster around the EPC cells after incubation with cells (MOI=60) for 60 min at 28℃. Chemical factors such as D-galactose, trypsin and proteinase K were able to interrupt 04-14 strain adhesion to EPC cells, but D-glucose, D-mannose, maltose and suorose had no effect on the adhesion of 04-14 strain. Therefore, it could infer that the adhesin of V.mimicus. may be some protein. The corresponding receptor possesses oligosaccharide chain structure, and the specificity is determined by D-galactose residue.Secondly, in order to identify the function of OmpU, a large number of recombinant fluorescent protein EGFP-OmpU were prepared. The recombinant fluorescent expression plasmid named pET-32a-EGFP-OmpUwas constructed by gene recombination technology and transformed into E.coli Rosetta to bemass-induced expression, then rHis-EGFP-OmpU was purified by Ni-Charged Resin affinity chromatography. It was found that the concentration and purity of the purified protein was 1.4mg/ml and 90% by SDS-PAGE analysis, respectively. rHis-EGFP-OmpU could emit green fluorescence under 488nm excitation light. Those results indicated that the recombinant fluorescent protein could fully meet the requirements of subsequent experiments.Then, the adhesion function of OmpU was identified with EPC cell model by indirect immunofiuorescence assay, in which puried anti-OmpU rabbit antibody as first antibody, goat anti-rabbit IgG-FITC as secondary antibody, DAPI as fluorescent dye nucleus. Post-incubated with rOmpU, EPC cells surface appeared bright green fluorescence and nuclear DNA stained with bule color, while EPC cells incubated with M199 and FITC appeared blue fluorescence only, without green fluorescence. Meanwhile, the adhesion inhibition test of rabbit anti-rOmpU antibody to strain 04-14 and competition binding test between rEGFP-OmpU and rOmpU to EPC cells were be conducted. It was found that rabbit anti-rOmpU antibody could inhibit the strain 04-14 adhesion to EPC cells, rOmpU EPC cells surface appeared bright green fluorescence after incubated with rEGFP-OmpU, but the green fluorescence on the cell surface weaken after incubated with rEGFP-OmpU and rOmpU. All of the results indicated that OmpU of V.mimicus possessed adhesion function.Finally, in order to determine the key receptor-binding domain of OmpU, six synthesis epitope peptides of OmpU were labeled with FITC in N-terminal, then the combination characteristics between different FITC-epitope peptide and EPC cells as well as competition binding between FITC-epitope peptide and respective epitope peptide to EPC cells were analyzed by flow cytometry. Results showed the percentage of positive cells fluorescently labeled was 5.51%,99.60%,3.93%,98.30%,6.35% and 13.10%, corresponding mean fluorescence intensity (MFI) was 18±0.42,650±11.93,16±0.37, 571±10.95,19±0.51 and 27±0.63, respectively, after co-incubation FITC-epitope peptide and EPC cells. Compared to the control level, the percentage of positive cells fluorescently labeled and MFI were significantly higher in treated groups with Epitope peptide 90-101 and Epitope peptide173-192, and could be inhibited by excessive corresponding unlabeled epitope peptide. Those results indicated the two epitope peptides was able to specific combine with EPC cells.90-101 and 173-192 amino acid areas in N-terminal of OmpU were the linear parts of the key receptor-binding domain.In conclusion, the present study clearly comfirmed that OmpU of V.mimicus possessed adhesion function, and 90-101 and 173-192 amino acid areas in N-terminal of OmpU were the linear parts of the key receptor-binding domain. Our data lay a good foundation for developing new adhesion antagonists based on key receptor-binding domain.