Abstract
Vibrio mimicus (V.mimicus) is a causative agent of ascites disease in aquatic animals. Our previous studies have demonstrated that the outer membrane protein U (OmpU) from V.mimicus is an immunoprotective antigen with six immunodominant linear B-cell epitopes. Although the N-terminus of OmpU contains potential binding motifs, it remained unclear whether OmpU possesses adhesion function. Here, the adhesive capacity of recombinant OmpU and V.mimicus to epithelioma papulosum cyprinid (EPC) cells was determined by immunofluorescence and adherence assay. The results showed that after co-incubated with rOmpU, an obvious visible green fluorescence could be observed on the EPC cell surface and the nuclei exhibited blue fluorescence; while the control cell surface did not show any signal, only nuclei exhibited blue fluorescence. The average number of wild-type strain adhered to each cell was 32.3 ± 4.5. The average adhesion number of OmpU gene deletion mutant was significantly reduced to 10.8 ± 0.5 (P < 0.01) and restored to 31.3 ± 2.8 by complement strain (P >0.05). Pretreatment of cells with rOmpU reduced the average adhesion number of wild-type strain to 9.7 ± 2.9 (P < 0.01). Likewise, binding was significantly decreased to 8.8 ± 3.2 (P < 0.01) due to blocking role of OmpU antibodies. To determine binding motifs of OmpU, six immunodominant B-cell epitope peptides labeled with FITC were employed in flow cytometry-based binding assay. Two FITC-labeled epitope peptides (aa90-101 and aa173-192) showed strong binding to EPC cells (the fluorescence positive cell rate was 99 ± 0.6% and 98 ± 0.3%, respectively), which could be specifically competed by excess corresponding unlabeled peptides, whereas the remaining four showed a low level of background binding. This is the first demonstration that OmpU possesses adhesion function and its N terminal 90–101 and 173–192 amino acid regions are critical sites for cell surface binding.
Highlights
Vibrio mimicus (V.mimicus), which resembles Vibrio cholera, is a common pathogen for humans and aquatic animals [1]
The results showed that after co-incubated with recombinant OmpU (rOmpU), an obvious visible green fluorescence could be observed on the epithelioma papulosum cyprinid (EPC) cell surface and the nuclei exhibited blue fluorescence; while the control cell surface did not show any signal, only nuclei exhibited blue fluorescence
We found that outer membrane protein U (OmpU) protein was highly homologous to the corresponding sequence of V.cholerae, and the N-terminus of the OmpU protein in V.mimicus contained motifs of the outer membrane protein adhesin of Haemophilus [17], and anti-OmpU antibodies could inhibit the adhesion of V. mimicus
Summary
Vibrio mimicus (V.mimicus), which resembles Vibrio cholera, is a common pathogen for humans and aquatic animals [1]. Due to inappropriate use and abuse of antibiotics in aquaculture industry, more and more drug resistant V.mimicus strains emerged, which led to the death of large numbers of aquatic animals. These serious impacts require new means of ascites disease prevention in aquaculture industry. The receptor triggers a range of signaling pathways that lead to bacterial invasion and colonization This process is a key step in the bacterial ability to evade the host immune system successfully establishing bacterial infection [5]. The identification of major adhesins and their critical functional domains creates opportunities for developing new strategies for effectively combating bacterial diseases, for example by creating adhesion antagonists and adhesin vaccines that block the initial bacterial infection [6]
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