Abstract
Vibrio mimicus is a pathogen that causes ascites disease in fish. We have previously demonstrated that the outer membrane protein U (OmpU) is an important adhesin in V. mimicus. Here eight specific OmpU-binding phage clones, which presented three different OmpU-binding peptides (designated P1, P2, P3), were screened from a commercially available phage displayed 12-mer peptide library using rOmpU protein as target. Then, synthetic OmpU-binding peptides were measured for their adhesion antagonistic activity and binding affinity via adhesion inhibition test and non-competitive ELISA, respectively. The results showed that after co-incubated with the mixture of rOmpU and P3, visible green fluorescence could be observed on the epithelioma papulosum cyprinidi (EPC) cells surface; while the EPC cells co-incubated with the mixture of rOmpU and P1/P2 exhibited little green fluorescence. The average adhesion number of V. mimicus 04–14 isolate before and after treatment with peptide was 21.4 ± 1.5, 20.8 ± 0.8 (irrelevant peptide), 20.2 ± 0.5 (P3), 5.1 ± 0.7 (P1) and 3.4 ± 0.8 (P2), respectively. There was a significant decrease in the adhesive level of 04–14 isolate treated with P1/ P2 compared to the untreated isolate (p<0.01). The affinity constants of P1 and P2 were (6.17 ± 0.19) × 108 L/mol and (1.24 ± 0.56) × 109 L/mol, respectively. Furthermore, protective effects of P1 and P2 on grass carps challenged with V. mimicus were preliminary detected. It was found there was delayed death of fish in the groups treated with P1/P2, and the survival rate of challenged fish improved with the increase of the dose of adhesion antagonistic peptide. Taken together, two novel OmpU-binding peptides, which possessed adhesion antagonistic activity, high affinity and a certain degree of antibacterial activity against V. mimicus, were screened and identified.
Highlights
Vibrio mimicus (V. mimicus) is a common pathogen found in aquaculture that can cause severe ascites disease in fish [1,2]
We have demonstrated that V. mimicus is an enteric pathogen and the outer membrane protein U (OmpU) is an important adhesin in V. mimicus
Rabbit anti-OmpU antibody and the preimmune serum were available from previous studies [10], and other materials were bought from legal biotechnology companies
Summary
Vibrio mimicus (V. mimicus) is a common pathogen found in aquaculture that can cause severe ascites disease in fish [1,2]. Novel OmpU-Binding Peptides with Adhesion Antagonistic Activity against Vibrio mimics products, and induces more and more drug resistant strains, which led to the death of many fish and economic losses to the aquaculture industry. Bioactive peptides can exert biological effects by highly selective binding to active site on a target protein in vitro and in vivo. They have several advantages, such as lower manufacturing costs, higher activity per unit mass, generally lower immunogenicity, little toxicity and better organ penetration. Creating adhesion antagonist that can block or antagonize V. mimicus adhesion to cells is a potential new strategy for the prevention and treatment of ascites disease in fish
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