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Screening a Phage Display Library for Two Novel OmpU-Binding Peptides with Adhesion Antagonistic Activity against
Lifang Qi1, Yan Liu1, Huizhu Tao1
1Key Laboratory of Zoonoses, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, P. R. China.
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.
Insights
Researchers identified two novel peptides that block Vibrio mimicus adhesion to fish cells. These OmpU-binding peptides show high affinity and offer protection against fish ascites disease.
Area of Science:
- Aquatic animal health
- Microbiology
- Biotechnology
Background:
- Vibrio mimicus causes ascites disease in fish.
- Outer membrane protein U (OmpU) is a key adhesin for V. mimicus.
Purpose of the Study:
- To screen for and characterize OmpU-binding peptides with adhesion antagonistic activity.
- To evaluate the protective effects of these peptides against V. mimicus infection in fish.
Main Methods:
- Screening of a phage displayed peptide library against recombinant OmpU (rOmpU).
- Assessing peptide adhesion antagonistic activity using adhesion inhibition tests and cell-based fluorescence.
- Measuring peptide binding affinity via non-competitive ELISA.
- Evaluating protective effects in grass carp challenged with V. mimicus.
Main Results:
- Two OmpU-binding peptides (P1 and P2) were identified, exhibiting significant adhesion antagonistic activity.
- P1 and P2 demonstrated high binding affinities to OmpU.
- Treatment with P1 and P2 delayed mortality and improved survival rates in grass carp challenged with V. mimicus.
Conclusions:
- Novel OmpU-binding peptides with adhesion antagonistic properties were successfully screened.
- These peptides show potential for controlling V. mimicus infections and ascites disease in fish.
