Development of a novel chitosan conjugated antimicrobial peptide as an alternative to antibiotics

Abstract

The invention relates to the development of a chitosan-conjugated antimicrobial peptide as an alternative to antibiotics, aimed at effectively combating fish diseases in aquaculture, particularly targeting bacterial and infectious pancreatic necrosis (IPN) diseases, without posing any harmful effects to the environment.

Description

[0001] DEVELOPMENT OF A NOVEL CHITOSAN CONJUGATED ANTIMICROBIAL PEPTIDE AS AN ALTERNATIVE TO ANTIBIOTICS

[0002] Technical Field

[0003] The invention relates to the development of a chitosan-conjugated antimicrobial peptide as an alternative to antibiotics, aimed at effectively combating fish diseases in aquaculture, mainly targeting bacterial and infectious pancreatic necrosis (IPN) diseases, without posing any harmful effects to the environment.

[0004] Background

[0005] In aquaculture, antibiotics and chemicals are commonly used for the treatment of bacterial diseases. However, this practice has raised significant concerns due to its harmful effects on the environment and associated public health risks. Additionally, the use of antibiotics in aquaculture has led to the emergence of antibiotic-resistant bacteria and the accumulation of antibiotic residues in aquatic products. These residues can pose significant barriers to the export of aquatic products. The United Nations General Assembly recognized the seriousness of this issue in 2016 by declaring Antimicrobial Resistance (AMR) as "the most urgent global risk." In response to this global imperative, the European Commission has developed an Action Plan emphasizing the need to develop alternatives to antibiotics. One of these promising alternatives is the use of antimicrobial peptides.

[0006] In the patent document W02020008254A1, antibodies developed against pathogens in aquaculture and their use are mentioned.

[0007] Upon examining the existing studies, there is a need for the development of an antimicrobial peptide capable of effectively combating fish diseases in aquaculture and treating bacterial and viral infections without causing any harmful effects on the environment.

[0008] Aim of the Invention

[0009] The aim of this invention is to develop an antimicrobial peptide that can effectively combat bacterial pathogens and IPN in aquaculture without causing any harmful effects on the environment. Detailed Description of the Invention

[0010] A novel peptide sequence conjugated with chitosan nanoparticles, WK14 (WKFHFAKIFIKYWK-NH2), has been developed within the scope of the invention. During the development of the antimicrobial peptide (AMP), 147 fish AMP sequences were obtained from AMP databases in FASTA file format. The sequences were aligned to discover common patterns or features that provide antimicrobial activity. Factors such as net charge, hydrophobicity, and secondary structure were examined to analyze the physicochemical properties of these new peptide sequences for antimicrobial activity were produced and optimized through rational design.

[0011] The sequences were later evaluated in the web-based Antimicrobial Peptides Collection (CAMPR3) database using Support Vector Machines (SVMs) algorithm. The SVM algorithm classified peptides as antimicrobial or non-antimicrobial based on the characteristics of the input sequences. The generated motifs were then scanned based on various physicochemical parameters, including amino acid length, positive net charge, hydrophobicity, and isoelectric point. The designed antimicrobial peptide was tailored considering polycationic and physicochemical parameters, including amino acid length (14 residues), positive net charge (+4 to +6), hydrophobicity (40% to 60%), and isoelectric point of 10. The positive net charge of AMPs aids in their interaction with negatively charged bacterial membranes, while their hydrophobicity facilitates penetration into the membrane, disrupting bacterial functions.

[0012] The peptide sequence of the invention was tested using both in silico and in vitro approaches, confirming its potential antimicrobial activity. Conjugation with chitosan was performed to impart stability and efficiency in biological systems The tests demonstrated that this new peptide exhibits strong antimicrobial properties and effectively combats various bacterial species commonly found in aquaculture. The peptide described in the invention holds considerable promise as an alternative to traditional antibiotics in aquaculture. One of the most significant advantages of the peptide is its ability to target multiple regions within bacterial cells, thereby reducing the likelihood of resistance development. This feature is crucial in addressing the growing concern of antimicrobial resistance on a global scale.

[0013] The development of the peptide described in the invention benefits significantly from the use of bioinformatics and in silico tools, which offer several advantages for various reasons. Scanning comprehensive databases accelerates the identification of potential peptide candidates, leading to valuable time and resource savings. This data-centric approach enables comprehensive analysis and data-driven decision-making by integrating biological and chemical information from various sources. The rational design approach guided by bioinformatics has served as a guiding principle in decision-making rather than relying on trial and error. These in silico prediction tools have facilitated the computational evaluation of the physicochemical and biological properties of the developed peptide, reducing the need for extensive laboratory work and ensuring successful outcomes.

[0014] The tests conducted have revealed that the WK 14 peptide possesses strong antimicrobial properties and is highly effective against various bacterial species responsible for diseases in fish. Particularly, it holds significant promise as an alternative to traditional antibiotics in aquaculture. One of the key advantages of the developed WK14 peptide is its ability to target multiple regions within bacterial cells, significantly reducing the likelihood of bacterial resistance development. This feature is crucial in addressing the growing concerns about antimicrobial resistance on a global scale. The subject antimicrobial peptide not only addresses the urgent issue of reducing dependence on antibiotics but also provides an environmentally friendly and safe solution for aquaculture practices. Furthermore, the antimicrobial peptide is fully compatible with the One Health approach, which considers the welfare of humans, animals, and the environment. WK 14, which offers an effective and sustainable solution for combating bacterial infections in aquaculture, supports the health and sustainability of aquatic ecosystems while preserving the health of humans and animals. The peptide sequence exhibits antimicrobial properties by disrupting the cell wall structures of both gram-positive and gram -negative bacteria.

Claims

CLAIMS1. The invention is a novel antimicrobial peptide suitable for effectively treating bacterial pathogens and infectious pancreatic necrosis (IPN) disease in aquaculture without causing any harmful effects on the environment, characterized by comprising a novel amino acid sequence as "WKFHFAKIFIKYWK".