Screening and Characterization of Antimicrobial peptides from fish mucus in three major Lakes of Uganda

Abstract

Background: Antimicrobial peptides (AMPs) constitute a broad range of bioactive compounds in diverse organisms, including fish. Currently, several peptide antibiotics have been approved for clinical use. Still, skin mucosal AMPs of African catfish and African lungfish are largely unexplored despite their potential in contributing novel drug candidates. Accordingly, the African Catfish Antimicrobial peptides (ACAPs) were investigated. Methods: Native peptides were extracted from fish mucus scrapings in IO % acetic acid (v/v) and ultra-filtered using a 5 kDa molecular weight cut-off membrane. The ultrafiltrate were purified using CIS Solid-Phase Extraction cartridges. Later, their antibacterial activity was determined using the Agar Well Diffusion and broth-dilution methods utilizing Staphylococcus aureus (ATCC 25923) and Eschenchia coli (ATCC 25922). Sephadex G-25 gel filtration was further utilized in bio—guided isolation Of the most active fractions prior to peptide identification using Orbitrap Fusion Lumos Tribrid Mass Spectrometry. Discriminant Analysis Machine and Deep learning algorithms were utilized in predicting antimicrobial scores of each peptide. Toxicity of thc AMPs was assessed using rabbit blood cells alongside HLPpred-Fuse and ToxinPred servers. The AMPs possible mode of action was assessed using SevenGo Duo SG-23 digital conductivity meter, QuantiChrom Alkaline phosphatase kit, and molecular docking engines prior to their chemical synthesis. Results: The skin mucus extracts from fish in three major lakes of Uganda exhibited antimicrobial activity on E. coli and S. aureus. Lake Albert's African catfish demonstrated the highest antimicrobial activity with the lowest MIC of 2.84 and 0.71 gg/mL on S aureus and E. coli, respectively. Sephadex G-25 peak I muss spectrometry revealed seven short peptides (11—16 amino acid residues) of high antimicrobial scores (0.561—0.905 units). In addition, these peptides had a low molecular weight (1005.57—1622.05 Da) and had a percentage hydrophobicity above 54%. Partially purified extracts were non-hemotoxic peptide ACAP-V demonstrated the highest binding and global energies against Monomeric sarcosine oxidase (PDB [d: 2gbO; - 9.40 kcal/mol) and NADH peroxidase (PDB Id: Inhp; BE: -88.84 kcal/mol) respectively. Synthesized ACAP-IV and ACAP-V had the MIC of 520.7 and 625.0 gg/mL on E. coli. Conclusion and Recommendation: Collectively, the 7 African catfish antimicrobial peptides were identified as potential novel drug candidates. Future research is recommended to validate their activity in vivo on a wider range of bacteria.