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Identification of antimicrobial peptides isolated from the skin mucus of African Catfish, Clarias gariepinus (Burchell, 1822)

ABSTRACT: Antimicrobial peptides (AMPs) constitute a broad range of bioactive compounds in diverse organisms, including fish. They are effector molecules for the innate immune response, against pathogens, tissue damage and infections. Still, AMPs from African Catfish, Clarias gariepinus skin mucus are largely unexplored despite their possible therapeutic role in combating antimicrobial resistance. In this study, African Catfish Antimicrobial peptides (ACAPs) were identified from the skin mucus of African Catfish, C. gariepinus. Native peptides were extracted from fish mucus scrapings in 10% acetic acid (v/v) and ultra-filtered using 5kDa molecular cut-off membrane. The extract was purified using C18 Solid Phase Extraction. The antibacterial activity was determined using the Agar Well Diffusion method and broth-dilution method utilizing Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922). Thereafter, 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. The skin mucus extracted from African Catfish from all the three major lakes of Uganda exhibited antimicrobial activity on E. coli and S. aureus. Lake Albert’s C. gariepinus demonstrated the best activity with the lowest MIC of 2.84 µg/mL and 0.71 µg/mL on S. aureus and E. coli respectively. Sephadex G-25 peak I mass spectrometry analysis alongside in silico analysis revealed seven short peptides (11-15 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 percentage hydrophobicity above 54%. Up to four of these antimicrobial peptides demonstrated α-helix structure conformation, rendering them amphipathic. The ﬁndings of this study indicate that novel antimicrobial peptides can be sourced from the skin mucus of C. gariepinus. Such antimicrobial peptides are potential alternatives to the traditional antibiotics and can be of great application to food and pharmaceutical industries; however, further studies are still needed to establish their drug-likeness and safety profiles.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Clarias Gariepinus

TISSUE(S): Skin Mucus

SUBMITTER: Didier Vertommen

LAB HEAD: Patrick Ogwang Engeu

PROVIDER: PXD029193 | Pride | 2021-12-01

REPOSITORIES: Pride

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Project description:Animal mucosal barriers constantly interact with the external environment and this interaction is markedly different in aquatic and terrestrial environments. Transitioning from water to land was a critical step in vertebrate evolution but the immune adaptations that mucosal barriers such as the skin underwent during that process are essentially unknown. Vertebrate animals such as the African lungfish have a bimodal life, switching from freshwater to terrestrial habitats when environmental conditions are not favorable. African lungfish skin mucus secretions contribute to the terrestrialization process by forming a cocoon that surrounds and protect the lungfish body. The goal of this study is to characterize the skin mucus immunoproteome of African lungfish, Protopterus dolloi, before and during the induction phase of terrestrialization as well as the immunoproteome of the gill mucus during the terrestrialization induction phase. Using LC-MS/MS, we identified a total of a total of 974 proteins using a lungfish Illumina RNA-seq database and 880 proteins using a lungfish 454 RNA-seq database for annotation in the three samples analyzed (control skin mucus, terrestrialized skin mucus and terrestrialized gill mucus). The terrestrialized skin mucus proteome was enriched in proteins with known antimicrobial functions such as histones and S100 proteins. In support, gene ontology analyses showed that the terrestrialized skin mucus proteome has predicted functions in processes such as viral process, defense response to Gram negative bacterium and tumor necrosis factor mediated signaling. Importantly, we observed a switch in immunoglobulin heavy chain secretion upon terrestrialization, with IgW1L and IgM1 present in control skin mucus and IgW1L, IgM1 and IgM2 in terrestrialized skin mucus. Combined, these results indicate an increase investment in the production of unique immune molecules in P. dolloi skin mucus in response to terrestrialization that likely better protect lungfish against external aggressors found in land.

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Identification of antimicrobial peptides isolated from the skin mucus of African Catfish, Clarias gariepinus (Burchell, 1822)

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