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

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.

抗菌肽（Antimicrobial Peptides, AMPs）是广泛存在于包括鱼类在内的各类生物中的一类生物活性化合物，作为先天免疫应答的效应分子，可对抗病原体侵袭、组织损伤与感染。尽管非洲鲶鱼（Clarias gariepinus）皮肤黏液来源的抗菌肽在对抗抗菌耐药性方面具备潜在治疗价值，但目前相关研究仍较为匮乏。
本研究从非洲鲶鱼（Clarias gariepinus）的皮肤黏液中分离得到非洲鲶鱼源抗菌肽（African Catfish Antimicrobial Peptides, ACAPs）。具体实验流程如下：从鱼黏液刮取物中提取天然肽段，采用10%（体积比）乙酸溶液作为提取溶剂，并通过分子量截留值为5kDa的超滤膜进行超滤分级；提取物经C18固相萃取完成纯化。采用琼脂孔穴扩散法与肉汤稀释法，以金黄色葡萄球菌（Staphylococcus aureus, ATCC 25923）和大肠埃希菌（Escherichia coli, ATCC 25922）为受试菌株，检测其抗菌活性。随后通过Sephadex G-25凝胶过滤层析进行生物导向分离，获取活性最强的组分，再利用Orbitrap Fusion Lumos Tribrid质谱仪进行肽段鉴定。
实验结果显示，采自乌干达三大主要湖泊的非洲鲶鱼皮肤黏液提取物，均对大肠埃希菌与金黄色葡萄球菌具有抗菌活性。其中，阿尔伯特湖来源的Clarias gariepinus活性最优，对金黄色葡萄球菌与大肠埃希菌的最低抑菌浓度（Minimum Inhibitory Concentration, MIC）分别低至2.84 µg/mL与0.71 µg/mL。对Sephadex G-25峰I组分进行质谱分析与虚拟（in silico）分析后，共鉴定得到7条短肽（氨基酸残基数为11~15），其抗菌评分介于0.561~0.905之间。此外，这些肽段分子量较低（1005.57~1622.05 Da），疏水占比均超过54%，其中4条具备α-螺旋结构构象，呈现两亲性。
本研究结果表明，可从Clarias gariepinus的皮肤黏液中获取新型抗菌肽。这类抗菌肽有望成为传统抗生素的潜在替代方案，在食品与制药工业中具备重要应用价值，但仍需开展进一步研究以明确其成药性与安全性特征。