Isolation and characterization of β-defensin-like protein 1 from epidermal mucus of fungal infected fish (Cyprinus carpio) and assessment of its antimicrobial potencies

Abstract

Fish epidermis is rich in different pharmacologically active substances, most of which play a crucial role in immunity. In the current study, β-defensin-like protein 1 with high in vitro antimicrobial activity was isolated and characterized from crude epidermal mucus extract of common carp, Cyprinus carpio L. The crude mucus was screened for antimicrobial activity against five bacterial and four fungal pathogens. Crude mucus exhibited varied antimicrobial activity against all the used pathogens. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude mucus extract revealed multiple prominent bands with molecular weights corresponding to 6.9 kDa, 14.5 kDa, 24 kDa, 27 kDa and 48 kDa. Using Sephadex G-50 gel filtration liquid chromatography in conjunction with mass spectroscopy (LC/MS), a single peak with a molecular weight of 6908 Da was isolated and characterized. This peptide showed potent antimicrobial activity against all the five bacterial and four fungal strains used. Leclercia adecarboxylata and Enterobacter kobei were most susceptible with minimum inhibition concentration (MIC) value of 0.017 mg/mL, while Aeromonas sobria was least susceptible with an MIC of 2.24 mg/mL. Among the fungal pathogens, Candida glabrata was most susceptible with MIC value of 0.14 mg/mL, while Aspergillus sp. was least susceptible with MIC value of 4.48 mg/mL. The activity was further confirmed by the time kill assay. The antimicrobial peptide sequence determined from mass spectra was “PQSILVLLVLVVLALHCKENEAVSFPWSCASLSGVCRQGVCLPSELYFGPLGCGKGFLCCVSHF”, which consisted of 64 amino acids. Protein Basic Local Alignment Search Tool (BLASTP) of this sequence revealed 100% homology with the β-defensin-like protein 1, which is the first report of this antimicrobial peptide from epidermal mucus of C. carpio from Kashmir waters.