Characterization of peptides and proteins from epidermal mucus of climbing perch Anabas testudineus (Bloch, 1972) with antibacterial and immunomodulatory properties

The skin of fish with its large mucosal surface contains a variety of biologically active compounds and antimicrobial peptides that are constitutively expressed to provide protection against potential pathogenic microbes. Climbing perch (Anabas testudineus) is an obligate air breather and tolerates extremely unfavourable water conditions. Its body is covered by a thick coat of mucus composed of mucopolysaccharides, lipids and proteins making the fish a suitable candidate to obtain bioactive compounds such as antibacterial proteins and peptides. Based on this background, the assessment of the antimicrobial and immunomodulatory effects of bioactive proteins and peptides in crude mucous extracts derived from epidermis of the climbing perch is an important step. Hence, the aim of this study was to determine the antimicrobial and immunomodulatory effects of the bioactive crude mucous extract from epidermis of climbing perch in order to test their efficacy both in vivo and in vitro. The bioactive crude mucous extract was prepared in aqueous and acidified form by heating moderately, followed by assessing their immune components as well as evaluating their antibacterial activity and determining some immune-related enzymes using well agar diffusion test, respectively. Proteomic analysis was also conducted to estimate the protein concentration including SDS-PAGE, haemolytic activity of the acidic crude extract as well as histopathological analysis. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) as well as NaCl2 tolerance of the bioactive crude were also conducted. Antibacterial activity was found to be statistically significant (P<0.05) showing maximum activity against P. aeruginosa ATCC10145. However, no haemolytic activity was observed for the acidified crude extract on horse RBCs. Total soluble protein concentration by OPA assay and SDS-PAGE profile revealed 13 bands (245- 11 kDa) distinguished using NuPAGE 4-12% Bis-Tris (Novex Invitrogen) gel. The MIC and MBC of mucous crude extract as determined by resazurin-based microliter dilution assay was found to have a similar value (2.5mg/ml) against multidrug resistance (MDR) P. aeruginosa ATCC10145 in addition to salt tolerance at physiological solution. Assessment of the immune-related enzymes activities of protease, lysozyme and esterase in bioactive crude indicated significant roles in disease defence mechanism. Similarly, antimicrobial proteins and peptides isolated by sepPak C18 and Ultrafiltration 30kDa and Superdex™peptide showed that the fractions in the first peak refer to antibacterial properties against P. aeruginosa ATCC 10145. Furthermore, bioactive fractions were run through Q-TOFLC/MS with subsequent digestion by trypsin to identify the antimicrobial proteins and peptides. This was the first time to identify novel ApolipoproteinA-1 (Antimicrobial proteins) and Haemoglobin subunit beta (Fragment) as well as β-actin (Fragment) and Elongation factor 1-α (Fragment) and Cytochrome-C oxidase subunit1(Fragment) in mucous crude extract derived from epidermal scraping of climbing perch. The de novo peptide AAGPKGPLGPR was selected among other de novo peptides as it was predicted to be antibacterial peptide according on its net charge (+2), low residues (11), the hydrophobic ratio of 27% and richness in proline and glycine. Following the proteomic analysis of bioactive crude and estimation of immune related enzymes activities, the efficacy of antimicrobial proteins and bioactive potential peptide in the crude (AMPPC) against P. aeruginosa ATCC 10145 was applied in an animal model. The inoculum size for MDR of P. aeruginosa ATCC 1014 was determined at 2×107 CFU/mouse using Reed and Muench method and oral administration AMPPC at 6.38mg/mouse was nontoxic to male ICR mice, with no change in physical state and body weight as compared to the control group, PBS only. Moreover, liver function enzymes, ALT and AST were not significantly different when compared to the control group mice given PBS only. In addition, kidney function indicator, creatinine and urea levels revealed no significant difference. Histological examination of liver and kidney manifested normal histological architectures. The efficacy test of AMPPC showed that mice injected with 2×107CFU/mouse of P. aeruginosa ATCC 10145 in 0.2 ml of AMPPC, CE and PBS solutions and evaluated after 48 hrs, indicated there were significant decreased (P<0.05) in mortality rate and bacteria count in organs and blood samples, liver enzymes (AST and ALT), renal functions (creatinine and urea levels) and proinflammatory cytokines TNF-α and IL-6 in treatment groups by bioactive crude mucous extract, as compared to non-treatment and antibiotic treatment groups. In conclusion, the epidermal mucous of the climbing perch are natural sources of antimicrobial proteins and peptides. Similarly, the proteomic analysis of bioactive crude extracts derived from the epidermal mucous may serve as an opening to further investigations to determine their potential medical application as antibacterial agent for preventing and elimination of P. aeruginosa sepsis.

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