| Trachinotus ovatus is a major economic species in cage culture in South China.However,with the expansion of the scale and density of T.ovatus culture,bacterial diseases have become one of the important factors limiting its development.The current misuse of chemical drugs such as antibiotics will not only cause drug resistance of pathogenic bacteria and pollute the water environment,but also affect the safety of aquatic products.Therefore,it is urgent to develop new green and efficient antibiotic alternatives.Antimicrobial peptides have become the best alternative for antibiotic replacement due to their broad-spectrum antimicrobial effects and environmentally friendly,drug-free residues,and less susceptible to drug resistance.In this study,we cloned and identified TroHepc1 and TroHepc2,the liver-expressed antimicrobial peptides of T.ovatus,and investigated their antimicrobial effects and mechanisms.The main results were as follows.Ⅰ、Identification and expression analysis of TroHepc1 and TroHepc2TroHepc1 and TroHepc2 have open reading frames(ORFs)of 273 bp and 255 bp that encode 90 and 84 amino acids,respectively.Sequence analysis showed that both TroHepc1 and TroHepc2 genes contain three parts: signal peptide,prodomain,and mature peptide which contains 8 cysteine residues.Multiple amino acid alignments revealed that TroHepc1 showed the highest homology of 93.33% with sea bass(Dicentrarchus labrax)and TroHepc2 showed the highest homology of 76.19% with the Hepcidin of starry flounders(Platichthys stellatus).The result of phylogenetic tree analysis showed that TroHepc1 and TroHepc2 were divided into two groups: HAMP1-type isoforms and HAMP2-type isoforms.TroHepc1 and TroHepc2 were respectively clustered with Hepcidin1 and Hepcidin2 of teleost,and closely related to Pagrus Auriga and Perca fluviatilis.The above results showed that TroHepc1 and TroHepc2 had high conservation.In addition,the expression profile of the tissues under normal physiological conditions showed that both TroHepc1 and TroHepc2 were expressed at extremely high levels in the liver,1160 and 2987-fold higher than the control,respectively,followed by high levels in other immune-related tissues including blood,spleen,and head kidney.The expression of TroHepc1 and TroHepc2 were significantly up-regulated in the liver,spleen,and head kidney after Vibrio harveyi challenge.The results of subcellular localization showed that TroHepc1 and TroHepc2 were distributed in both cytoplasm and nucleus.Ⅱ、Antibacterial immunity and mechanisms of TroHepc1 and TroHepc2To investigate the antibacterial effects and mechanisms of TroHepc1 and TroHepc2,we constructed TroHepc1 and TroHepc2 eukaryotic expression vectors named pTroHepc1 and pTroHepc2,respectively.Following the V.harveyi challenge,in vivo results showed that over-expression of TroHepc1 and TroHepc2 could significantly enhance the ability to resist V.harveyi.In contrast,the bacterial load in the tissues of the interference group increased significantly compared to the control group after the interference of TroHepc1 and TroHepc2 expression,indicating that TroHepc1 and TroHepc2 play an important role in the process of resistance to V.harveyi infestation in fish.The respiratory burst,acid phosphatase(ACP),alkaline phosphatase(AKP),and superoxide dismutase(SOD)activity assay of over-expression TroHepc1 and TroHepc2 macrophage activities were significantly increased compared with the control group,indicating that TroHepc1 and TroHepc2 could enhanced macrophages activation and play an important role in innate immunity.Quantitative real-ime PCR(qRT-PCR)results showed that over-expression of TroHepc1 and TroHepc2 significantly upregulated the expression of IL-1β,IL-6,IL-8,TNF-α,IFN-γ,CCL4 and CXCL9 mRNA levels in liver,and head kidney,indicating that TroHepc1 and TroHepc2 can induce the expression of immune-related factors.To further investigate the mechanism of antibacterial immunity of TroHepc1 and TroHepc2,we further cloned and analyzed their upstream regulatory regions and amplified 1841 bp and 1927 bp sequences,respectively,and found that nuclear factor kappa-B(NF-κB)binding sites existed in this region.The expression levels of TroHepc1 and TroHepc2 were significantly downregulated in the liver,and head kidney compared to the control,with NF-κB inhibited followed by V.harveyi challenge in vivo analysis.The results indicated that NF-κB can regulate the expression of TroHepc1 and TroHepc2.The results from Dual-Luciferase Reporter Assay showed that the region where NF-κB mainly regulates the promoters of TroHepc1 and TroHepc2 may be located approximately-399 bp ～-411 bp and-254 bp～-47 bp upstream of the start site,respectively.The predicted transcription factor binding sites which presented in both TroHepc1 and TroHepc2 regions.The above results suggest that TroHepc1 and TroHepc2 can effectively activate macrophage activity and enhance the expression of some immune-related genes,which can play an important role in antibacterial immunity through the regulatory role of NF-κB.Ⅲ、Antibacterial activity and mechanism of TroHepc1 and TroHepc2-derived peptidesTo further analyze whether TroHepc1 and TroHepc2 have antimicrobial activity,we performed further analysis of their amino acid sequences.Predicted amino acids 1-26(TroHepc1-26)and 1-21(TroHepc1-21)of the TroHepc1 gene and amino acids 1-22 of the TroHepc2 gene(TroHepc2-22)may have antimicrobial peptide activity,so we chemically synthesized its derivative peptide.The analysis of erythrocyte hemolysis rate in mice showed that the synthetic peptides TroHepc1-21 and TroHepc2-22 have very low cytotoxicity to mouse erythrocytes and are highly safe for subsequent experiments.Bacterial inhibition zone assay,minimum inhibitory concentration(MIC),and minimum bactericidal concentration(MBC)assays showed that TroHepc1-21 and TroHepc2-22 were highly effective against Staphylococcus aureus,Streptococcus agalactiae,V.harveyi and Edwardsiella piscicida,while TroHepc1-26 had weaker antibacterial activity.Growth curve assay showed that TroHepc1-21 and TroHepc2-22 could inhibit the growth of S.aureus,S.agalactiae,V.harveyi and E.piscicida with concentration-dependent.The mechanism of TroHepc1-21 and TroHepc2-22 damage to bacterial cell membranes was further investigated by membrane depolarization assay,propidium iodide(PI)staining assay,and scanning electron microscopy(SEM)observation.The results showed that TroHepc1-21 and TroHepc2-22 significantly induced S.aureus,S.agalactiae,V.harveyi and E.piscicida cell membrane depolarization,and then change the permeability of bacterial cell membrane,induce changes in cell morphology and structure,showing cell shrinkage,cell membrane rupture and leakage of cell contents.Gel retardation assay showed that TroHepc1-21 and TroHepc2-22 the synthetic peptide could bind to bacterial genomic DNA and subsequently cause its degradation after penetrating the bacterial membrane.Bacterial genomic DNA can be completely degraded at high concentrations of peptides;however,migration of bacterial genomic DNA can be completely or partially blocked at low concentrations of peptides.To further analyze the antibacterial effects of the derivative peptides,in vivo experiments were also conducted in this study.The results showed that the bacterial load was significantly reduced in the liver,spleen,and head kidney tissues of T.ovatus injected with synthetic derived peptides TroHepc1-21 and TroHepc2-22 compared to the control group.Moreover,the expression levels of these related immune genes,such as IL-6,TNFα,IL-1β,IL-8,IFN-γ,and TLR1 were significantly up-regulated.The above results indicated that the derived peptides TroHepc1-21 and TroHepc2-22 had broad-spectrum antimicrobial peptide activity and could regulate inflammatory cytokines and activate immune-related signaling pathways,stimulating the immune response of the organism.TroHepc1 and TroHepc2 might be able to play a role in the antibacterial immune response of T.ovatus through their antimicrobial peptide fragments.In conclusion,the antibacterial peptides TroHepc1 and TroHepc2 can activate immune cells and immune factors,and their derived peptides could be used as a potential immunomodulator and an anti-microbial therapeutic agent in the aquaculture of T.ovatus.Our results of this research contribute to insight into the antimicrobial effect and immune mechanism of Hepcidin,which provide theoretical support for the development and application of novel antibacterial peptides in the aquaculture industry. |
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