Molecular And Functional Characterization Of β-defensin In Large Yellow Croaker(larimichthys Crocea)

Large yellow croaker(Larimichthys crocea)is an economically important marine fish species in China.The aquaculture production of large yellow croaker reached approximately 198 thousand tons in 2018,which was the highest annual production among maricultured fish in China.In recent years,the infectious diseases caused by bacteria are becoming more and more severe,resulting in great economic losses.The drugs were often used for disease prevention and treatment in large yellow croaker industry,however,problems arose with extensive use of drugs,including food safety issues,environment safety issues,and resistance to drugs.β-defensin is a cysteine-rich cationic antibacterial peptide that is widely present in animals and plants and active against a wide range of bacteria,fungi and viruses.The study of β-defensin and its potential function in large yellow croaker will help to prevent pathogenic bacterial invasion.The open reading frame(ORF)of LycBD is 195 nucleotides,encoding a protein of 64 amino acids(aa).The deduced LycBD protein contains a20-aa signal peptide and a 44-aa mature polypeptide,which possesses the typical arrangement of six conserved cysteine residues,contributing to form three disulfide bonds.LycBD was constitutively expressed in all tissues examined,with the highest levels in spleen and head kidney.After induction with Vibrio alginolyticus,the expression levels of LycBD were significantly up-regulated in spleen,head kidney and gills,suggesting that LycBD may play an important role in anti-bacterial response of large yellow croaker.The mature peptide of LycBD was synthesized and its antimicrobial activity was evaluated by using minimum inhibitory concentration(MIC)assays.The synthetic mature peptide of LycBD had an antimicrobial effect on seven gram-negative bacteria(Vibrio alginolyticus,V.parahaemolyticus,V.harveyi,V.campbellii,Pseudomonas plecoglossicida,Aeromonas hydrophila and Escherichia coli)and two gram-positive bacteria(Bacillus subtilis and Staphylococcus aureus).Synthesized LycBD had more potent inhibitory effect on V.campbellii with a MIC value of 4 μM,followed by V.parahaemolyticus with a MIC value of 8 μM.The MIC value of Synthesized LycBD to V.alginolyticus,B.subtilis and V.harveyi was 16μM,and the MIC value of LycBD to P.plecoglossicida was 64 μM.Transmission electron microscopy(TEM)images showed that the membrane of P.plecoglossicida was destroyed by synthesized LycBD,leading to bacterial content outflow and the bacteria death.Besides,synthetic LycBD could up-regulated the expression levels of IL-1β,TNF-α2 and IL-8 in macrophages from head kidney of large yellow croaker,while down-regulated the expression level of IL-10,suggesting that LycBD may have immunoregulatory function on macrophage activation.In conclusion,a β-defensin homologue(LycBD)was identified in large yellow croaker,with conserved structural domains.LycBD is highly expressed in the important immune organs,and its synthetic peptides have significant antibacterial activity and regulate the expression of inflammatory factors,indicating that LycBD may play important roles in anti-bacterial response of large yellow croaker.These results will help to understand the function and related mechanism of β-defensins in fish,and also provide new ideas for disease prevention of large yellow croaker.