Expression And Functional Characteristics Of β-Defensins In Grass Carp(Ctenopharyngodon Idella)

Antimicrobial peptides(AMPs),also known as host defense peptides or antimicrobial peptides,are important substances in the body’s natural immune response.They are widely distributed in vertebrates,mainly expressed in skin and mucosal epithelium,and are the first line of defense against infection.play an important role.β-defensins(BDs)are cationic AMPs rich in cysteine,which widely exist in animals and plants including teleosts,and are an important part of fish innate immunity.In this study,three BD genes(Ci BD-1,Ci BD-2,and Ci BD-3)were identified in grass carp(Ctenopharyngodon idella,Ci).Bioinformatics methods were used to analyze their differences and conserved features with other species.Real-time fluorescent quantitative PCR(q RT-PCR)was performed to analyze the expression differences of the three BD genes in different tissues.The primary head kidney leukocytes(HKLs)were isolated and stimulated with Polyinosinic acid-polycytidylic acid[poly(I:C)],phorbol ester(Phorbol12-myristate 13-acetate,PMA),lipopolysaccharide(Lipopolysaccharide,LPS),Phytohaemagglutinin(PHA),and heat-inactivated Aeromonas hydrophila(A.hydrophila)to observe the expression changes of Ci BDs.The expression regulation of Ci BDs was also detected in the gills,head kidney,and hindgut of grass carp under the infection of A.hydrophila.Recombinant polymorphism with high purity and strong activity was obtained from HEK293-F,and it was used to determine the minimum inhibitory concentration(MIC)and antibacterial kinetics of different bacteria susceptible to aquatic organisms.Finally,the inhibitory effect of Ci BDs on the replication of Spring Viremia of Carp Virus(SVCV)was analyzed by q RT-PCR.The specific results are as follows:(1)Analyze the grass carp genome to obtain three Ci BD genes and verify their coding sequences.Phylogenetic tree analysis showed that fish BDs clustered on three distinct clades.Ci BD-1 was placed in the BD-1 group,distinct from the clade consisting of the BD-2 and BD-3 groups.Although the BD-2 and BD-3 groups were separate,they formed an extended branch with a bootstrap value of 100%.Multiple sequence alignment showed that 6 cysteine residues were well preserved in fish BDs,which were predicted to form 3pairs of disulfide bonds(Cys~1-Cys~5,Cys~2-Cys~4,Cys~3-Cys~6).Different from the 2 exons/1intron gene structure of mammalian BD genes,all fish BDs genes have 3 exons.The second exon encodes 4 cysteines,and the third exon encodes 2 consecutive cysteines.(2)The expression of Ci BDs genes in six grass carp tissues was analyzed by q RT-PCR.All three Ci BD genes were constantly expressed in skin,liver,spleen,head kidney,hindgut and muscle.Compared with other tissues,the expression of Ci BD-1 and Ci BD-3was highest in the skin,while the highest expression of Ci BD-2 was detected in the spleen.All of this Ci BDs,expression levels were lowest in muscle.In addition,the expression level of Ci BD-1 was much higher than that of Ci BD-2 and Ci BD-3 in the same tissue.(3)Stimulate freshly isolated progenitors with LPS,poly(I:C),PHA,PMA,heat-inactivated A.hydrophila,or inflammatory cytokines(IL-1β,IFN-γ,IL-22,and IL-10).Substitute HKLs.Clearly,all three Ci BDs could be induced by LPS,poly(I:C),PHA,and heat-inactivated A.hydrophila,and none of the Ci BDs was upregulated by PMA and IL-10;stimulation of HKLs by IL-1βand IFN-γresulted in upregulation of Ci BDs,IL-22induced the expression of Ci BD-1,but not Ci BD-2 and Ci BD-3.All three Ci BDs were upregulated in the gills,head kidney and hindgut at different time points of A.hydrophila infection.(4)The expression and purity of Ci BDs recombinant proteins were analyzed by confocal microscope,Western blot,and Tricine SDS-PAGE.The antibacterial activity of Ci BDs peptides against five bacterial pathogens,including four Gram-negative bacteria(A.hydrophila,Edwardsiella tarda,Flavobacterium columnare,and Klebsiella pneumoniae)and a Gram-positive bacterium(Staphylococcus aureus),was analyzed by the MIC method.All three Ci BDs were effective in inhibiting the growth of A.hydrophila and F.columnare,among which Ci BD-3(6.25μg/m L)was more effective against A.hydrophila(12.5μg/m L for Ci BD-1 and Ci BD-2)and had stronger antibacterial activity against F.columnare(6.25μg/m L).Ci BDs were less effective against E.tarda and K.pneumoniae(MIC value:25μg/m L).Additionally,higher MIC values were observed for Ci BDs against S.aureus.(5)To assess the effect of Ci BDs transfection on EPC cells and its influence on SVCV replication,we conducted q RT-PCR analysis.Ci BD-1 significantly inhibited SVCV replication,and the interaction between Ci BD-1 and SVCV-G was shown by co-immunoprecipitation and confocal microscopy.This study compared the expression and antibacterial activity of three BDs in grass carp.The results showed that Ci BDs could be upregulated by PAMPs(such as PHA,LPS and poly(I:C))and cytokines(such as IL-1βand IFN-γ).Ci BDs are upregulated in response to A.hydrophila infection.Recombinant Ci BDs peptides derived from HEK293-F cells have strong antibacterial activity against Gram-negative and Gram-positive fish bacterial pathogens.In addition,the antiviral activity of Ci BD-1 was preliminarily investigated.Through our research,we seek to gain insight into how fish respond to microbial threats and encourage the implementation of antibiotic-minimizing strategies in aquaculture.