Screening And Functional Analysis Of Antimicrobial Peptides In Chinese Horseshoe Crab

Chinese horseshoe crab(Tachypleus tridentatus),an ancient marine invertebrate lacking adaptive immunity system,relies only on innate immune mechanisms including humoral immunity and cellular responses during pathogen defense.Humoral immunity mainly includes a series of hemolymph-dependent reactions such as the complement system,hemolymph agglutination,melanization reaction and the production of antimicrobial peptides,which are the main components of the congenital immune defense system of Chinese horseshoe crabs.In this study,a large number of candidate antimicrobial peptides were screened in the hemolymph of Chinese horseshoe crab through multi-omics analysis of transcriptome and proteome.And gene cloning,expression and function analysis of the candidate antimicrobial peptides including Tatritin,Cp AMP and H2 A,were conducted to explore their expression patterns and biological activity in vitro and in vivo.The main results of this study were shown blow:1.Comprehensive analysis of the immune response mechanism in Chinese horseshoe crab hemolymph by combined transcriptome and proteome analysis showed that the signaling pathways of complement and coagulation cascade,Toll,NF-κB,C-type lectin receptor,JAK-STAT and MAPK,and antimicrobial substances such as antimicrobial peptides and lectins,were significantly activated after bacteria challenge.These results suggested that Gram-negative bacteria can activate the hemolymph coagulation cascade and antimicrobial substance release through the above pathways.In addition,Toll and NFk B signaling pathways were involved in the immune response of Chinese horseshoe crab hemolymph through an integral signaling cascade,regulating the expression of downstream immune factors such as antimicrobial peptides.Using the multi-omics high-throughput data of Chinese horseshoe crab,120 non-redundant candidate antimicrobial peptides were screened in Chinese horseshoe crab.And the candidate antimicrobial peptides including Tatritin,Cp AMP and H2 A were analysed by bioinformatics and antibacterial related function prediction.The characteristics of small molecule,positive charge and amphiphilicity confirm their potential as antimicrobial peptides.2.The antimicrobial peptide Tatritin was mainly expressed in the hemolymph,muscle and gill of Chinese horseshoe crab,and the Tatritin expression level was significantly induced in hemolymph and gill after bacteria infection.The preprotein of Tatritin consists of a signal peptide(21 aa)and a mature cysteine-rich peptide(47 aa)with a predicted molecular weight of approximately 5.6 k Da and theoretical isoelectric point(p I)of 9.99.Tatritin has a α-helical structure in N-terminal and an antiparallel amphipathic β-sheet structure in the cysteine-stabilized C-terminal region.Chemically synthesized Tatritin peptide had broad-spectrum antibacterial activity against Gram-negative and-positive bacteria and fungi,and also had significant inhibitory effects on antibiotic-resistant strains.Analysis of antibacterial mechanism showed that Tatritin can recognize and inhibit pathogenic microorganisms by directly binding to LPS,DNA and chitin.In addition,injection of the antimicrobial peptide Tatritin can significantly reduce the mortality after bacteria infection in zebrafish.Due to its broad-spectrum antibacterial activity and sensitivity to drug-resistant strains in vivo and in vitro,Tatritin can be used as a novel antibacterial drug for the treatment of bacteria infections in animals or as an animal immune enhancer.Cytotoxicity analysis showed that the Tatritin had certain cytotoxicity at high concentrations,especially to tumor cells.Tatritin could inhibit tumor cell proliferation and promote apoptosis by interfering with mitochondrial activity and regulating intracellular ROS levels.3.Antibacterial peptides Cp AMP and H2 A were widely expressed in various tissues of Chinese horseshoe crab,and the expression level in muscle,heart and gill tissues was significantly activated after bacterial challenge.The preprotein of Cp AMP contains 68 amino acid residues,which contain a signal peptide(21 aa)in N-terminal and a mature peptide composed of 47 amino acid residues in C-terminal.Cp AMP has high homology with LMW-P15 isolated from the cuticular protein of Chinese horseshoe crab,and only the signal peptide region has a little difference.There was no signal peptide in H2 A,and its mature peptide was a hydrolysis product with 16 amino acid residues(H2A16)of the Nterminal sequence in the preprotein.The H2A-based phylogenetic analysis revealed that the Chinese horseshoe crab was closely related to Drosophila melanogaster and Nephila pilipes.Antibacterial activity analysis showed that the antibacterial peptides Cp AMP and H2 A had broad-spectrum antibacterial activities aganist Gram-negative and-positive bacteria,and also had inhibitory ability against antibiotic-resistant strains.Cp AMP showed stronger antibacterial activity than the antibiotic enrofloxacin in several strains,and H2 A had slightly weaker antibacterial activity.Compared with enrofloxacin,antibacterial activity of H2 A was stronger only in drug-resistant E.coli.In zebrafish,the antimicrobial peptide Cp AMP can significantly fight bacterial infection and improve the survival rate of diseased zebrafish,achieving similar effects as the antibiotic enrofloxacin.However,Cp AMP had no effect for bacterial infection in the therapy exprement of zebrafish.