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| Identification, Expression and Preliminary Antimicrobial Mechanism of Liver-expressed Antimicrobial Peptide 2 Gene in Black Scraper (Thamnaconus modestus) |
| HU Yi-Fan, WEI Bo-Jue, ZHANG Yi-Rong, LI Chang-Hong*, CHEN Jiong |
| Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315832, China |
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Abstract Liver-expressed antimicrobial peptide 2 (LEAP-2) is a cationic antimicrobial peptide that plays a crucial role in the innate immunity of animals. The cDNA sequences of TmLEAP-2A and TmLEAP-2C were identified through transcriptome sequencing in black scraper (Thamnaconus modestus) in this study. Sequence analysis indicated that TmLEAP-2A and TmLEAP-2C encoded 103 and 79 amino acids, respectively, with molecular weights of 11.18 and 9.14 kD. TmLEAP-2A and TmLEAP-2C exhibited the highest amino acid homology with LEAP-2A and LEAP-2C from the large yellow croaker (Larimichthys crocea), at 72.8% and 62.5% respectively. Phylogenetic tree analysis showed that TmLEAP-2A and TmLEAP-2C had the closest evolutionary correlation with LEAP-2A and LEAP-2C in large yellow croaker respectively. qPCR analysis showed that both TmLEAP-2A and TmLEAP-2C were predominately expressed in the liver. Further, their expression levels were significantly induced by infection with Vibrio alginolyticus in liver of black scraper. TmLEAP-2A mature peptide (TmLEAP-2AP46) only exhibited inhibitory activity against V. vulnificus, while TmLEAP-2C mature peptide (TmLEAP-2CP39) exhibited inhibitory activity against both V. alginolyticus and V. vulnificus. Further research demonstrated that TmLEAP-2CP39 exhibited a hydrolytic effect on the genome of V. alginolyticus. Following incubation with TmLEAP-2CP39, V. alginolyticus showed increased uptake of propidium iodide, indicating a significant reduction in the proportion of viable cells. Scanning electron microscopy (SEM) observations revealed that treatment with TmLEAP-2CP39 resulted in altered morphology of V. alginolyticus, characterized by surface wrinkles and the presence of depressions or holes on most bacterial cells. Additionally, transmission electron microscopy (TEM) analysis revealed that, after treatment with TmLEAP-2CP39, the cell membrane of V. alginolyticus exhibited pronounced wrinkling, significant separation from the cytoplasm, rupturing of the membrane, and notable leakage of cellular contents. Overall, TmLEAP-2A and TmLEAP-2C were found to be closely associated with V. alginolyticus infection; however, only TmLEAP-2C demonstrated antibacterial activity against V. alginolyticus in vitro. TmLEAP-2C exerted direct antibacterial effects by disrupting the integrity of the bacterial cell membrane and hydrolyzing genomic DNA (gDNA) within V. alginolyticus cells. These findings not only enhance the understanding of fish antimicrobial peptides but also provide a theoretical foundation for the research and development of fish-derived antimicrobial peptide drugs.
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Received: 11 October 2024
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Corresponding Authors:
*lichanghong0716@163.com
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[1] 苏锦祥, 李春生. 2002. 中国动物志: 硬骨鱼纲-鲀形目、海蛾鱼目、喉盘鱼目、鮟鱇目[M]. 科学出版社, 北京. pp. 128-129.
(Su J X, Li C S.2002. Fauna of China: Osteichthyes-Tetraodontiformes, Pegasiformes, Gobiesociformes, Lophiiformes[M]. Science Press, Beijing, pp. 128-129.)
[2] 王维, 李长红, 詹萍萍, 等. 2022, 一株引起大弹涂鱼出血症的维氏气单胞菌的分离及鉴定[J].农业生物技术学报, 30(02): 344-355.
(Wang W, Li C H, Zhan P P, et al.2022. Isolation and identification of a Aeromonas veronii strain causing the hemorrhagic disease of great blue-spotted mudskipper (Boleophthalmus pectinirostris)[J]. Journal of Agricultural Biotechnology, 30(02): 344-355.)
[3] 张艺榕, 李长红, 陈炯. 2021. 花鲈C型凝集素受体CD302基因的分子鉴定及其在哈维氏弧菌感染下的表达[J]. 农业生物技术学报, 29(03): 424-434.
(Molecular identification of C-type lectin receptor gene CD302 from Japanese sea bass(Lateolabrax japonicus) and its expression upon Vibrio harveyi infection[J]. Journal of Agricultural Biotechnology, 29(03): 424-434.)
[4] Brogden K A.2005. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacterial?[J]. Nature Reviews Microbiology, 3(3): 238-250.
[5] Casterlow S, Li H, Gilbert E R, et al.2011. An antimicrobial peptide is downregulated in the small intestine of Eimeria maxima-infected chickens[J]. Poultry Science Journal, 90(6): 1212-1219.
[6] Chen J, Chen Q, Lu X J, et al.2016. The protection effect of LEAP-2 on the mudskipper (Boleophthalmus pectinirostris) against Edwardsiella tarda infection is associated with its immunomodulatory activity on monocytes/macrophages[J]. Fish and Shellfish Immunology, 59: 66-76.
[7] Chen J, Lu Y P, Dai Q M, et al.2019. Host defense peptide LEAP-2 contributes to monocyte/macrophage polarization in barbel steed (Hemibarbus labeo)[J]. Fish and Shellfish Immunology, 87: 184-192.
[8] Ding F F, Li C H, Chen J.2019. Molecular characterization of the NK-lysin in a teleost fish, Boleophthalmus pectinirostris: Antimicrobial activity and immunomodulatory activity on monocytes/macrophages[J]. Fish and Shellfish Immunology, 92: 256-264.
[9] Drayton M, Deisinger J P, Ludwig K C.2021. Host defense peptides: Dual antimicrobial and immunomodulatory action[J]. International Journal of Molecular Sciences, 22(20): 11172.
[10] Hocquellet A, Odaert B, Cabanne C, et al.2010. Structure-activity relationship of human liver-expressed antimicrobial peptide 2[J]. Peptides, 31(1): 58-66.
[11] Hong Y, Truong A D, Lee J, et al.2019. Identification of duck liver-expressed antimicrobial peptide 2 and characterization of its bactericidal activity[J]. Asian-Australasian Journal of Animal Sciences, 32(7): 1052-1061.
[12] Howard A, Townes C, Milona P, et al.2010. Expression and functional analyses of liver expressed antimicrobial peptide-2 (LEAP-2) variant forms in human tissues[J]. Cellular Immunology, 261(2): 128-133.
[13] Hwang S D, Joo M S, Hwang J Y, et al.2019. Molecular characterization and gene expression data of liver expressed antimicrobial peptide-2 (LEAP-2) isolated from rock bream (Oplegnathus fasciatus)[J]. Data in Brief, 26: 104538.
[14] Jang W S, Kim H K, Lee K Y, et al.2006. Antifungal activity of synthetic peptide derived from halocidin, antimicrobial peptide from the tunicate, Halocynthia aurantium[J]. FEBS Letters, 580(5): 1490-1496.
[15] Kim C H, Kim E J, Nam Y K.2019. Subfunctionalization and evolution of liver-expressed antimicrobial peptide 2 (LEAP2) isoform genes in Siberian sturgeon (Acipenser baerii), a primitive chondrostean fish species[J]. Fish and Shellfish Immunology, 93: 161-173.
[16] Krause A, Sillard R, Kleemeier B, et al.2003. Isolation and biochemical characterization of LEAP-2, a novel blood peptide expressed in the liver[J]. Protein Science, 12(1): 143-152.
[17] Kumar S, Stecher G, Tamura K.2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Molecular Biology and Evolution, 33(7): 1870-1874.
[18] Li H X, Lu X J, Li C H, et al.2014. Molecular characterization and functional analysis of two distinct liver-expressed antimicrobial peptide 2 (LEAP-2) genes in large yellow croaker (Larimichthys crocea)[J]. Fish and Shellfish Immunology, 38(2): 330-339.
[19] Li H X, Lu X J, Li C H, et al.2015. Molecular characterization of the liver-expressed antimicrobial peptide 2 (LEAP-2) in a teleost fish, Plecoglossus altivelis: Antimicrobial activity and molecular mechanism[J]. Molecular Immunology, 65(2): 406-415.
[20] Liang T, Ji W, Zhang G R, et al.2013. Molecular cloning and expression analysis of liver-expressed antimicrobial peptide 1 (LEAP-1) and LEAP-2 genes in the blunt snout bream (Megalobrama amblycephala)[J]. Fish and Shellfish Immunology, 35(2): 553-563.
[21] Liu F, Li J L, Yue G H, et al.2010. Molecular cloning and expression analysis of the liver-expressed antimicrobial peptide 2 (LEAP-2) gene in grass carp[J]. Veterinary Immunology and Immunopathology, 133(2-4): 133-143.
[22] Liu T, Gao Y, Wang R, et al.2014. Characterization, evolution and functional analysis of the liver-expressed antimicrobial peptide 2 (LEAP-2) gene in miiuy croaker[J]. Fish and Shellfish Immunology, 41(2): 191-199.
[23] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method[J]. Methods, 25(4): 402-408.
[24] Mazurkiewicz-Pisarek A, Baran J, Ciach T.2023. Antimicrobial peptides: Challenging journey to the pharmaceutical, biomedical, and cosmeceutical use[J]. International Journal of Molecular Sciences, 24(10): 9031.
[25] Mba I E, Nweze E I.2022. Antimicrobial peptides therapy: An emerging alternative for treating drug-resistant bcteria[J]. Yale Journal of Biology and Medicine, 95(4): 445-463.
[26] Nogrado K, Adisakwattana P, Reamtong O.2022. Antimicrobial peptides: On future antiprotozoal and anthelminthic applications[J]. Acta Tropica, 235: 106665.
[27] Sang Y, Ramanathan B, Minton J E, et al.2006. Porcine liver-expressed antimicrobial peptides, hepcidin and LEAP-2: Cloning and induction by bacterial infection[J]. Developmental and Comparative Immunology, 30(4): 357-366.
[28] Shata M T M, Abdel-Hameed E A, Hetta H F, et al.2013. Immune activation in HIV/HCV-infected patients is associated with low-level expression of liver expressed antimicrobial peptide-2 (LEAP-2)[J]. Journal of Clinical Pathology, 66(11): 967-975.
[29] Smolarczyk R, Cichoń T, Szala S.2009. Peptides: A new class of anticancer drugs[J]. Postepy Higieny I Medycyny Doswiadczalnej, 63: 360-368.
[30] Townes C L, Michailidis G, Hall J.2009. The interaction of the antimicrobial peptide cLEAP-2 and the bacterial membrane[J]. Biochemical and Biophysical Research Communications, 387(3): 500-503.
[31] Townes C L, Michailidis G, Nile C J, et al.2004. Induction of cationic chicken liver-expressed antimicrobial peptide 2 in response to Salmonella enterica infection[J]. Infection and Immunity, 72(12): 6987-6993.
[32] Zhang D, He Y, Ye Y, et al.2019. Little antimicrobial peptides with big therapeutic roles[J]. Protein and Peptide Letters, 26(8): 564-578.
[33] Zhang Y A, Zou J, Chang C I, et al.2004. Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout[J]. Veterinary Immunology and Immunopathology, 101(3-4): 259-269. |
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