大弹涂鱼皮肤黏液的抑菌活性和蛋白质组学
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摘要
皮肤黏液是鱼类免疫防御的第一道防线。大弹涂鱼皮肤黏液对其免疫防御、渗透压维持以及适应水陆生活等方面具有重要意义。为深入了解大弹涂鱼皮肤黏液的蛋白质分子组成,对其皮肤黏液开展了抑菌活性分析和蛋白质组学分析。通过电刺激法收集大弹涂鱼皮肤黏液,采用打孔法比较了皮肤黏液和血清的抑菌活性差异;进一步利用鳗弧菌对大弹涂鱼进行诱导,采用生长曲线抑制法分析和比较了诱导前后皮肤黏液的抑菌活性差异。利用Shotgun质谱技术,结合大弹涂鱼皮肤转录组数据库,对大弹涂鱼皮肤黏液开展了蛋白质组学分析;进一步利用String软件对所鉴定的蛋白质开展了蛋白质相互作用预测。大弹涂鱼皮肤黏液具有广谱抑菌活性,鳗弧菌诱导后的大弹涂鱼皮肤黏液与诱导前相比,对部分革兰氏阴性菌的抑菌活性明显上升,但对其他菌株的抑菌活性差异不明显。从大弹涂鱼皮肤黏液中共鉴定各类蛋白质分子97种,基本分子组成与其他硬骨鱼类皮肤黏液蛋白相似,但也有少数蛋白如泛素蛋白、胸腺素蛋白等未在其他鱼类皮肤黏液中报道。此外,其他鱼类中所鉴定到的部分蛋白如热休克蛋白、抗菌肽等在大弹涂鱼皮肤黏液中未能鉴定到。大弹涂鱼皮肤黏液中已鉴定的蛋白多数具有与免疫功能相关的结构域,且存在一个以肌动蛋白为核心的相互作用网络。大弹涂鱼皮肤黏液具有明显的广谱抑菌活性,其蛋白质组成与其他鱼类皮肤黏液具有一定的相似性。本研究为深入了解大弹涂鱼皮肤黏液的分子多样性及功能机制奠定了基础。
Fish skin mucus serves as the first line of defense against pathogens and external stressors. The mudskipper Boleophthalmus pectinirostris inhabit intertidal mudflats containing abundant and diverse microbial population, thus, the skin together with the mucus of B. pectinirostris are very important for the immune defense,osmotic pressure maintenance and adaptation to amphibian life. For exploring the proteomic profile of the skin mucus and understanding the molecular mechanism of B. pectinirostris adaptation to amphibious environments,the antibacterial activity was determined by agar diffusion plate method for the mucus and the serum samples. In addition, a growth curve inhibition method was used to compare the antibacterial activities of B. pectinirostris mucus before and after vibrio induction. Furthermore, the proteomic profile of natural B. pectinirostris mucus was identified by Shotgun mass spectrometry technology combining with skin transcriptome searching. The interaction network analysis of the identified proteins from mucus was performed by String software. Skin mucus was collected from B. pectinirostris after electrical stimulation. The mucus of B. pectinirostris showed a broad spectrum of antimicrobial activity more than serum of the same species, indicating that the mucus has a stronger antimicrobial activity than the serum. After vibrio induction, the antibacterial activity of mucus was slightly stronger for some gram-negative bacteria than that of un-induced mucus. A total of 97 proteins were identified from natural mucus of B. pectinirostris with a similar results from studies of other fish species, including actins,keratins, apolipoproteins, transferrins, calmodulins, ubiquitins, pentraxins, and various enzymes. However, some proteins, such as ubiquitin-like proteins and thymosin, were identified first from fish mucus. The identified proteins can be clustered into structural proteins, enzymes, material transport related proteins, immune proteins,and other proteins. Most of these proteins are known to be involved in immune and/or stress responses. Protein interaction analysis showed strong interactions among the identified proteins, such as actin, myosin, cofilin,filamin, apolipoprotein, transferrin, calmodulin, and superoxide dismutase. The proteomic profile established in this study could not only provide knowledge on the routes involved in mucosal innate immunity, but also put forward a non-invasive technique based on locating immune markers with a potential use for prevention and/or diagnosis of fish diseases.
Fish skin mucus serves as the first line of defense against pathogens and external stressors. The mudskipper Boleophthalmus pectinirostris inhabit intertidal mudflats containing abundant and diverse microbial population, thus, the skin together with the mucus of B. pectinirostris are very important for the immune defense,osmotic pressure maintenance and adaptation to amphibian life. For exploring the proteomic profile of the skin mucus and understanding the molecular mechanism of B. pectinirostris adaptation to amphibious environments,the antibacterial activity was determined by agar diffusion plate method for the mucus and the serum samples. In addition, a growth curve inhibition method was used to compare the antibacterial activities of B. pectinirostris mucus before and after vibrio induction. Furthermore, the proteomic profile of natural B. pectinirostris mucus was identified by Shotgun mass spectrometry technology combining with skin transcriptome searching. The interaction network analysis of the identified proteins from mucus was performed by String software. Skin mucus was collected from B. pectinirostris after electrical stimulation. The mucus of B. pectinirostris showed a broad spectrum of antimicrobial activity more than serum of the same species, indicating that the mucus has a stronger antimicrobial activity than the serum. After vibrio induction, the antibacterial activity of mucus was slightly stronger for some gram-negative bacteria than that of un-induced mucus. A total of 97 proteins were identified from natural mucus of B. pectinirostris with a similar results from studies of other fish species, including actins,keratins, apolipoproteins, transferrins, calmodulins, ubiquitins, pentraxins, and various enzymes. However, some proteins, such as ubiquitin-like proteins and thymosin, were identified first from fish mucus. The identified proteins can be clustered into structural proteins, enzymes, material transport related proteins, immune proteins,and other proteins. Most of these proteins are known to be involved in immune and/or stress responses. Protein interaction analysis showed strong interactions among the identified proteins, such as actin, myosin, cofilin,filamin, apolipoprotein, transferrin, calmodulin, and superoxide dismutase. The proteomic profile established in this study could not only provide knowledge on the routes involved in mucosal innate immunity, but also put forward a non-invasive technique based on locating immune markers with a potential use for prevention and/or diagnosis of fish diseases.
引文
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[27] Terrazzano G, Rubino V, Damiano S, et al. T cell activation induces CuZn superoxide dismutase(SOD)-1intracellular re-localization, production and secretion[J].Biochimica et Biophysica Acta(BBA)-Molecular Cell Research,2014, 1843(2):265-274.
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[33] Du Clos T W. Pentraxins:Structure, function, and role in inflammation[J]. ISRN Inflammation,2013, 2013:379040.
[34] Patel D M, Brinchmann M F. Skin mucus proteins oflumpsucker(Cyclopterus lumpus)[J]. Biochemistry and Biophysics Reports, 2017, 9:217-225.
[35] Rombout J H W M, Taverne N, van de Kamp M, et al.Differences in mucus and serum immunoglobulin of carp(Cyprinus carpio L[J]. Developmental&Comparative Immunology, 1993, 17(4):309-317.
[36] Zhang Y A, Salinas I, Li J, et al. IgT, a primitive immunoglobulin class specialized in mucosal immunity[J]. Nature Immunology, 2010, 11(9):827-835.
[37] Coscia M R, Simoniello P, Giacomelli S, et al. Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii[J]. Fish&Shellfish Immunology, 2014, 39(2):206-214.
[38] Sheng X Z, Xu G J, Tang X Q, et al. Monoclonal antibodies recognizing mucus immunoglobulin and surface immunoglobulin-positive cells of flounder(Paralichthys olivaceus)[J]. Veterinary Immunology and Immunopathology, 2012, 145(1-2):143-150.
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[42] Johnston L D, Brown G, Gauthier D, et al. Apolipoprotein A-I from striped bass(Morone saxatilis)demonstrates antibacterial activity in vitro[J]. Comparative Biochemistry and Physiology-Part B:Biochemistry and Molecular Biology,2008, 151(2):167-175.
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[2] Ingram G A. Substances involved in the natural resistance of fish to infection-a review[J]. Journal of Fish Biology,1980, 16(1):23-60.
[3] Nigam A K, Kumari U, Mittal S, et al. Comparative analysis of innate immune parameters of the skin mucous secretions from certain freshwater teleosts, inhabiting different ecological niches[J]. Fish Physiology and Biochemistry, 2012, 38(5):1245-1256.
[4] Esteban M A. An overview of the immunological defen-ses in fish skin[J]. ISRN Immunology, 2012, 2012:853470.
[5]Ellis A E. Innate host defense mechanisms of fish against viruses and bacteria[J]. Developmental and Comparative Immunology, 2001, 25(8-9):827-839.
[6]Verdugo P. Goblet cells secretion and mucogenesis[J].Annual Review of Physiology,1990, 52:157-176.
[7]Strous G J, Dekker J. Mucin-type glycoproteins[J].Critical Reviews in Biochemistry and Molecular Biology, 1992, 27(1-2):57-92.
[8]Alexander J B, Ingram G A. Noncellular nonspecific defence mechanisms of fish[J]. Annual Review of Fish Diseases, 1992,2:249-279.
[9] Ensign L M, Schneider C, Suk J S, et al. Mucus penetrating nanoparticles:biophysical tool and method of drug and gene delivery[J]. Advanced Materials, 2012,24(28):3887-3894.
[10] Tyler M J, Stone D J M, Bowie J H. A novel method for the release and collection of dermal, glandular secretions from the skin of frogs[J]. Journal of Pharmacological and Toxicological Methods, 1992, 28(4):199-200.
[11]张东玲,关瑞章,黄文树,等.pH和色谱柱对日本鳗鲡肝脏抗菌肽分离纯化效果的影响及抗菌活性检测[J].水产学报,2013, 37(4):614-621.Zhang D L, Guan R Z, Huang W S, et al. Effects of pH and chromatographic column on isolation, purification of antibacterial peptide from Japanese eel liver, and determination of antibacterial activity[J]. Journal of Fisheries of China,2013, 37(4):614-621(in Chinese).
[12]严林飞,安昕,包苗苗,等.大黄鱼过氧化氢酶基因的克隆及其对鳗弧菌感染的响应[J].水产学报,2017,41(5):641-648.Yan L F, An X, Bao M M, et al. Expression of catalase in Larimichthys crocea after infection of Vibrio anguillarum[J]. Journal of Fisheries of China, 2017, 41(5):641-648(in Chinese).
[13] Liao Z, Bao L F, Fan M H, et al. In-depth proteomic analysis of nacre, prism, and myostracum of Mytilus shell[J]. Journal of Proteomics, 2015, 122:26-40.
[14]张毓霞,石戈,王日昕,等.大弹涂鱼皮肤转录组测序及抗菌肽基因分析[J].生命科学研究,2018, 22(1):26-35.Zhang Y X, Shi G, Wang R X, et al. Transcriptome of Boleophthalmus pectinirostris skin and analysis of anti-microbial peptide related unigenes[J]. Life Science Research,2018, 22(1):26-35(in Chinese).
[15] Guardiola F A, Cuesta A, Arizcun M, et al. Comparative skin mucus and serum humoral defence mechanisms in the teleost gilthead seabream(Sparus aurata)[J]. Fish&Shellfish Immunology, 2014, 36(2):545-551.
[16] Subramanian S,Mackinnon S L,Ross N W. A comparative study on innate immune parameters in the epidermal mucus of various fish species[J]. Comparative Biochemistry and Physiology-Part B:Biochemistry and Molecular Biology, 2007, 148(3):256-263.
[17] Cordero H, Morcillo P, Cuesta A, et al. Differential proteome profile of skin mucus of gilthead seabream(Sparus aurata)after probiotic intake and/or overcrowding stress[J]. Journal of Proteomics, 2016, 132:41-50.
[18] Cordero H, Brinchmann M F, Cuesta A, et al. Skin mucus proteome map of European sea bass(Dicentrarchus labrax)[J]. Proteomics, 2015, 15(23-24):4007-4020.
[19] Valdenegro-Vega V A, Crosbie P, Bridle A, et al.Differentially expressed proteins in gill and skin mucus of Atlantic salmon(Salmo salar)affected by amoebic gill disease[J]. Fish&Shellfish Immunology, 2014,40(1):69-77.
[20] Rajan B, Lokesh J, Kiron V, et al. Differentially expressed proteins in the skin mucus of Atlantic cod(Gadus morhua)upon natural infection with Vibrio anguillarum[J]. BMC Veterinary Research, 2013, 9:103.
[21] Molle V, Campagna S, Bessin Y, et al. First evidence of the pore-forming properties of a keratin from skin mucus of rainbow trout(Oncorhynchus mykiss, formerly Salmo gairdneri)[J]. Biochemical Journal, 2008, 411(1):33-40.
[22] Provan F, Jensen L B, Uleberg K E, et al. Proteomic analysis of epidermal mucus from sea lice-infected Atlantic salmon, Salmo salar L.[J]. Journal of Fish Diseases, 2013, 36(3):311-321.
[23] Easy R H,Ross N W. Changes in Atlantic salmon(Salmo salar)epidermal mucus protein composition profiles following infection with sea lice(Lepeophtheirus salmonis)[J]. Comparative Biochemistry and Physiology-Part D:Genomics and Proteomics, 2009,4(3):159-167.
[24] Sandiford S L, Dong Y M, Pike A, et al. Cytoplasmic actin is an extracellular insect immune factor which issecreted upon immune challenge and mediates phagocytosis and direct killing of bacteria, and is a Plasmodium Antagonist[J]. PLoS Pathogens,2015, 11(2):e1004631.
[25] Jurado J, Fuentes-Almagro C A, Guardiola F A, et al.Proteomic profile of the skin mucus of farmed gilthead seabream(Sparus aurata)[J]. Journal of Proteomics,2015, 120:21-34.
[26] Chong K, Joshi S, Jin L T, et al. Proteomics profiling of epidermal mucus secretion of a cichlid(Symphysodon aequifasciata)demonstrating parental care behavior[J].Proteomics, 2006, 6(7):2251-2258.
[27] Terrazzano G, Rubino V, Damiano S, et al. T cell activation induces CuZn superoxide dismutase(SOD)-1intracellular re-localization, production and secretion[J].Biochimica et Biophysica Acta(BBA)-Molecular Cell Research,2014, 1843(2):265-274.
[28] Gongora M C, Lob H E, Landmesser U, et al. Loss of extracellular superoxide dismutase leads to acute lung damage in the presence of ambient air:a potential mechanism underlying adult respiratory distress syndrome[J]. American Journal of Pathology, 2008, 173(4):915-926.
[29] Long W, Guo H Y, Zhang N, et al. Molecular characterization and functional analysis of a peroxiredoxin 1cDNA from golden pompano(Trachinotus ovatus)[J].Developmental&Comparative Immunology, 2015,51(2):261-270.
[30] Riddell J R, Wang X Y, Minderman H, et al.Peroxiredoxin 1 stimulates secretion of proinflammatory cytokines by binding to TLR4[J]. The Journal of Immunology, 2010, 184(2):1022-1030.
[31] Suzuki Y, Tasumi S, Tsutsui S, et al. Molecular diversity of skin mucus lectins in fish[J]. Comparative Biochemistry and Physiology-Part B:Biochemistry and Molecular Biology, 2003, 136(4):723-730.
[32] Tsutsui S, Yamaguchi M, Hirasawa A, et al. Common skate(Raja kenojei)secretes pentraxin into the cutaneous secretion:the first skin mucus lectin in cartilaginous fish[J]. The Journal of Biochemistry, 2009, 146(2):295-306.
[33] Du Clos T W. Pentraxins:Structure, function, and role in inflammation[J]. ISRN Inflammation,2013, 2013:379040.
[34] Patel D M, Brinchmann M F. Skin mucus proteins oflumpsucker(Cyclopterus lumpus)[J]. Biochemistry and Biophysics Reports, 2017, 9:217-225.
[35] Rombout J H W M, Taverne N, van de Kamp M, et al.Differences in mucus and serum immunoglobulin of carp(Cyprinus carpio L[J]. Developmental&Comparative Immunology, 1993, 17(4):309-317.
[36] Zhang Y A, Salinas I, Li J, et al. IgT, a primitive immunoglobulin class specialized in mucosal immunity[J]. Nature Immunology, 2010, 11(9):827-835.
[37] Coscia M R, Simoniello P, Giacomelli S, et al. Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii[J]. Fish&Shellfish Immunology, 2014, 39(2):206-214.
[38] Sheng X Z, Xu G J, Tang X Q, et al. Monoclonal antibodies recognizing mucus immunoglobulin and surface immunoglobulin-positive cells of flounder(Paralichthys olivaceus)[J]. Veterinary Immunology and Immunopathology, 2012, 145(1-2):143-150.
[39] Guardiola F A, Cuesta A, Abellan E, et al. Comparative analysis of the humoral immunity of skin mucus from several marine teleost fish[J]. Fish&Shellfish Immunology, 2014,40(1):24-31.
[40] Xu Z, Parra D, Gomez D, et al. Teleost skin, an ancient mucosal surface that elicits gut-like immune responses[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(32):13097-13102.
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