Differentially expressed proteins in the skin mucus of Atlantic cod (Gadus morhua) upon natural infection with Vibrio anguillarum

BMC Veterinary Research

Monica F Brinchmann¹  Viswanath Kiron¹  Jep Lokesh¹  Binoy Rajan¹ 

[1] Faculty of Biosciences and Aquaculture, University of Nordland, Bodø 8049, Norway

关键词: Bath challenge;    Vibrio anguillarum;    Atlantic cod;    Skin mucosa;    Comparative proteome;   

Others  :  1119535
DOI  :  10.1186/1746-6148-9-103

 received in 2013-01-26, accepted in 2013-05-08,  发布年份 2013

【 摘 要 】

Background

Vibriosis caused by V. anguillarum is a commonly encountered disease in Atlantic cod farms and several studies indicate that the initiation of infection occurs after the attachment of the pathogen to the mucosal surfaces (gut, skin and gills) of fish. Therefore it is necessary to investigate the role of different mucosal components in fish upon V. anguillarum infection. The present study has two parts; in the first part we analyzed the differential expression of skin mucus proteins from Atlantic cod naturally infected with V. anguillarum using two dimensional gel electrophoresis coupled with mass spectrometry. In the second part, a separate bath challenge experiment with V. anguillarum was conducted to assess the mRNA levels of the genes in skin tissue, corresponding to the selected proteins identified in the first part.

Results

Comparative proteome analysis of skin mucus of cod upon natural infection with V. anguillarum revealed key immune relevant proteins like calpain small subunit 1, glutathione-S-transferase omega 1, proteasome 26S subunit, 14-kDa apolipoprotein, beta 2-tubulin, cold inducible RNA binding protein, malate dehydrogenase 2 (mitochondrial) and type II keratin that exhibited significant differential expression. Additionally a number of protein spots which showed large variability amongst individual fish were also identified. Some of the proteins identified were mapped to the immunologically relevant JNK (c-Jun N-terminal kinases) signalling pathway that is connected to cellular events associated with pathogenesis. A bath challenge experiment with V. anguillarum showed differential expression of beta 2-tubulin, calpain small subunit 1, cold inducible RNA binding protein, flotillin1, and glutathione S-transferase omega 1 transcripts in the skin tissue of cod during early stages of infection.

Conclusions

Differentially expressed proteins identified in the cod skin mucus point towards their possible involvement in V. anguillarum pathogenesis. The role of some of these proteins in vibriosis in cod described in this paper can be considered unconventional with respect to their established functions in higher vertebrates. Based on the differential expression of these proteins they are possibly important components of fish defence against bacteria and innate immunity at large. The feasibility of utilizing these proteins/genes as markers of bacterial infection or stress in cod needs to be explored further.

【 授权许可】

   
2013 Rajan et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Star B, Nederbragt AJ, Jentoft S, Grimholt U, Malmstrom M, Gregers TF, Rounge TB, Paulsen J, Solbakken MH, Sharma A: The genome sequence of Atlantic cod reveals a unique immune system. Nature 2011, 477:207-210.
• [2]Pilstrom L, Warr GW, Stromberg S: Why is the antibody response of Atlantic cod so poor? The search for a genetic explanation. Fish Sci 2005, 71:961-971.
• [3]Esteban MA: An overview of the immunological defenses in fish skin. ISRN Immunol 2012.
• [4]Larsen JL, Pedersen K, Dalsgaard I: Vibrio anguillarum serovars associated with vibriosis in fish. J Fish Dis 1994, 17:259-267.
• [5]Pedersen K, Grisez L, Houdt R, Tiainen T, Ollevier F, Larsen JL: Extended serotyping scheme for Vibrio anguillarum with the definition and characterization of seven provisional O-serogroups. Curr Microbiol 1999, 38:183-189.
• [6]Frans I, Michiels CW, Bossier P, Willems KA, Lievens B, Rediers H: Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and prevention. J Fish Dis 2011, 34:643-661.
• [7]Engelsen AR, Sandlund N, Fiksdal IU, Bergh O: Immunohistochemistry of Atlantic cod larvae Gadus morhua experimentally challenged with Vibrio anguillarum. Dis Aquat Organ 2008, 80:13-20.
• [8]Mikkelsen H, Lund V, Larsen R, Seppola M: Vibriosis vaccines based on various sero-subgroups of Vibrio anguillarum O2 induce specific protection in Atlantic cod (Gadus morhua L.) juveniles. Fish Shellfish Immunol 2011, 30:330-339.
• [9]Harris JB, LaRocque RC, Qadri F, Ryan ET, Calderwood SB: Cholera. Lancet 2012, 379:2466-2476.
• [10]Rombout JWHM, Blok LJ, Lamers CHJ, Egberts E: Immunization of carp (Cyprinus carpio) with a Vibrio anguillarum bacterin: Indications for a common mucosal immune system. Dev Comp Immunol 1986, 10:341-351.
• [11]O’Toole R, Lundberg S, Fredriksson S-Å, Jansson A, Nilsson B, Wolf-Watz H: The chemotactic response of Vibrio anguillarum to fish intestinal mucus is mediated by a combination of multiple mucus components. J Bacteriol 1999, 181:4308-4317.
• [12]O'Toole R, von Hofsten J, Rosqvist R, Olsson P-E, Wolf-Watz H: Visualisation of Zebrafish infection by GFP-labelled Vibrio anguillarum. Microb Pathog 2004, 37:41-46.
• [13]Spanggaard B, Huber I, Nielsen J, Nielsen T, Gram L: Proliferation and location of Vibrio anguillarum during infection of rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 2000, 23:423-427.
• [14]Lindell K, Fahlgren A, Hjerde E, Willassen N-P, Fällman M, Milton DL: Lipopolysaccharide O-antigen prevents phagocytosis of Vibrio anguillarum by rainbow trout (Oncorhynchus mykiss) skin epithelial cells. PLoS One 2012, 7:e37678.
• [15]Vervarcke S, Ollevier F, Kinget R, Michoel A: Mucosal response in African catfish after administration of Vibrio anguillarum O2 antigens via different routes. Fish Shellfish Immunol 2005, 18:125-133.
• [16]Rock JL, Nelson DR: Identification and characterization of a hemolysin gene cluster in Vibrio anguillarum. Infect Immun 2006, 74:2777-2786.
• [17]Øverbye A, Brinchmann MF, Seglen PO: Proteomic analysis of membrane-associated proteins from rat liver autophagosomes. Autophagy 2007, 3:300-322.
• [18]Fernandes JMO, Ruangsri J, Kiron V: Atlantic cod piscidin and its diversification through positive selection. PLoS One 2010, 5:e9501.
• [19]Subramanian S, MacKinnon SL, Ross NW: A comparative study on innate immune parameters in the epidermal mucus of various fish species. Comp Biochem Physiol B 2007, 148:256-263.
• [20]Shephard KL: Functions for fish mucus. Rev Fish Biol Fish 1994, 4:401-429.
• [21]Suzuki Y, Tasumi S, Tsutsui S, Okamoto M, Suetake H: Molecular diversity of skin mucus lectins in fish. Comp Biochem Physiol B 2003, 136:723-730.
• [22]Rajan B, Fernandes JMO, Caipang CMA, Kiron V, Rombout JHWM, Brinchmann MF: Proteome reference map of the skin mucus of Atlantic cod (Gadus morhua) revealing immune competent molecules. Fish Shellfish Immunol 2011, 31:224-231.
• [23]Ruangsri J, Fernandes JMO, Brinchmann M, Kiron V: Antimicrobial activity in the tissues of Atlantic cod (Gadus morhua L.). Fish Shellfish Immunol 2010, 28:879-886.
• [24]Denkin SM, Nelson DR: Induction of protease activity in Vibrio anguillarum by gastrointestinal mucus. Appl Environ Microbiol 1999, 65:3555-3560.
• [25]Vlahopoulos S, Zoumpourlis VC: JNK: A key modulator of intracellular signaling. Biochemistry (Mosc) 2004, 69:844-854.
• [26]Sorimachi H, Hata S, Ono Y: Calpain chronicle-an enzyme family under multidisciplinary characterization. Proc Jpn Acad Ser B Phys Biol Sci 2011, 87:287-327.
• [27]Saido TC, Sorimachi H, Suzuki K: Calpain: C: new perspectives in molecular diversity and physiological-pathological involvement. FASEB J 1994, 8:814-822.
• [28]Ishikawa H, Nakagawa Y, Shimizu K, Nishihara H, Matsusue Y, Nakamura T: Inflammatory cytokines induced down-regulation of m-calpain mRNA expression in fibroblastic synoviocytes from patients with osteoarthritis and rheumatoid arthritis. Biochem Biophys Res Commun 1999, 266:341-346.
• [29]Inami M: The immune system of Atlantic cod: with emphasis on intestinal immunology. University of Tromsø, Faculty of Biosciences, Fisheries and Economics: PhD thesis; 2011.
• [30]Lokesh J, Fernandes JMO, Korsnes K, Bergh Ø, Brinchmann MF, Kiron V: Transcriptional regulation of cytokines in the intestine of Atlantic cod fed yeast derived mannan oligosaccharide or β-glucan and challenged with Vibrio anguillarum. Fish Shellfish Immunol 2012, 33:626-631.
• [31]Matsushita Y, Shimada Y, Kawara S, Takehara K, Sato S: Autoantibodies directed against the protease inhibitor calpastatin in psoriasis. Clin Exp Immunol 2005, 139:355-362.
• [32]Salem M, Nath J, Killefer J: Cloning of the calpain regulatory subunit cDNA from fish reveals a divergent domain-V. Anim Biotechnol 2004, 15:145-157.
• [33]Wellmann S, Bührer C, Moderegger E, Zelmer A, Kirschner R, Koehne P, Fujita J, Seeger K: Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism. J Cell Sci 2004, 117:1785-1794.
• [34]Pan F, Zarate J, Choudhury A, Rupprecht R, Bradley TM: Osmotic stress of salmon stimulates upregulation of a cold inducible RNA binding protein (CIRP) similar to that of mammals and amphibians. Biochimie 2004, 86:451-461.
• [35]Minárik P, Tomásková N, Kollárová M, Antalík M: Malate dehydrogenases–structure and function. Gen Physiol Biophys 2002, 21:257-265.
• [36]Cripps RA, Reish DJ: The effect of environmental stress on the activity of malate dehydrogenase and lactate dehydrogenase in Neanthes arenaceodentata (Annelida: Polychaeta). Comp Biochem Physiol B 1973, 46:123-133.
• [37]Addis M, Cappuccinelli R, Tedde V, Pagnozzi D, Viale I, Meloni M, Salati F, Roggio T, Uzzau S: Influence of Moraxella sp. colonization on the kidney proteome of farmed gilthead sea breams (Sparus aurata L.). Proteome Sci 2010, 8:50.
• [38]Coulombe PA, Omary MB: ‘Hard’ and ‘soft’ principles defining the structure, function and regulation of keratin intermediate filaments. Curr Opin Cell Biol 2002, 14:110-122.
• [39]Molle V, Campagna S, Bessin Y, Ebran N, Saint N, Molle G: First evidence of the pore-forming properties of a keratin from skin mucus of rainbow trout (Oncorhynchus mykiss, formerly Salmo gairdneri). Biochem J 2008, 411:33-40.
• [40]Rogel MR, Jaitovich A, Ridge KM: The role of the ubiquitin proteasome pathway in keratin intermediate filament protein degradation. Proc Am Thorac Soc 2010, 7:71-76.
• [41]Hammond JW, Cai D, Verhey KJ: Tubulin modifications and their cellular functions. Curr Opin Cell Biol 2008, 20:71-76.
• [42]Zhang J, Li F, Jiang H, Yu Y, Liu C, Li S, Wang B, Xiang J: Proteomic analysis of differentially expressed proteins in lymphoid organ of Fenneropenaeus chinensis response to Vibrio anguillarum stimulation. Fish Shellfish Immunol 2010, 29:186-194.
• [43]Choudhury M, Yamada S, Komatsu M, Kishimura H, Ando S: Homologue of mammalian apolipoprotein A-II in non-mammalian vertebrates. Acta Biochim Biophys Sin 2009, 41:370-378.
• [44]Concha MI, Smith VJ, Castro K, Bastías A, Romero A, Amthauer RJ: Apolipoproteins A-I and A-II are potentially important effectors of innate immunity in the teleost fish Cyprinus carpio. Eur J Biochem 2004, 271:2984-2990.
• [45]Sarropoulou E, Sepulcre P, Poisa-Beiro L, Mulero V, Meseguer J, Figueras A, Novoa B, Terzoglou V, Reinhardt R, Magoulas A: Profiling of infection specific mRNA transcripts of the European seabass Dicentrarchus labrax. BMC Genomics 2009, 10:157. BioMed Central Full Text
• [46]Burmeister C, Lüersen K, Heinick A, Hussein A, Domagalski M, Walter RD, Liebau E: Oxidative stress in Caenorhabditis elegans: protective effects of the Omega class glutathione transferase (GSTO-1). FASEB J 2008, 22:343-354.
• [47]Olsvik PA, Nordtug T, Altin D, Lie KK, Overrein I, Hansen BH: Transcriptional effects on glutathione S-transferases in first feeding Atlantic cod (Gadus morhua) larvae exposed to crude oil. Chemosphere 2010, 79:905-913.
• [48]Li C, Su X, Li Y, Li T, Sun C, Zhou T, Liu H: Two classes of glutathione S-transferase genes with different response profiles to bacterial challenge in Venerupis philippinarum. Fish Shellfish Immunol 2012, 32:219-222.
• [49]Zhao D, Chen L, Qin C, Zhang H, Wu P, Zhang F: A delta-class glutathione transferase from the Chinese mitten crab Eriocheir sinensis: cDNA cloning, characterization and mRNA expression. Fish Shellfish Immunol 2010, 29:698-703.
• [50]Canesi L, Barmo C, Fabbri R, Ciacci C, Vergani L, Roch P, Gallo G: Effects of vibrio challenge on digestive gland biomarkers and antioxidant gene expression in Mytilus galloprovincialis. Comp Biochem Physiol C 2010, 152:399-406.
• [51]Kloetzel P-M, Ossendorp F: Proteasome and peptidase function in MHC-class-I-mediated antigen presentation. Curr Opin Immunol 2004, 16:76-81.
• [52]Feng CY, Johnson SC, Hori TS, Rise M, Hall JR, Gamperl AK, Hubert S, Kimball J, Bowman S, Rise ML: Identification and analysis of differentially expressed genes in immune tissues of Atlantic cod stimulated with formalin-killed, atypical Aeromonas salmonicida. Physiol Genomics 2009, 37:149-163.
• [53]Bricknell IR, Bron JE, Bowden TJ: Diseases of gadoid fish in cultivation: a review. ICES J Mar Sci 2006, 63:253-266.
• [54]Samuelsen OB, Bergh Ø: Efficacy of orally administered florfenicol and oxolinic acid for the treatment of vibriosis in cod (Gadus morhua). Aquaculture 2004, 235:27-35.
• [55]Gygi SP, Rochon Y, Franza BR, Aebersold R: Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 1999, 19:1720-1730.
• [56]Sigh J, Lindenstrøm T, Buchmann K: Expression of pro-inflammatory cytokines in rainbow trout (Oncorhynchus mykiss) during an infection with Ichthyophthirius multifiliis. Fish Shellfish Immunol 2004, 17:75-86.
• [57]Caipang CMA, Lazado CC, Brinchmann MF, Rombout JHWM, Kiron V: Differential expression of immune and stress genes in the skin of Atlantic cod (Gadus morhua). Comp Biochem Physiol D 2011, 6:158-162.