Fisheries Science

, Volume 83, Issue 3, pp 489–498 | Cite as

Exploration of the antibacterial proteins in pearl oyster Pinctada fucata induced by bacterial inoculation

  • Haisheng Lin
  • Shoichiro Ishizaki
  • Yuji Nagashima
  • Kiyohito Nagai
  • Kaoru Maeyama
  • Shugo Watabe
Original Article Chemistry and Biochemistry


The aim of this research was to characterize immune-related antibacterial substances from pearl oyster Pinctada fucata induced by bacterial invasion. Bacteria inoculation was performed by injecting 0.1 ml of 1.0 × 1012 colony-forming units/ml Vibrio parahaemolyticus into adductor muscle. Acidic extracts were prepared with 0.1% trifluoroacetic acid from different tissues after 8 h of injection, and antibacterial activity against V. parahaemolyticus was determined via the microdilution broth method. The acidic extracts from gills of inoculated oysters (AEg) showed stronger antibacterial activity than those from non-inoculated ones. Based on this result, antibacterial proteins were purified from AEg via two-step gel filtration chromatography, followed by high-performance liquid chromatography using a TSkgel G3000 column. Protein components were analyzed by both sodium dodecyl sulfate and native polyacrylamide gel electrophoresis. As a result, two antibacterial proteins, APg-1 (with a molecular mass of approximately 210 kDa) and APg-2 (of approximately 30 kDa), were obtained from AEg. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and partial amino acid sequences revealed that these proteins might be novel antibacterial proteins. These results indicate that antibacterial proteins are potentially upregulated in the gill of pearl oysters or released therefrom for defense against bacterial invasion.


Innate immunity Bacterial invasion Gill antibacterial protein High-performance liquid chromatography 



This study was partially supported by the Sasakawa Scientific Research Grant from the Japan Science Society (28-317).


  1. 1.
    Sperstad SV, Haug T, Blencke HM, Styrvold OB, Li C, Stensvag K (2011) Antimicrobial peptides from marine invertebrates: challenges and perspectives in marine antimicrobial peptide discovery. Biotechnol Adv 29:519–530CrossRefPubMedGoogle Scholar
  2. 2.
    Seo JK, Lee MJ, Nam BH, Park NG (2013) cgMolluscidin, a novel dibasic residue repeat rich antimicrobial peptide, purified from the gill of the Pacific oyster, Crassostrea gigas. Fish Shellfish Immunol 35:480–488CrossRefPubMedGoogle Scholar
  3. 3.
    Defer D, Bourgougnon N, Fleury Y (2009) Screening for antibacterial and antiviral activities in three bivalve and two gastropod marine molluscs. Aquaculture 293:1–7CrossRefGoogle Scholar
  4. 4.
    Allam B, Raftos D (2015) Immune responses to infectious diseases in bivalves. J Invertebr Pathol 131:121–136CrossRefPubMedGoogle Scholar
  5. 5.
    Wang L, Qiu L, Zhou Z, Song L (2013) Research progress on the mollusc immunity in China. Dev Comp Immunol 39:2–10CrossRefPubMedGoogle Scholar
  6. 6.
    Adhya M, Singha B, Chatterjee BP (2009) Purification and characterization of an N-acetylglucosamine specific lectin from marine bivalve Macoma birmanica. Fish Shellfish Immunol 27:1–8CrossRefPubMedGoogle Scholar
  7. 7.
    Itoh N, Takahashi KG (2009) A novel peptidoglycan recognition protein containing a goose-type lysozyme domain from the Pacific oyster, Crassostrea gigas. Mol Immunol 46:1768–1774CrossRefPubMedGoogle Scholar
  8. 8.
    Song X, Wang H, Xin L, Xu J, Jia Z, Wang L, Song L (2016) The immunological capacity in the larvae of Pacific oyster Crassostrea gigas. Fish Shellfish Immunol 49:461–469CrossRefPubMedGoogle Scholar
  9. 9.
    Seo JK, Stephenson J, Noga EJ (2011) Multiple antibacterial histone H2B proteins are expressed in tissues of American oyster. Comp Biochem Physiol B Biochem Mol Biol 158:223–229CrossRefPubMedGoogle Scholar
  10. 10.
    Canesi L, Gallo G, Gavioli M, Pruzzo C (2002) Bacteria-hemocyte interactions and phagocytosis in marine bivalves. Microsc Res Tech 57:469–476CrossRefPubMedGoogle Scholar
  11. 11.
    Muroga K, Takahashi KG (2007) Review: overview on the studies of humoral defense factors in bivalve molluscs. Fish Pathol 42:1–17CrossRefGoogle Scholar
  12. 12.
    Bachere E, Rosa RD, Schmitt P, Poirier AC, Merou N, Charriere GM, Destoumieux-Garzon D (2015) The new insights into the oyster antimicrobial defense: cellular, molecular and genetic view. Fish Shellfish Immunol 46:50–64CrossRefPubMedGoogle Scholar
  13. 13.
    Jain D, Nair DT, Swaminathan GJ, Abraham EG, Nagaraju J, Salunke DM (2001) Structure of the induced antibacterial protein from tasar silkworm, Antheraea mylitta. Implications for molecular evolution. J Biol Chem 276:41377–41382CrossRefPubMedGoogle Scholar
  14. 14.
    Li YM, Xiang Q, Zhang QH, Huang YD, Su ZJ (2012) Overview on the recent study of antimicrobial peptides: origins, functions, relative mechanisms and application. Peptides 37:207–215CrossRefPubMedGoogle Scholar
  15. 15.
    Defoirdt T, Boon N, Sorgeloos P, Verstraete W, Bossier P (2007) Alternatives to antibiotics to control bacterial infections: luminescent vibriosis in aquaculture as an example. Trends Biotechnol 25:472–479CrossRefPubMedGoogle Scholar
  16. 16.
    Nagai K (2013) A history of the cultured pearl industry. Zool Sci 30:783–793CrossRefPubMedGoogle Scholar
  17. 17.
    Li J, Chen JH, Zhang Y, Yu ZN (2013) Expression of allograft inflammatory factor-1 (AIF-1) in response to bacterial challenge and tissue injury in the pearl oyster, Pinctada martensii. Fish Shellfish Immunol 34:365–371CrossRefPubMedGoogle Scholar
  18. 18.
    Morizane T, Takimoto S, Nishikawa S, Matsuyama N, Tyohno K, Uemura S, Fujita Y, Yamashita H, Kawakami H, Koizumi Y, Uchimura Y, Ichikawa M (2001) Mass mortalities of Japanese pearl oyster in Uwa Sea, Ehime in 1997–1999. Fish Pathol 36:207–216CrossRefGoogle Scholar
  19. 19.
    Gomez-Mendikute A, Elizondo M, Venier P, Cajaraville MP (2005) Characterization of mussel gill cells in vivo and in vitro. Cell Tissue Res 321:131–140CrossRefPubMedGoogle Scholar
  20. 20.
    Wang Z, Jian J, Lu Y, Wang B, Wu Z (2011) A tandem-repeat galectin involved in innate immune response of the pearl oyster Pinctada fucata. Mar Genom 4:229–236CrossRefGoogle Scholar
  21. 21.
    Anju A, Jeswin J, Thomas PC, Vijayan KK (2013) Molecular cloning, characterization and expression analysis of F-type lectin from pearl oyster Pinctada fucata. Fish Shellfish Immunol 35:170–174CrossRefPubMedGoogle Scholar
  22. 22.
    Cui SG, Zhang DC, Jiang SG, Pu HL, Hu YT, Guo HY, Chen MQ, Su TF, Zhu CY (2011) A macrophage migration inhibitory factor like oxidoreductase from pearl oyster Pinctada fucata involved in innate immune responses. Fish Shellfish Immunol 31:173–181CrossRefPubMedGoogle Scholar
  23. 23.
    Nagashima Y, Kikuchi N, Shimakura K, Shiomi K (2003) Purification and characterization of an antibacterial protein in the skin secretion of rockfish Sebastes schlegeli. Comp Biochem Physiol C Toxicol Pharmacol 136:63–71CrossRefPubMedGoogle Scholar
  24. 24.
    Bringans S, Eriksen S, Kendrick T, Gopalakrishnakone P, Livk A, Lock R, Lipscombe R (2008) Proteomic analysis of the venom of Heterometrus longimanus (Asian black scorpion). Proteomics 8:1081–1096CrossRefPubMedGoogle Scholar
  25. 25.
    Kuchel RP, Raftos DA, Birch D, Vella N (2010) Haemocyte morphology and function in the Akoya pearl oyster, Pinctada imbricata. J Invertebr Pathol 105:36–48CrossRefPubMedGoogle Scholar
  26. 26.
    Liu WG, Huang XD, Wang Q, Zhao M, Wu SZ, He MX (2013) Gene cloning and function analysis of cytokine-induced suppressor of cytokine signaling (SOCS) from pearl oyster Pinctada fucata. Fish Shellfish Immunol 34:849–854CrossRefPubMedGoogle Scholar
  27. 27.
    Espinosa EP, Winnicki S, Allam B (2013) Early host-pathogen interactions in a marine bivalve: Crassostrea virginica pallial mucus modulates Perkinsus marinus growth and virulence. Dis Aquat Org 104:237–247CrossRefGoogle Scholar
  28. 28.
    Allam B, Carden WE, Ward JE, Ralph G, Winnicki S, Espinosa EP (2013) Early host-pathogen interactions in marine bivalves: evidence that the alveolate parasite Perkinsus marinus infects through the oyster mantle during rejection of pseudofeces. J Invertebr Pathol 113:26–34CrossRefPubMedGoogle Scholar
  29. 29.
    Pruzzo C, Gallo G, Canesi L (2005) Persistence of vibrios in marine bivalves: the role of interactions with haemolymph components. Environ Microbiol 7:761–772CrossRefPubMedGoogle Scholar
  30. 30.
    Seo JK, Lee MJ, Go HJ, Do Kim G, Do Jeong H, Nam BH, Park NG (2013) Purification and antimicrobial function of ubiquitin isolated from the gill of Pacific oyster, Crassostrea gigas. Mol Immunol 53:88–98CrossRefPubMedGoogle Scholar
  31. 31.
    Sun JC, Ugolini S, Vivier E (2014) Immunological memory within the innate immune system. EMBO J 33:1295–1303PubMedPubMedCentralGoogle Scholar
  32. 32.
    Sadd BM, Schmid-Hempel P (2006) Insect immunity shows specificity in protection upon secondary pathogen exposure. Curr Biol 16:1206–1210CrossRefPubMedGoogle Scholar
  33. 33.
    Roth O, Sadd BM, Schmid-Hempel P, Kurtz J (2009) Strain-specific priming of resistance in the red flour beetle, Tribolium castaneum. Proc R Soc Lond B Biol Sci 276:145–151CrossRefGoogle Scholar
  34. 34.
    Fredrick WS, Ravichandran S (2012) Hemolymph proteins in marine crustaceans. Asian Pac J Trop Biomed 2:496–502CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Japanese Society of Fisheries Science 2017

Authors and Affiliations

  • Haisheng Lin
    • 1
    • 2
  • Shoichiro Ishizaki
    • 1
  • Yuji Nagashima
    • 1
  • Kiyohito Nagai
    • 3
  • Kaoru Maeyama
    • 4
  • Shugo Watabe
    • 5
  1. 1.Graduate School of Marine Science and TechnologyTokyo University of Marine Science and TechnologyMinatoJapan
  2. 2.College of Food Science and TechnologyGuangdong Ocean UniversityZhanjiangChina
  3. 3.Pearl Research Laboratory, Mikimoto Company LimitedShimaJapan
  4. 4.Mikimoto Pharmaceutical Company LimitedIseJapan
  5. 5.School of Marine BiosciencesKitasato UniversitySagamiharaJapan

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