Screening and Purification of an Antimicrobial Peptide from the Gill of the Manila Clam Ruditapes philippinarum

바지락(Ruditapes philippinarum) 아가미로부터 항균 펩타이드의 탐색 및 정제

Seo, Jung-Kil

  • Received : 2016.02.29
  • Accepted : 2016.04.07
  • Published : 2016.04.30


This study screened the biological activity of an acidified gill extract of the Manila clam Ruditapes philippinarum including antimicrobial, hemolytic, membrane permeabilization, and DNA-binding activity, and purified the antimicrobial material. The acidified gill extract showed potent antimicrobial activity against Bacillus subtilis and Escherichia coli without significant hemolytic activity, but showed no membrane permeabilization or DNA-binding ability. An antimicrobial material was purified from the acidified gill extract using C18 reversed-phase and cation-exchange high-performance liquid chromatography (HPLC). Treatment of the purified material with trypsin completely abolished all of the antibacterial activity against Bacillus subtilis, suggesting that the purified material is a proteinaceous antibiotic. The molecular weight of the purified material was 2571.9 Da, but no primary structural information was obtained due to N-terminal blocking. A future study should confirm the primary structure. Our results suggest that the Manila clam gill contains proteinaceous antibiotics that have a role in first-line defense. This information could be used to better understand the Manila clam innate immune system.


Manila clam;Ruditapes philippinarum;Antimicrobial peptide;Innate defense;Purification


  1. Gueguen Y, Herpin A, Aumelas A, Garnier J, Fievet J, Escoubas JM, Bulet P, Gonzalez M, Lelong C, Favrel P and Bachère E. 2006. Characterization of a defensin from the oyster Crassostrea gigas. Recombinant production, folding, solution structure, antimicrobial activities, and gene expression. J Biol Chem 281, 313-323.
  2. Alfred RL, Palombo EA, Panozzo JF and Bhave M. 2013. The antimicrobial domains of wheat puroindolines are cell-penetrating peptides with possible intracellular mechanism of action. PLoS One 8, e75488.
  3. Andreu D and Rivas L. 1998. Animal antimicrobial peptides: an overview. Biopolymers 147, 415-433.
  4. Charlet M, Chernysh S, Philippe H, Hetru C, Hoffmann JA and Bulet P. 1996. Innate immunity: Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusk, Mytilus edulis. J Biol Chem 271, 21808-21813.
  5. Destoumieux D, Bulet P, Loew D, Van DA, Rodriguez J and Bachere E. 1997. Penaeidins, a new family of antimicrobial peptides isolated from the shrimp Penaeus vannamei (Decapoda). J Biol Chem 272, 28398-28406.
  6. De Zoysa M, Nikapitiya C, Whang I, Lee JS and Lee J. 2009. Abhisin: a potential antimicrobial peptide derived from histone H2A of disk abalone (Haliotis discus discus). Fish Shellfish Immunol 27, 639-646.
  7. Hancock REW and Diamond G. 2000. The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol 8, 402-410.
  8. Hsu CH, Chen C, Jou ML, Lee AYL, Lin YC, Yu YP, Huang WT and Wu SH. 2005. Structural and DNA-binding studies on the bovine antimicrobial peptide, indolicidin, evidence for multiple conformations involved in binding to membranes and DNA. Nucl Acids Res 33, 4053-4064.
  9. Iijima R, Kisugi J and Yamazaki M. 2003. A novel antimicrobial peptide from the sea hare Dolabella auricularia. Dev Comp Immunol 27, 305-311.
  10. Izadpanah A and Gallo RL. 2005. Antimicrobial peptides. J Am Acad Dermatol 52, 381-390.
  11. Li Y. 2009. The role of antimicrobial peptides in cardiovascular physiology and disease. Biochem Biophys Res Commun 390, 363-367.
  12. Kang YS, Kim YM, Park KL, Cho SK, Choi KS and Cho M. 2006. Analysis of EST and lectin expression in hemocytes of Manila clams (Ruditapes phylippinarum) (Bivalvia: Mollusca) infected with Perkinsus olseni. Dev Comp Immunol 30, 1119-1131.
  13. Khoo L1, Robinette DW and Noga EJ. 1999. Callinectin, an antibacterial peptide from blue crab, Callinectes sapidus, hemocytes. Mar Biotechnol 1, 44-51.
  14. Lehrer RI, Rosenman M, Harwig SSL, Jackson R and Eisenhaur P. 1991. Ultrasensitive assay for endogenous antimicrobial polypeptides. J Immunol Methods 137, 167-173.
  15. Mitta G, Hubert F, Dyrynda EA, Boudry P and Roch P. 2000. Mytilin B and MGD2, two antimicrobial peptides of marine mussels: gene structure and expression analysis. Dev Comp Immunol 24, 381-393.
  16. Mitta G, Hubert F, Noel T and Roch P. 1999. Myticin, a novel cysteine-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis. Eur J Biochem 265, 71-78.
  17. Miyata T, Tokunaga F, Yoneya T, Yoshikawa K, Iwanaga S, Niwa M, Takao T and Shimonishi Y. 1989. Antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin II, and polyphemusins I and II: chemical structures and biological activity. J Biochem 106, 663-668.
  18. Nam BH, Seo JK, Lee MJ, Kim YO, Kim DG, An CM and Park NG. 2015. Functional analysis of Pacific oyster (Crassostrea gigas) β-thymosin: Focus on antimicrobial activity. Fish Shellfish Immunol 45, 167-174.
  19. Saito T, Kawabata S, Shigenaga T, Takayenoki Y, Cho J, Nakajima H, Hirata M and Iwanaga S. 1995. A novel big defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence, and antibacterial activity. J Biochem 117, 1131-1137.
  20. Ovchinnikova TV, Aleshina GM, Balandin SV, Krasnosdembskaya AD, Markelov ML, Frolova EI, Leonova YF, Tagaev AA, Krasnodembsky EG and Kokryakov VN. 2004. Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina. FEBS Lett 577, 209-214.
  21. Park KI, Paillard C, Chevalier P and Choi KS. 2006. Report on the occurrence of brown ring disease (BRD) in Manila clam, Ruditapes philippinarum, on the west coast of Korea. Aquaculture 255, 610-613.
  22. Relf JM, Chisholm JRS, Kemp GD and Smith VJ. 1999. Purification and characterization of a cystein-rich 11.5-kDa antibacterial protein from the granular haemocytes of the shore crab, Carcinus maenas. Eur J Biochem 264, 350-357.
  23. Seo JK, Crawford JM, Stone KL and Noga EJ. 2005. Purification of a novel arthropod defensin from the American oyster, Crassostrea virginica. Biochem Biophys Res Commun 338, 1998-2004.
  24. Seo JK, Lee MJ, Go HJ, Kim GD, Jeong HD, Nam BH and Park NG. 2013. Purification and antimicrobial function of ubiquitin isolated from the gill of Pacific oyster, Crassostrea gigas. Mol Immunol 53, 88-98.
  25. Seo JK, Lee MJ, Go HJ, Kim YJ and Park NG. 2014. Antimicrobial function of the GAPDH-related antimicrobial peptide in the skin of skipjack tuna, Katsuwonus pelamis. Fish Shellfish Immunol 36, 571-581.
  26. Seo JK, Lee MJ, Nam BH and 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, 480-488.
  27. Stark M, Liu LP and Deber CM. 2002. Cationic hydrophobic peptides with antimicrobial activity. Antimicrob Agents Chemother 46, 3585-3590.
  28. Shai Y. 2002. Mode of action of membrane active antimicrobial peptides. Biopolymers 66, 236-248.
  29. Silphaduang U and Noga EJ. 2001. Peptide antibiotics in mast cells of fish. Nature 414, 268-269.
  30. Skerlavaj B, Romeo D and Gennaro R. 1990. Rapid membrane permeabilization and inhibition of vital functions of Gram-negative bacteria by bactenecins. Infect Immun 58, 3724-3730.
  31. Tincu JA and Taylor SW. 2004. Antimicrobial peptides from marine invertebrates. Antimicrob Agents Chemother 48, 3645-3654.
  32. van''t Hof W, Veerman EC, Helmerhorst EJ and Amerongen AV. 2001. Antimicrobial peptides: properties and applicability. Biol Chem 382, 597-619.
  33. Zasloff M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415, 389-395.
  34. Zhang L, Yang D, Wang Q, Yuan Z, Wu H5, Pei D, Cong M, Li F, Ji C and Zhao J. 2015. A defensin from clam Venerupis philippinarum: Molecular characterization, localization, antibacterial activity, and mechanism of action. Dev Comp Immunol 51, 29-38.
  35. Zhao J, Li C, Chen A, Li L, Su X and Li T. 2010. Molecular characterization of a novel big defensin from clam, Venerupis philippinarum. PLoS One 5, e13480