Journal of Membrane Biology

, Volume 216, Issue 2–3, pp 61–71 | Cite as

The Bacterial Peptide Pheromone Plantaricin A Permeabilizes Cancerous, but not Normal, Rat Pituitary Cells and Differentiates between the Outer and Inner Membrane Leaflet

  • Sverre L. Sand
  • Trude M. Haug
  • Jon Nissen-Meyer
  • Olav Sand


Plantaricin A (PlnA) is a 26-mer peptide pheromone with membrane-permeabilizing, strain-specific antibacterial activity, produced by Lactobacillus plantarum C11. We investigated the membrane-permeabilizing effects of PlnA on cultured cancerous and normal rat anterior pituitary cells using patch-clamp techniques and microfluorometry (fura-2). Cancerous cells displayed massive permeabilization within 5 s after exposure to 10–100 μm PlnA. The membrane depolarized to nearly 0 mV, and the membrane resistance decreased to a mere fraction of the initial value after less than 1 min. In outside-out membrane patches, 10 μm PlnA induced membrane currents reversing at 0 mV, which is compatible with an unspecific conductance increase. The d and l forms of the peptide had similar potency, indicating a nonchiral mechanism for the membrane-permeabilizing effect. Surprisingly, inside-out patches were insensitive to 1 mm PlnA. Primary cultures of normal rat anterior pituitary cells were also insensitive to the peptide. Thus, PlnA differentiates between plasma membranes and membrane leaflets. Microfluorometric recordings of [Ca2+]i and cytosolic concentration of fluorochrome verified the rapid permeabilizing effect of PlnA on cancerous cells and the insensitivity of normal pituitary cells.


Antimicrobial peptide Plantaricin A Lactobacillus plantarum Anterior pituitary cells Membrane permeabilization Patch clamp 


  1. Anderssen EL, Diep DB, Nes IF, Eijsink VG, Nissen-Meyer J (1998) Antagonistic activity of Lactobacillus plantarum C11: two new two-peptide bacteriocins, plantaricins EF and JK, and the induction factor plantaricin A. Appl Environ Microbiol 64:2269–2272PubMedGoogle Scholar
  2. Balasubramanian K, Schroit AJ (2003) Aminophospholipid asymmetry: a matter of life and death. Annu Rev Physiol 65:701–734PubMedCrossRefGoogle Scholar
  3. Bauer R, Dicks LM (2005) Mode of action of lipid II-targeting lantibiotics. Int J Food Microbiol 101:201–216PubMedCrossRefGoogle Scholar
  4. Benkirane N, Friede M, Guichard G, Briand JP, Van Regenmortel MH, Muller S (1993) Antigenicity and immunogenicity of modified synthetic peptides containing d-amino acid residues. Antibodies to a d-enantiomer do recognize the parent l-hexapeptide and reciprocally. J Biol Chem 268:26279–26285PubMedGoogle Scholar
  5. Breukink E, de Kruijff B (1999) The lantibiotic nisin, a special case or not? Biochim Biophys Acta 1462:223–234PubMedCrossRefGoogle Scholar
  6. Brotz H, Josten M, Wiedemann I, Schneider U, Gotz F, Bierbaum G, Sahl HG (1998) Role of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics. Mol Microbiol 30:317–327PubMedCrossRefGoogle Scholar
  7. Cappelli G, Paladini S, D’Agata A (1999) Tumor markers in the diagnosis of pancreatic cancer. Tumori 85:S19–S21PubMedGoogle Scholar
  8. Castano S, Desbat B, Delfour A, Dumas JM, da Silva A, Dufourcq J (2005) Study of structure and orientation of mesentericin Y105, a bacteriocin from gram-positive Leuconostoc mesenteroides, and its Trp-substituted analogues in phospholipid environments. Biochim Biophys Acta 1668:87–98PubMedCrossRefGoogle Scholar
  9. Chen HM, Leung KW, Thakur NN, Tan A, Jack RW (2003) Distinguishing between different pathways of bilayer disruption by the related antimicrobial peptides cecropin B, B1 and B3. Eur J Biochem 270:911–920PubMedCrossRefGoogle Scholar
  10. Chen HM, Wang W, Smith D, Chan SC (1997) Effects of the anti-bacterial peptide cecropin B and its analogs, cecropins B-1 and B-2, on liposomes, bacteria, and cancer cells. Biochim Biophys Acta 1336:171–179PubMedGoogle Scholar
  11. Cruciani RA, Barker JL, Zasloff M, Chen HC, Colamonici O (1991) Antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation. Proc Natl Acad Sci USA 88:3792–3796PubMedCrossRefGoogle Scholar
  12. Diep DB, Havarstein LS, Nes IF (1995) A bacteriocin-like peptide induces bacteriocin synthesis in Lactobacillus plantarum C11. Mol Microbiol 18:631–639PubMedCrossRefGoogle Scholar
  13. Diep DB, Havarstein LS, Nissen-Meyer J, Nes IF (1994) The gene encoding plantaricin A, a bacteriocin from Lactobacillus plantarum C11, is located on the same transcription unit as an agr-like regulatory system. Appl Environ Microbiol 60:160–166PubMedGoogle Scholar
  14. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148:2207–2216PubMedGoogle Scholar
  15. Fimland G, Johnsen L, Dalhus B, Nissen-Meyer J (2005) Pediocin-like antimicrobial peptides (class IIa bacteriocins) and their immunity proteins: biosynthesis, structure, and mode of action. J Pept Sci 11:688–696PubMedCrossRefGoogle Scholar
  16. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch 391:85–100CrossRefGoogle Scholar
  17. Hancock RE, Chapple DS (1999) Peptide antibiotics. Antimicrob Agents Chemother 43:1317–1323PubMedGoogle Scholar
  18. Hauge HH, Mantzilas D, Moll GN, Konings WN, Driessen AJ, Eijsink VG, Nissen-Meyer J (1998) Plantaricin A is an amphiphilic alpha-helical bacteriocin-like pheromone which exerts antimicrobial and pheromone activities through different mechanisms. Biochemistry 37:16026–16032PubMedCrossRefGoogle Scholar
  19. Huang HW (2000) Action of antimicrobial peptides: two-state model. Biochemistry 39:8347–8352PubMedCrossRefGoogle Scholar
  20. Jacob L, Zasloff M (1994) Potential therapeutic applications of magainins and other antimicrobial agents of animal origin. Ciba Found Symp 186:197–216PubMedGoogle Scholar
  21. Kristiansen PE, Fimland G, Mantzilas D, Nissen-Meyer J (2005) Structure and mode of action of the membrane-permeabilizing antimicrobial peptide pheromone plantaricin A. J Biol Chem 280:22945–22950PubMedCrossRefGoogle Scholar
  22. Lehrer RI, Lichtenstein AK, Ganz T (1993) Defensins: antimicrobial and cytotoxic peptides of mammalian cells. Annu Rev Immunol 11:105–128PubMedCrossRefGoogle Scholar
  23. Leuschner C, Hansel W (2004) Membrane disrupting lytic peptides for cancer treatments. Curr Pharm Des 10:2299–2310PubMedCrossRefGoogle Scholar
  24. Lichtenstein A (1991) Mechanism of mammalian cell lysis mediated by peptide defensins. Evidence for an initial alteration of the plasma membrane. J Clin Invest 88:93–100PubMedCrossRefGoogle Scholar
  25. Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, Green DR (1995) Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 182:1545–1556PubMedCrossRefGoogle Scholar
  26. Matsuzaki K (1998) Magainins as paradigm for the mode of action of pore forming polypeptides. Biochim Biophys Acta 1376:391–400PubMedGoogle Scholar
  27. Matsuzaki K (1999) Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim Biophys Acta 1462:1–10PubMedCrossRefGoogle Scholar
  28. Matsuzaki K, Sugishita K, Fujii N, Miyajima K (1995) Molecular basis for membrane selectivity of an antimicrobial peptide, magainin 2. Biochemistry 34:3423–3429PubMedCrossRefGoogle Scholar
  29. Miyagi T, Wada T, Yamaguchi K, Hata K (2004) Sialidase and malignancy: a minireview. Glycoconj J 20:189–198PubMedCrossRefGoogle Scholar
  30. Nissen-Meyer J, Nes IF (1997) Ribosomally synthesized antimicrobial peptides: their function, structure, biogenesis, and mechanism of action. Arch Microbiol 167:67–77CrossRefGoogle Scholar
  31. Oren Z, Shai Y (1998) Mode of action of linear amphipathic alpha-helical antimicrobial peptides. Biopolymers 47:451–463PubMedCrossRefGoogle Scholar
  32. Papo N, Shai Y (2003) New lytic peptides based on the d,l-amphipathic helix motif preferentially kill tumor cells compared to normal cells. Biochemistry 42:9346–9354PubMedCrossRefGoogle Scholar
  33. Parker MW, Feil SC (2005) Pore-forming protein toxins: from structure to function. Prog Biophys Mol Biol 88:91–142PubMedCrossRefGoogle Scholar
  34. Rao LV, Tait JF, Hoang AD (1992) Binding of annexin V to a human ovarian carcinoma cell line (OC-2008). Contrasting effects on cell surface factor VIIa/tissue factor activity and prothrombinase activity. Thromb Res 67:517–531PubMedCrossRefGoogle Scholar
  35. Raval GN, Patel DD, Parekh LJ, Patel JB, Shah MH, Patel PS (2003) Evaluation of serum sialic acid, sialyltransferase and sialoproteins in oral cavity cancer. Oral Dis 9:119–128PubMedCrossRefGoogle Scholar
  36. Sablon E, Contreras B, Vandamme E (2000) Antimicrobial peptides of lactic acid bacteria: mode of action, genetics and biosynthesis. Adv Biochem Eng Biotechnol 68:21–60PubMedGoogle Scholar
  37. Shai Y (1999) Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta 1462:55–70PubMedCrossRefGoogle Scholar
  38. Shai Y (2002) Mode of action of membrane active antimicrobial peptides. Biopolymers 66:236–248PubMedCrossRefGoogle Scholar
  39. Sobko AA, Kotova EA, Antonenko YN, Zakharov SD, Cramer WA (2006) Lipid dependence of the channel properties of a colicin E1-lipid toroidal pore. J Biol Chem 281:14408–14416PubMedCrossRefGoogle Scholar
  40. Sugimura M, Donato R, Kakkar VV, Scully MF (1994) Annexin V as a probe of the contribution of anionic phospholipids to the procoagulant activity of tumour cell surfaces. Blood Coagul Fibrinolysis 5:365–373PubMedGoogle Scholar
  41. Tashjian AH Jr, Yasumura Y, Levine L, Sato GH, Parker ML (1968) Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 82:342–352PubMedCrossRefGoogle Scholar
  42. Thennarasu S, Lee DK, Poon A, Kawulka KE, Vederas JC, Ramamoorthy A (2005) Membrane permeabilization, orientation, and antimicrobial mechanism of subtilosin A. Chem Phys Lipids 137:38–51PubMedCrossRefGoogle Scholar
  43. Tossi A, Sandri L, Giangaspero A (2000) Amphipathic, alpha-helical antimicrobial peptides. Biopolymers 55:4–30PubMedCrossRefGoogle Scholar
  44. Utsugi T, Schroit AJ, Connor J, Bucana CD, Fidler IJ (1991) Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes. Cancer Res 51:3062–3066PubMedGoogle Scholar
  45. Wiedemann I, Breukink E, van Kraaij C, Kuipers OP, Bierbaum G, de Kruijff B, Sahl HG (2001) Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J Biol Chem 276:1772–1779PubMedGoogle Scholar
  46. Williamson P, Schlegel RA (1994) Back and forth: the regulation and function of transbilayer phospholipid movement in eukaryotic cells. Mol Membr Biol 11:199–216PubMedCrossRefGoogle Scholar
  47. Yang L, Harroun TA, Weiss TM, Ding L, Huang HW (2001) Barrel-stave model or toroidal model? A case study on melittin pores. Biophys J 81:1475–1485PubMedCrossRefGoogle Scholar
  48. Ye JS, Zheng XJ, Leung KW, Chen HM, Sheu FS (2004) Induction of transient ion channel-like pores in a cancer cell by antibiotic peptide. J Biochem 136:255–259PubMedCrossRefGoogle Scholar
  49. Zasloff M (1987) Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci USA 84:5449–5453PubMedCrossRefGoogle Scholar
  50. Zelezetsky I, Pacor S, Pag U, Papo N, Shai Y, Sahl HG, Tossi A (2005) Controlled alteration of the shape and conformational stability of alpha-helical cell-lytic peptides: effect on mode of action and cell specificity. Biochem J 390:177–188PubMedCrossRefGoogle Scholar
  51. Zhao H, Sood R, Jutila A, Bose S, Fimland G, Nissen-Meyer J, Kinnunen PK (2006) Interaction of the antimicrobial peptide pheromone plantaricin A with model membranes: implications for a novel mechanism of action. Biochim Biophys Acta 1758:1461–1474PubMedCrossRefGoogle Scholar
  52. Zwaal RF, Comfurius P, Bevers EM (2005) Surface exposure of phosphatidylserine in pathological cells. Cell Mol Life Sci 62:971–988PubMedCrossRefGoogle Scholar
  53. Zwaal RF, Schroit AJ (1997) Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 89:1121–1132PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Sverre L. Sand
    • 1
  • Trude M. Haug
    • 1
  • Jon Nissen-Meyer
    • 1
  • Olav Sand
    • 1
  1. 1.Department of Molecular BiosciencesUniversity of OsloOsloNorway

Personalised recommendations