Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structure and function of a unique pore-forming protein from a pathogenic acanthamoeba


Human pathogens often produce soluble protein toxins that generate pores inside membranes, resulting in the death of target cells and tissue damage. In pathogenic amoebae, this has been exemplified with amoebapores of the enteric protozoan parasite Entamoeba histolytica. Here we characterize acanthaporin, to our knowledge the first pore-forming toxin to be described from acanthamoebae, which are free-living, bacteria-feeding, unicellular organisms that are opportunistic pathogens of increasing importance and cause severe and often fatal diseases. We isolated acanthaporin from extracts of virulent Acanthamoeba culbertsoni by tracking its pore-forming activity, molecularly cloned the gene of its precursor and recombinantly expressed the mature protein in bacteria. Acanthaporin was cytotoxic for human neuronal cells and exerted antimicrobial activity against a variety of bacterial strains by permeabilizing their membranes. The tertiary structures of acanthaporin's active monomeric form and inactive dimeric form, both solved by NMR spectroscopy, revealed a currently unknown protein fold and a pH-dependent trigger mechanism of activation.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: Activity of acanthaporin on membranes.
Figure 2: The three-dimensional structure of the monomeric acanthaporin.
Figure 3: Molecular organization of acanthaporin in solution and after lipid interaction.
Figure 4: The three-dimensional structure of the acanthaporin dimer.
Figure 5: Model of action of acanthaporin toward membranes.

Accession codes

Primary accessions

NCBI Reference Sequence

Protein Data Bank


  1. Leippe, M. Antimicrobial and cytolytic polypeptides of amoeboid protozoa—effector molecules of primitive phagocytes. Dev. Comp. Immunol. 23, 267–279 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Herbst, R. et al. Pore-forming polypeptides of the pathogenic protozoon Naegleria fowleri. J. Biol. Chem. 277, 22353–22360 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Herbst, R., Marciano-Cabral, F. & Leippe, M. Antimicrobial and pore-forming peptides of free-living and potentially highly pathogenic Naegleria fowleri are released from the same precursor molecule. J. Biol. Chem. 279, 25955–25958 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Hecht, O. et al. Solution structure of the pore-forming protein of Entamoeba histolytica. J. Biol. Chem. 279, 17834–17841 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Kolter, T., Winau, F., Schaible, U.E., Leippe, M. & Sandhoff, K. Lipid-binding proteins in membrane digestion, antigen presentation, and antimicrobial defense. J. Biol. Chem. 280, 41125–41128 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Liepinsh, E., Andersson, M., Ruysschaert, J.M. & Otting, G. Saposin fold revealed by the NMR structure of NK-lysin. Nat. Struct. Biol. 4, 793–795 (1997).

    Article  CAS  PubMed  Google Scholar 

  7. Anderson, D.H. et al. Granulysin crystal structure and a structure-derived lytic mechanism. J. Mol. Biol. 325, 355–365 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Leippe, M., Bruhn, H., Hecht, O. & Grötzinger, J. Ancient weapons: the three-dimensional structure of amoebapore A. Trends Parasitol. 21, 5–7 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Marciano-Cabral, F. & Cabral, G. Acanthamoeba spp. as agents of disease in humans. Clin. Microbiol. Rev. 16, 273–307 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Martinez, A.J. Free-living amebas: infection of the central nervous system. Mt. Sinai J. Med. 60, 271–278 (1993).

    CAS  PubMed  Google Scholar 

  11. Visvesvara, G.S. Amebic meningoencephalitides and keratitis: challenges in diagnosis and treatment. Curr. Opin. Infect. Dis. 23, 590–594 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Greub, G. & Raoult, D. Microorganisms resistant to free-living amoebae. Clin. Microbiol. Rev. 17, 413–433 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Ellman, G.L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77 (1959).

    Article  CAS  PubMed  Google Scholar 

  14. Holm, L., Kaariainen, S., Rosenstrom, P. & Schenkel, A. Searching protein structure databases with DaliLite v.3. Bioinformatics 24, 2780–2781 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Madej, T., Gibrat, J.F. & Bryant, S.H. Threading a database of protein cores. Proteins 23, 356–369 (1995).

    Article  CAS  PubMed  Google Scholar 

  16. Gibrat, J.F., Madej, T. & Bryant, S.H. Surprising similarities in structure comparison. Curr. Opin. Struct. Biol. 6, 377–385 (1996).

    Article  CAS  PubMed  Google Scholar 

  17. Andrä, J., Herbst, R. & Leippe, M. Amoebapores, archaic effector peptides of protozoan origin, are discharged into phagosomes and kill bacteria by permeabilizing their membranes. Dev. Comp. Immunol. 27, 291–304 (2003).

    Article  PubMed  Google Scholar 

  18. Yeaman, M.R. & Yount, N.Y. Mechanisms of antimicrobial peptide action and resistance. Pharmacol. Rev. 55, 27–55 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Rötzschke, O., Lau, J.M., Hofstatter, M., Falk, K. & Strominger, J.L. A pH-sensitive histidine residue as control element for ligand release from HLA-DR molecules. Proc. Natl. Acad. Sci. USA 99, 16946–16950 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Baumann, G. & Mueller, P. A molecular model of membrane excitability. J. Supramol. Struct. 2, 538–557 (1974).

    Article  CAS  PubMed  Google Scholar 

  21. Shai, Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta 1462, 55–70 (1999).

    Article  CAS  PubMed  Google Scholar 

  22. Yang, L., Harroun, T.A., Weiss, T.M., Ding, L. & Huang, H.W. Barrel-stave model or toroidal model? A case study on melittin pores. Biophys. J. 81, 1475–1485 (2001).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Anderluh, G. & Lakey, J.H. Disparate proteins use similar architectures to damage membranes. Trends Biochem. Sci. 33, 482–490 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Jenssen, H., Hamill, P. & Hancock, R.E. Peptide antimicrobial agents. Clin. Microbiol. Rev. 19, 491–511 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 415, 389–395 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Gutsmann, T. et al. Interaction of amoebapores and NK-lysin with symmetric phospholipid and asymmetric lipopolysaccharide/phospholipid bilayers. Biochemistry 42, 9804–9812 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Leippe, M., Andrä, J., Nickel, R., Tannich, E. & Müller-Eberhard, H.J. Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore formation in bacterial cytoplasmic membranes. Mol. Microbiol. 14, 895–904 (1994).

    Article  CAS  PubMed  Google Scholar 

  28. Schägger, H. & von Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368 (1987).

    Article  PubMed  Google Scholar 

  29. Frohman, M.A., Dush, M.K. & Martin, G.R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Scotto-Lavino, E., Du, G. & Frohman, M.A. 3′ end cDNA amplification using classic RACE. Nat. Protoc. 1, 2742–2745 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. LaVallie, E.R. et al. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Bio/Technology 11, 187–193 (1993).

    CAS  Google Scholar 

  32. Dingley, A.J., Lorenzen, I. & Grötzinger, J. NMR analysis of viral protein structures. Methods Mol. Biol. 451, 441–462 (2008).

    Article  CAS  PubMed  Google Scholar 

  33. Markley, J.L. et al. Recommendations for the presentation of NMR structures of proteins and nucleic acids. J. Mol. Biol. 280, 933–952 (1998).

    Article  CAS  PubMed  Google Scholar 

  34. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  PubMed  Google Scholar 

  35. Goddard, T. & Kneller, D. SPARKY 3 (University of San Francisco, California, USA.)

  36. Johnson, B.A. Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol. Biol. 278, 313–352 (2004).

    CAS  PubMed  Google Scholar 

  37. Güntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353–378 (2004).

    PubMed  Google Scholar 

  38. Schwieters, C.D., Kuszewski, J.J., Tjandra, N. & Clore, G.M. The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 65–73 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Linge, J.P., Williams, M.A., Spronk, C.A., Bonvin, A.M. & Nilges, M. Refinement of protein structures in explicit solvent. Proteins 50, 496–506 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. Koradi, R., Billeter, M. & Wuthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55, 29–32 (1996).

    Article  CAS  PubMed  Google Scholar 

  41. Andrä, J. & Leippe, M. Pore-forming peptide of Entamoeba histolytica. Significance of positively charged amino acid residues for its mode of action. FEBS Lett. 354, 97–102 (1994).

    Article  PubMed  Google Scholar 

  42. Montal, M. & Mueller, P. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc. Natl. Acad. Sci. USA 69, 3561–3566 (1972).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Dougherty, R.M., Galli, C., Ferro-Luzzi, A. & Iacono, J.M. Lipid and phospholipid fatty acid composition of plasma, red blood cells, and platelets and how they are affected by dietary lipids: a study of normal subjects from Italy, Finland, and the USA. Am. J. Clin. Nutr. 45, 443–455 (1987).

    Article  CAS  PubMed  Google Scholar 

  44. Bruhn, H., Riekens, B., Berninghausen, O. & Leippe, M. Amoebapores and NK-lysin, members of a class of structurally distinct antimicrobial and cytolytic peptides from protozoa and mammals: a comparative functional analysis. Biochem. J. 375, 737–744 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Boman, H.G., Nilsson-Faye, I., Paul, K. & Rasmuson, T. Jr. Insect immunity. I. Characteristics of an inducible cell-free antibacterial reaction in hemolymph of Samia cynthia pupae. Infect. Immun. 10, 136–145 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Leippe, M., Ebel, S., Schoenberger, O.L., Horstmann, R.D. & Müller-Eberhard, H.J. Pore-forming peptide of pathogenic Entamoeba histolytica. Proc. Natl. Acad. Sci. USA 88, 7659–7663 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


The authors acknowledge support in measuring the 750-MHz NMR spectra at The Netherlands Foundation for Chemical Research (SON) NMR Large Scale Facility in Utrecht, The Netherlands, funded by the European Union (contract number RII3-026145). We thank F. Buck, Institute for Cell Biochemistry and Clinical Neurobiology, University of Hamburg, for protein sequencing, C. Ott for expert technical assistance during purification of the natural acanthaporin and H. Ließegang for technical help during antibacterial activity testing. This work was supported by German Research Council (DFG) grant LE 1075/2-4 to M.L. and J.G. and the excellence cluster 306 'Inflammation at Interfaces'.

Author information

Authors and Affiliations



M.L. conceived the study and purified the natural protein. M.M. performed the majority of the experiments. F.D.S., R.W., A.J.D. and H.W. contributed to structure determination and data analysis. C.-W.H. and A.T. assigned the disulfide bond connectivity. A.K. and T.G. completed the planar lipid bilayer experiments. M.S., R.H. and I.L. performed molecular biology and initial protein expression. M.S. contributed to antibacterial assays. F.M.-C. cultured and passaged the amoebae and made the amoebic extracts. C.G. performed the initial mass spectrometry measurements of the protein. M.L. and J.G. directed experiments. M.M. and M.L. wrote the manuscript.

Corresponding author

Correspondence to Matthias Leippe.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Note and Supplementary Results (PDF 1648 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Michalek, M., Sönnichsen, F., Wechselberger, R. et al. Structure and function of a unique pore-forming protein from a pathogenic acanthamoeba. Nat Chem Biol 9, 37–42 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing