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

Abstract

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.

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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.

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Acknowledgements

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'.

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Contributions

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.

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Correspondence to Matthias Leippe.

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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). https://doi.org/10.1038/nchembio.1116

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