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Amphotericin forms an extramembranous and fungicidal sterol sponge

Nature Chemical Biology volume 10, pages 400–406 (2014)Cite this article

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Abstract

For over 50 years, amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells. In contrast, we report that amphotericin exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings reveal that extraction of a polyfunctional lipid underlies the resistance-refractory antimicrobial action of amphotericin and suggests a roadmap for separating its cytocidal and membrane-permeabilizing activities. This new mechanistic understanding is also guiding development of what are to our knowledge the first derivatives of amphotericin that kill yeast but not human cells.

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Figure 1: Models for the structure and function of AmB in the presence of lipid bilayers.
Figure 2: AmB primarily exists as large extramembranous aggregates.
Figure 3: Direct visualization of large extramembranous aggregates of AmB by TEM.
Figure 4: AmB extracts Erg from lipid bilayers.
Figure 5: AmB extracts Erg from and thereby kills yeast cells.

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References

  1. Cannon, R.D. et al. Candida albicans drug resistance—another way to cope with stress. Microbiology 153, 3211–3217 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Mora-Duarte, J. et al. Comparison of caspofungin and amphotericin B for invasive candidiasis. N. Engl. J. Med. 347, 2020–2029 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Monk, B.C. & Goffeau, A. Outwitting multidrug resistance to antifungals. Science 321, 367–369 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Ermishkin, L.N., Kasumov, K.M. & Potzeluyev, V.M. Single ionic channels induced in lipid bilayers by polyene antibiotics amphotericin-B and nystatine. Nature 262, 698–699 (1976).

    Article  CAS  PubMed  Google Scholar 

  5. Andreoli, T.E. & Monahan, M. The interaction of polyene antibiotics with thin lipid membranes. J. Gen. Physiol. 52, 300–325 (1968).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cass, A., Finkelstein, A. & Krespi, V. The ion permeability induced in thin lipid membranes by the polyene antibiotics nystatin and amphotericin B. J. Gen. Physiol. 56, 100–124 (1970).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Finkelstein, A. & Holz, R. Aqueous pores created in thin lipid membranes by the polyene antibiotics nystatin and amphotericin B. Membranes 2, 377–408 (1973).

    CAS  PubMed  Google Scholar 

  8. Andreoli, T.E. Structure and function of amphotericin B–cholesterol pores in lipid bilayer membranes. Ann. NY Acad. Sci. 235, 448–468 (1974).

    Article  CAS  PubMed  Google Scholar 

  9. de Kruijff, B. & Demel, R.A. Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. III. Molecular structure of polyene antibiotic–cholesterol complexes. Biochim. Biophys. Acta 339, 57–70 (1974).

    Article  CAS  PubMed  Google Scholar 

  10. Bolard, J. How do the polyene macrolide antibiotics affect the cellular membrane-properties. Biochim. Biophys. Acta 864, 257–304 (1986).

    Article  CAS  PubMed  Google Scholar 

  11. Volmer, A.A., Szpilman, A.M. & Carreira, E.M. Synthesis and biological evaluation of amphotericin B derivatives. Nat. Prod. Rep. 27, 1329–1349 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Cereghetti, D.M. & Carreira, E.M. Amphotericin B: 50 years of chemistry and biochemistry. Synthesis 2006, 914–942 (2006).

    Google Scholar 

  13. Chéron, M. et al. Quantitative structure-activity relationships in amphotericin B derivatives. Biochem. Pharmacol. 37, 827–836 (1988).

    Article  PubMed  Google Scholar 

  14. Croatt, M.P. & Carreira, E.M. Probing the role of the mycosamine C2′-OH on the activity of amphotericin B. Org. Lett. 13, 1390–1393 (2011).

    Article  CAS  PubMed  Google Scholar 

  15. Baginski, M., Resat, H. & Borowski, E. Comparative molecular dynamics simulations of amphotericin B–cholesterol/ergosterol membrane channels. Biochim. Biophys. Acta 1567, 63–78 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Murata, M. et al. Ion channel complex of antibiotics as viewed by NMR. Pure Appl. Chem. 81, 1123–1129 (2009).

    Article  CAS  Google Scholar 

  17. Matsumori, N., Sawada, Y. & Murata, M. Large molecular assembly of amphotericin B formed in ergosterol-containing membrane evidenced by solid-state NMR of intramolecular bridged derivative. J. Am. Chem. Soc. 128, 11977–11984 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Matsuoka, S. et al. Membrane interaction of amphotericin B as single-length assembly examined by solid state NMR for uniformly 13C-enriched agent. Bioorg. Med. Chem. 14, 6608–6614 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Umegawa, Y. et al. Head-to-tail interaction between amphotericin B and ergosterol occurs in hydrated phospholipid membrane. Biochemistry 51, 83–89 (2012).

    Article  CAS  PubMed  Google Scholar 

  20. Verkleij, A.J. et al. Freeze-etch electron microscopy of erythrocytes, Acholeplasma laidlawii cells and liposomal membranes after action of filipin and amphotericin B. Biochim. Biophys. Acta 291, 577–581 (1973).

    Article  CAS  PubMed  Google Scholar 

  21. Milhaud, J., Ponsinet, V., Takashi, M. & Michels, B. Interactions of the drug amphotericin B with phospholipid membranes containing or not ergosterol: new insight, into them role of ergosterol. Biochim. Biophys. Acta 1558, 95–108 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. Mouri, R. et al. Complex formation of amphotericin B in sterol-containing membranes as evidenced by surface plasmon resonance. Biochemistry 47, 7807–7815 (2008).

    Article  CAS  PubMed  Google Scholar 

  23. Katzung, B.G., Masters, S.B. & Trevor, A.J. Basic and Clinical Pharmacology 12th edn. (McGraw-Hill Education, Europe, 2012).

  24. Fernandez-Lopez, S. et al. Antibacterial agents based on the cyclic D,l-α-peptide architecture. Nature 412, 452–455 (2001).

    CAS  PubMed  Google Scholar 

  25. Gray, K.C. et al. Amphotericin primarily kills yeast by simply binding ergosterol. Proc. Natl. Acad. Sci. USA 109, 2234–2239 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Palacios, D.S., Anderson, T.M. & Burke, M.D. A post-PKS oxidation of the amphotericin B skeleton predicted to be critical for channel formation is not required for potent antifungal activity. J. Am. Chem. Soc. 129, 13804–13805 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Palacios, D.S. et al. Synthesis-enabled functional group deletions reveal key underpinnings of amphotericin B ion channel and antifungal activities. Proc. Natl. Acad. Sci. USA 108, 6733–6738 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Jin, H., McCaffery, J.M. & Grote, E. Ergosterol promotes pheromone signaling and plasma membrane fusion in mating yeast. J. Cell Biol. 180, 813–826 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kato, M. & Wickner, W. Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion. EMBO J. 20, 4035–4040 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Heese-Peck, A. et al. Multiple functions of sterols in yeast endocytosis. Mol. Biol. Cell 13, 2664–2680 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. te Welscher, Y.M. et al. Polyene antibiotic that inhibits membrane transport proteins. Proc. Natl. Acad. Sci. USA 109, 11156–11159 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Solomon, I. Relaxation processes in a system of 2 spins. Phys. Rev. 99, 559–565 (1955).

    Article  CAS  Google Scholar 

  33. Nadaud, P.S., Helmus, J.J., Hofer, N. & Jaroniec, C.P. Long-range structural restraints in spin-labeled proteins probed by solid-state nuclear magnetic resonance spectroscopy. J. Am. Chem. Soc. 129, 7502–7503 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Sankaram, M.B. & Thompson, T.E. Cholesterol-induced fluid-phase immiscibility in membranes. Proc. Natl. Acad. Sci. USA 88, 8686–8690 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Hsueh, Y.W. et al. Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols. Biophys. J. 92, 1606–1615 (2007).

    Article  CAS  PubMed  Google Scholar 

  36. Watson, K., Bertoli, E. & Griffiths, D.E. Phase transitions in yeast mitochondrial membranes—effect of temperature on energies of activation of respiratory enzymes of Saccharomyces cerevisiae. Biochem. J. 146, 401–407 (1975).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ejsing, C.S. et al. Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. Proc. Natl. Acad. Sci. USA 106, 2136–2141 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Chen, L. et al. Constant-time through-bond 13C correlation spectroscopy for assigning protein resonances with solid-state NMR spectroscopy. J. Am. Chem. Soc. 128, 9992–9993 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Takegoshi, K., Nakamura, S. & Terao, T. 13C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem. Phys. Lett. 344, 631–637 (2001).

    Article  CAS  Google Scholar 

  40. Hohwy, M., Rienstra, C.M., Jaroniec, C.P. & Griffin, R.G. Fivefold symmetric homonuclear dipolar recoupling in rotating solids: application to double quantum spectroscopy. J. Chem. Phys. 110, 7983–7992 (1999).

    Article  CAS  Google Scholar 

  41. Huster, D., Yao, X.L. & Hong, M. Membrane protein topology probed by 1H spin diffusion from lipids using solid-state NMR spectroscopy. J. Am. Chem. Soc. 124, 874–883 (2002).

    Article  CAS  PubMed  Google Scholar 

  42. Lange, A., Luca, S. & Baldus, M. Structural constraints from proton-mediated rare-spin correlation spectroscopy in rotating solids. J. Am. Chem. Soc. 124, 9704–9705 (2002).

    Article  CAS  PubMed  Google Scholar 

  43. Tang, M., Waring, A.J. & Hong, M. Intermolecular packing and alignment in an ordered beta-hairpin antimicrobial peptide aggregate from 2D solid-state NMR. J. Am. Chem. Soc. 127, 13919–13927 (2005).

    Article  CAS  PubMed  Google Scholar 

  44. Hohwy, M. et al. Local structure and relaxation in solid-state NMR: Accurate measurement of amide N-H bond lengths and H-N-H bond angles. J. Am. Chem. Soc. 122, 3218–3219 (2000).

    Article  CAS  Google Scholar 

  45. Haas, A. A quantitative assay to measure homotypic vacuole fusion in vitro. Methods Cell Sci. 17, 283–294 (1995).

    Article  Google Scholar 

  46. Yancey, P.G. et al. Cellular cholesterol effect mediated by cyclodextrins—demonstration of kinetic pools and mechanism of efflux. J. Biol. Chem. 271, 16026–16034 (1996).

    Article  CAS  PubMed  Google Scholar 

  47. Wilcock, B.C., Endo, M.M., Uno, B.E. & Burke, M.D. C2′-OH of amphotericin B plays an important role in binding the primary sterol of human cells but not yeast cells. J. Am. Chem. Soc. 135, 8488–8491 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kang, C.K. et al. Visualization analysis of the vacuole-targeting fungicidal activity of amphotericin B against the parent strain and an ergosterol-less mutant of Saccharomyces cerevisiae. Microbiology 159, 939–947 (2013).

    Article  CAS  PubMed  Google Scholar 

  49. Vincent, B.M. et al. Fitness trade-offs restrict the evolution of resistance to amphotericin B. PLoS Biol. 11, e1001692 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Sanglard, D. et al. Candida albicans mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents. Antimicrob. Agents Chemother. 47, 2404–2412 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Pangborn, A.B. et al. Safe and convenient procedure for solvent purification. Organometallics 15, 1518–1520 (1996).

    CAS  Google Scholar 

  52. Seo, S. et al. Biosynthesis of sitosterol, cycloartenol, and 24-methylenecycloartanol in tissue-cultures of higher-plants and of ergosterol in yeast from [1,2-13C2]- acetate and [2-13C-2H3]-acetate and [5-13C2-2H2] MVA. J. Chem. Soc. Perkin Trans. I 2407–2414 (1988).

  53. Comellas, G., Lopez, J.J., Nieuwkoop, A.J., Lemkau, L.R. & Rienstra, C.M. Straightforward, effective calibration of SPINAL-64 decoupling results in the enhancement of sensitivity and resolution of biomolecular solid-state NMR. J. Magn. Reson. 209, 131–135 (2011).

    Article  CAS  PubMed  Google Scholar 

  54. Morcombe, C.R. & Zilm, K.W. Chemical shift referencing in MAS solid state NMR. J. Magn. Reson. 162, 479–486 (2003).

    Article  CAS  PubMed  Google Scholar 

  55. Rienstra, C.M. et al. Determination of multiple torsion-angle constraints in U-13C,15N-labeled peptides: 3D 1H-15N-13C-1H dipolar chemical shift NMR spectroscopy in rotating solids. J. Am. Chem. Soc. 124, 11908–11922 (2002).

    Article  CAS  PubMed  Google Scholar 

  56. Ohi, M., Li, Y., Cheng, Y. & Walz, T. Negative staining and image classification—powerful tools in modern electron microscopy. Biol. Proced. Online 6, 23–34 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Winkler, J.K., Rennick, K.A., Eller, F.J. & Vaughn, S.F. Phytosterol and tocopherol components in extracts of corn distiller's dried grain. J. Agric. Food Chem. 55, 6482–6486 (2007).

    Article  CAS  PubMed  Google Scholar 

  58. Shervani, Z. et al. Aggregation of polyene antibiotics as studied by electronic absorption and circular dichroism spectroscopies. Colloid. Surface. B 7, 31–38 (1996).

    Article  CAS  Google Scholar 

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Acknowledgements

We gratefully acknowledge P.J. Hergenrother and E. Oldfield for helpful discussions, and we thank J.J. Lopez for preliminary spin diffusion SSNMR experiments. Portions of this work were supported by the US National Institutes of Health (NIH) (R01GM080436 and F30DK081272) and the University of Illinois at Urbana-Champaign (Centennial Scholar Award to C.M.R.). M.D.B. is a Howard Hughes Medical Institute Early Career Scientist. M.C.C. is an American Heart Association Predoctoral Fellow. T.M.A. is a Ruth L. Kirchstein NIH National Research Service Award Predoctoral Fellow. The Gonen lab is funded by the Howard Hughes Medical Institute.

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Author notes

  1. Thomas M Anderson and Mary C Clay: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Thomas M Anderson, Mary C Clay, Grant S Hisao, Marcus D Tuttle, Andrew J Nieuwkoop, Nashrah Maryum, Shu Wang, Brice E Uno, Chad M Rienstra & Martin D Burke

  2. Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Alexander G Cioffi, Katrina A Diaz, Erin L Wildeman, Chad M Rienstra & Martin D Burke

  3. Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Gemma Comellas & Chad M Rienstra

  4. Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Shu Wang & Martin D Burke

  5. Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA

    Tamir Gonen

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Contributions

T.M.A., M.C.C., A.G.C., K.A.D., A.J.N., G.C., T.G., C.M.R. and M.D.B. designed research. T.M.A., N.M. and A.G.C. prepared [U-13C]AmB and [13C]Erg. T.M.A., M.C.C., A.G.C., G.S.H., A.J.N., G.C. and B.E.U. prepared samples for SSNMR. M.C.C., A.J.N., G.C., G.S.H., M.D.T. and C.M.R. acquired SSNMR data. A.G.C. and T.G. performed microscopy. K.A.D. performed cell-based assays. T.M.A., M.C.C., A.G.C., K.A.D., G.S.H., M.D.T., A.J.N., G.C., S.W., B.E.U., E.L.W., T.G., C.M.R. and M.D.B. analyzed data. T.M.A., M.C.C., A.G.C., K.A.D., C.M.R. and M.D.B. wrote the paper.

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Correspondence to Chad M Rienstra or Martin D Burke.

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Supplementary information

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Supplementary Results, Supplementary Figures 1–16 and Supplementary Tables 1–3. (PDF 7590 kb)

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Biosynthesis and characterization of U-13C-Amphotericin Biosynthesis (DOCX 45 kb)

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Anderson, T., Clay, M., Cioffi, A. et al. Amphotericin forms an extramembranous and fungicidal sterol sponge. Nat Chem Biol 10, 400–406 (2014). https://doi.org/10.1038/nchembio.1496

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