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Nucleation and growth of a bacterial functional amyloid at single-fiber resolution

Nature Chemical Biology volume 13, pages 902–908 (2017)Cite this article

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Abstract

Curli are functional amyloids produced by proteobacteria like Escherichia coli as part of the extracellular matrix that holds cells together into biofilms. The molecular events that occur during curli nucleation and fiber extension remain largely unknown. Combining observations from curli amyloidogenesis in bulk solutions with real-time in situ nanoscopic imaging at the single-fiber level, we show that curli display polar growth, and we detect two kinetic regimes of fiber elongation. Single fibers exhibit stop-and-go dynamics characterized by bursts of steady-state growth alternated with periods of stagnation. At high subunit concentrations, fibers show constant, unperturbed burst growth. Curli follow a one-step nucleation process in which monomers contemporaneously fold and oligomerize into minimal fiber units that have growth characteristics identical to those of the mature fibrils. Kinetic data and interaction studies of curli fibrillation in the presence of the natural inhibitor CsgC show that the inhibitor binds curli fibers and predominantly acts at the level of fiber elongation.

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Figure 1: CsgA fibrillation in bulk solution.
Figure 2: Curli show polar growth kinetics.
Figure 3: Curli display stop-and-go growth kinetics.
Figure 4: Curli originate from one-step, direct nucleation events.
Figure 5: CsgC binds curli and halts curli elongation.
Figure 6: Integrated model for curli amyloidogenesis.

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References

  1. Chiti, F. & Dobson, C.M. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333–366 (2006).

    Article  CAS  Google Scholar 

  2. Knowles, T.P.J., Vendruscolo, M. & Dobson, C.M. The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 15, 384–396 (2014).

    CAS  PubMed  Google Scholar 

  3. Fowler, D.M., Koulov, A.V., Balch, W.E. & Kelly, J.W. Functional amyloid—from bacteria to humans. Trends Biochem. Sci. 32, 217–224 (2007).

    Article  CAS  Google Scholar 

  4. Chapman, M.R. et al. Role of Escherichia coli curli operons in directing amyloid fiber formation. Science 295, 851–855 (2002).

    Article  CAS  Google Scholar 

  5. Dueholm, M.S. et al. Functional amyloid in Pseudomonas. Mol. Microbiol. 77, 1009–1020 (2010).

    CAS  PubMed  Google Scholar 

  6. Romero, D., Aguilar, C., Losick, R. & Kolter, R. Amyloid fibers provide structural integrity to Bacillus subtilis biofilms. Proc. Natl. Acad. Sci. USA 107, 2230–2234 (2010).

    Article  CAS  Google Scholar 

  7. Schwartz, K., Syed, A.K., Stephenson, R.E., Rickard, A.H. & Boles, B.R. Functional amyloids composed of phenol soluble modulins stabilize Staphylococcus aureus biofilms. PLoS Pathog. 8, e1002744 (2012).

    Article  CAS  Google Scholar 

  8. Oli, M.W. et al. Functional amyloid formation by Streptococcus mutans. Microbiology 158, 2903–2916 (2012).

    Article  CAS  Google Scholar 

  9. Alteri, C.J. et al. Mycobacterium tuberculosis produces pili during human infection. Proc. Natl. Acad. Sci. USA 104, 5145–5150 (2007).

    Article  CAS  Google Scholar 

  10. Toyama, B.H. & Weissman, J.S. Amyloid structure: conformational diversity and consequences. Annu. Rev. Biochem. 80, 557–585 (2011).

    Article  CAS  Google Scholar 

  11. Eichner, T. & Radford, S.E. A diversity of assembly mechanisms of a generic amyloid fold. Mol. Cell 43, 8–18 (2011).

    Article  CAS  Google Scholar 

  12. Watanabe-Nakayama, T. et al. High-speed atomic force microscopy reveals structural dynamics of amyloid β1-42 aggregates. Proc. Natl. Acad. Sci. USA 113, 5835–5840 (2016).

    Article  CAS  Google Scholar 

  13. Bucciantini, M. et al. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416, 507–511 (2002).

    Article  CAS  Google Scholar 

  14. Kayed, R. et al. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486–489 (2003).

    Article  CAS  Google Scholar 

  15. Tipping, K.W., van Oosten-Hawle, P., Hewitt, E.W. & Radford, S.E. Amyloid fibres: inert end-stage aggregates or key players in disease? Trends Biochem. Sci. 40, 719–727 (2015).

    Article  CAS  Google Scholar 

  16. Olsén, A., Jonsson, A. & Normark, S. Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli. Nature 338, 652–655 (1989).

    Article  Google Scholar 

  17. Collinson, S. K. et al. Salmonella enteritidis agfBAC operon encoding thin, aggregative fimbriae. J. Bacteriol. 178, 662–667 (1996).

    Article  CAS  Google Scholar 

  18. Dueholm, M.S., Albertsen, M., Otzen, D. & Nielsen, P.H. Curli functional amyloid systems are phylogenetically widespread and display large diversity in operon and protein structure. PLoS One 7, e51274 (2012).

    Article  CAS  Google Scholar 

  19. Van Gerven, N., Klein, R.D., Hultgren, S.J. & Remaut, H. Bacterial amyloid formation: structural insights into curli biogensis. Trends Microbiol. 23, 693–706 (2015).

    Article  CAS  Google Scholar 

  20. Hawthorne, W., Rouse, S., Sewell, L. & Matthews, S.J. Structural insights into functional amyloid inhibition in Gram -ve bacteria. Biochem. Soc. Trans. 44, 1643–1649 (2016).

    Article  CAS  Google Scholar 

  21. Wang, X., Smith, D.R., Jones, J.W. & Chapman, M.R. In vitro polymerization of a functional Escherichia coli amyloid protein. J. Biol. Chem. 282, 3713–3719 (2007).

    Article  CAS  Google Scholar 

  22. Shu, Q. et al. Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation. Proc. Natl. Acad. Sci. USA 113, 7130–7135 (2016).

    Article  CAS  Google Scholar 

  23. Wang, X., Hammer, N.D. & Chapman, M.R. The molecular basis of functional bacterial amyloid polymerization and nucleation. J. Biol.Chem. 283, 21530–21539 (2008).

    Article  CAS  Google Scholar 

  24. Wang, X. & Chapman, M.R. Sequence determinants of bacterial amyloid formation. J. Mol. Biol. 380, 570–580 (2008).

    Article  CAS  Google Scholar 

  25. Evans, M.L. et al. The bacterial curli system possesses a potent and selective inhibitor of amyloid formation. Mol. Cell 57, 445–455 (2015).

    Article  CAS  Google Scholar 

  26. Taylor, J.D. et al. Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones. Sci. Rep. 6, 24656 (2016).

    Article  CAS  Google Scholar 

  27. Ando, T., Uchihashi, T. & Scheuring, S. Filming biomolecular processes by high-speed atomic force microscopy. Chem. Rev. 114, 3120–3188 (2014).

    Article  CAS  Google Scholar 

  28. Xiao, J. & Dufrêne, Y.F. Optical and force nanoscopy in microbiology. Nat. Microbiol. 1, 16186 (2016).

    Article  CAS  Google Scholar 

  29. Cherny, I. & Gazit, E. Amyloids: not only pathological agents but also ordered nanomaterials. Angew. Chem. Int. Ed. Engl. 47, 4062–4069 (2008).

    Article  CAS  Google Scholar 

  30. Knowles, T.P.J. & Buehler, M.J. Nanomechanics of functional and pathological amyloid materials. Nat. Nanotechnol. 6, 469–479 (2011).

    Article  CAS  Google Scholar 

  31. Van Gerven, N. et al. Secretion and functional display of fusion proteins through the curli biogenesis pathway. Mol. Microbiol. 91, 1022–1035 (2014).

    Article  CAS  Google Scholar 

  32. Chen, A.Y. et al. Synthesis and patterning of tunable multiscale materials with engineered cells. Nat. Mater. 13, 515–523 (2014).

    Article  CAS  Google Scholar 

  33. Nguyen, P.Q., Botyanszki, Z., Tay, P.K.R. & Joshi, N.S. Programmable biofilm-based materials from engineered curli nanofibres. Nat. Commun. 5, 4945 (2014).

    Article  CAS  Google Scholar 

  34. Shammas, S.L. et al. A mechanistic model of tau amyloid aggregation based on direct observation of oligomers. Nat. Commun. 6, 7025 (2015).

    Article  CAS  Google Scholar 

  35. Hung, C. et al. Escherichia coli biofilms have an organized and complex extracellular matrix structure. MBio 4, e00645–13 (2013).

    PubMed  PubMed Central  Google Scholar 

  36. Tian, P. et al. Structure of a functional amyloid protein subunit computed using sequence variation. J. Am. Chem. Soc. 137, 22–25 (2015).

    Article  CAS  Google Scholar 

  37. Cohen, S.I.A. et al. Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism. Proc. Natl. Acad. Sci. USA 110, 9758–9763 (2013).

    CAS  Google Scholar 

  38. Arosio, P., Knowles, T.P.J. & Linse, S. On the lag phase in amyloid fibril formation. Phys. Chem. Chem. Phys. 17, 7606–7618 (2015).

    Article  CAS  Google Scholar 

  39. Goldsbury, C., Kistler, J., Aebi, U., Arvinte, T. & Cooper, G.J.S. Watching amyloid fibrils grow by time-lapse atomic force microscopy. J. Mol. Biol. 285, 33–39 (1999).

    Article  CAS  Google Scholar 

  40. Kellermayer, M.S.Z., Karsai, A., Benke, M., Soós, K. & Penke, B. Stepwise dynamics of epitaxially growing single amyloid fibrils. Proc. Natl. Acad. Sci. USA 105, 141–144 (2008).

    Article  CAS  Google Scholar 

  41. Ban, T. et al. Direct observation of Abeta amyloid fibril growth and inhibition. J. Mol. Biol. 344, 757–767 (2004).

    Article  CAS  Google Scholar 

  42. Ferkinghoff-Borg, J. et al. Stop-and-go kinetics in amyloid fibrillation. Phys. Rev. E 82, 010901 (2010).

    Article  Google Scholar 

  43. Wördehoff, M.M. et al. Single fibril growth kinetics of α-synuclein. J. Mol. Biol. 427 6 Part B: 1428–1435 (2015).

    Article  Google Scholar 

  44. Hoyer, W., Cherny, D., Subramaniam, V. & Jovin, T.M. Rapid self-assembly of α-synuclein observed by in situ atomic force microscopy. J. Mol. Biol. 340, 127–139 (2004).

    Article  CAS  Google Scholar 

  45. Ban, T., Hamada, D., Hasegawa, K., Naiki, H. & Goto, Y. Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence. J. Biol. Chem. 278, 16462–16465 (2003).

    Article  CAS  Google Scholar 

  46. Colvin, M.T. et al. Atomic resolution structure of monomorphic Aβ42 amyloid fibrils. J. Am. Chem. Soc. 138, 9663–9674 (2016).

    Article  CAS  Google Scholar 

  47. Wälti, M.A. et al. Atomic-resolution structure of a disease-relevant Aβ(1-42) amyloid fibril. Proc. Natl. Acad. Sci. USA 113, E4976–E4984 (2016).

    Article  Google Scholar 

  48. Berne, B.J. & Pecora, R. Dynamic Light Scattering: With Applications to Chemistry, Biology, and Physics (Courier Dover Publications, 1976).

  49. Marsh, J.A. & Forman-Kay, J.D. Sequence determinants of compaction in intrinsically disordered proteins. Biophys. J. 98, 2383–2390 (2010).

    Article  CAS  Google Scholar 

  50. Beloin, C. et al. Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression. Mol. Microbiol. 51, 659–674 (2004).

    Article  CAS  Google Scholar 

  51. Pallesen, L., Poulsen, L.K., Christiansen, G. & Klemm, P. Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences. Microbiology 141, 2839–2848 (1995).

    Article  CAS  Google Scholar 

  52. Tseng, Q. et al. Spatial organization of the extracellular matrix regulates cell-cell junction positioning. Proc. Natl. Acad. Sci. USA 109, 1506–1511 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

M.S. and N.V.G. acknowledge financial support by the FWO under projects G0H5316N and 1516215N, respectively. H.R. acknowledges financial support by VIB project grant PRJ9 and ERC grant 649082 BAS-SBBT. I.V.B. is a recipient of a PhD fellowship by the IWT. Work at the Université Catholique de Louvain was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. 693630), the National Fund for Scientific Research (FNRS) and the FNRS-WELBIO (grant no. WELBIO-CR-2015A-05).

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Author notes

  1. Mike Sleutel, Imke Van den Broeck and Nani Van Gerven: These authors contributed equally to this work.

Authors and Affiliations

  1. Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

    Mike Sleutel, Imke Van den Broeck, Nani Van Gerven, Wim Jonckheere & Han Remaut

  2. Structural and Molecular Microbiology, Structural Biology Research Center, VIB, Brussels, Belgium

    Mike Sleutel, Imke Van den Broeck, Nani Van Gerven, Wim Jonckheere & Han Remaut

  3. Institute of Life Sciences, Université Catholique de Louvain, Louvain-la-Neuve, Belgium

    Cécile Feuillie, Claire Valotteau & Yves F Dufrêne

  4. Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Belgium

    Yves F Dufrêne

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Contributions

I.V.B., W.J. and N.V.G. produced protein samples, performed bulk fibrillation, CsgC activity experiments, and TEM. I.V.B., N.V.G. and M.S. performed the biochemical assays. M.S. performed and analyzed AFM measurements with help from C.F., C.V. and Y.F.D. M.S. and H.R. supervised the study, analyzed data and wrote the paper, with contributions from all authors.

Corresponding authors

Correspondence to Mike Sleutel or Han Remaut.

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Competing interests

N.V.G. and H.R. are inventors on PCT/EP2014/079319, describing the use of amyloid peptides for protein display.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–8 (PDF 7698 kb)

41589_2017_BFnchembio2413_MOESM2_ESM.mov

In situ imaging of CsgA fiber nucleation and growth on mica using Nanowizard III AFM (JPK): [CsgA] = 360 nM in 10 mM MES pH 6.0, 13 s/frame, total time: 13 min, 3100 nm x 3100 nm, Tapping Mode (vertical deflection channel) (MOV 2683 kb)

41589_2017_BFnchembio2413_MOESM3_ESM.mov

In situ imaging of CsgA fiber nucleation and growth on mica using Nanowizard III AFM (JPK): [CsgA] = 90 nM in 10 mM MES pH 6.0, 5 s/frame, total time: 20 min, 500 nm x 500 nm, Tapping Mode (error channel) (MOV 2739 kb)

41589_2017_BFnchembio2413_MOESM4_ESM.mov

In situ imaging of CsgA fiber nucleation and growth on mica using Nanowizard III AFM (JPK): [CsgA] = 90 nM in 10 mM MES pH 6.0, 506 frames, 2.5 s/frame, total time: 21 min, 450 nm x 450 nm, Tapping Mode (error channel) (MOV 10173 kb)

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Sleutel, M., Van den Broeck, I., Van Gerven, N. et al. Nucleation and growth of a bacterial functional amyloid at single-fiber resolution. Nat Chem Biol 13, 902–908 (2017). https://doi.org/10.1038/nchembio.2413

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