Structure of S-layer protein Sap reveals a mechanism for therapeutic intervention in anthrax

Abstract

Anthrax is an ancient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis. At present, anthrax mostly affects wildlife and livestock, although it remains a concern for human public health—primarily for people who handle contaminated animal products and as a bioterrorism threat due to the high resilience of spores, a high fatality rate of cases and the lack of a civilian vaccination programme1,2. The cell surface of B. anthracis is covered by a protective paracrystalline monolayer—known as surface layer or S-layer—that is composed of the S-layer proteins Sap or EA1. Here, we generate nanobodies to inhibit the self-assembly of Sap, determine the structure of the Sap S-layer assembly domain (SapAD) and show that the disintegration of the S-layer attenuates the growth of B. anthracis and the pathology of anthrax in vivo. SapAD comprises six β-sandwich domains that fold and support the formation of S-layers independently of calcium. Sap-inhibitory nanobodies prevented the assembly of Sap and depolymerized existing Sap S-layers in vitro. In vivo, nanobody-mediated disruption of the Sap S-layer resulted in severe morphological defects and attenuated bacterial growth. Subcutaneous delivery of Sap inhibitory nanobodies cleared B. anthracis infection and prevented lethality in a mouse model of anthrax disease. These findings highlight disruption of S-layer integrity as a mechanism that has therapeutic potential in S-layer-carrying pathogens.

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Fig. 1: X-ray structure of B. anthracis SapAD.
Fig. 2: Single-domain antibodies inhibit Sap S-layer formation and affect bacterial growth.
Fig. 3: S-layer assembly inhibitory Nbs affect B. anthracis cell morphology.
Fig. 4: Clearance of B. anthracis infection through NbsSAI treatment.

Data availability

Coordinates and structure factors of the SapAD–NbsAF684–NbsAF694 and SapAD–NbsAF683–NbsAF694 complexes have been deposited in PDB under accession codes 6HHU and 6QX4, respectively. All other data are available in the manuscript or the Supplementary Information.

References

  1. 1.

    Jernigan, D. B. et al. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg. Infect. Dis. 8, 1019–1028 (2002).

    PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Sweeney, D. A., Hicks, C. W., Cui, X., Li, Y. & Eichacker, P. Q. Anthrax infection. Am. J. Respir. Crit. Care Med. 184, 1333–1341 (2011).

    PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Couture-Tosi, E. et al. Structural analysis and evidence for dynamic emergence of Bacillus anthracis S-layer networks. J. Bacteriol. 184, 6448–6456 (2002).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Collier, R. J. & Young, J. A. Anthrax toxin. Annu. Rev. Cell Dev. Biol. 19, 45–70 (2003).

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Zwartouw, H. T. & Smith, H. Polyglutamic acid from Bacillus anthracis grown in vivo; structure and aggressin activity. Biochem. J. 63, 437–442 (1956).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Makino, S., Uchida, I., Terakado, N., Sasakawa, C. & Yoshikawa, M. Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis. J. Bacteriol. 171, 722–730 (1989).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Weiner, Z. P. & Glomski, I. J. Updating perspectives on the initiation of Bacillus anthracis growth and dissemination through its host. Infect. Immun. 80, 1626–1633 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 8.

    Candela, T. & Fouet, A. Bacillus anthracis CapD, belonging to the gamma-glutamyltranspeptidase family, is required for the covalent anchoring of capsule to peptidoglycan. Mol. Microbiol. 57, 717–726 (2005).

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Sara, M. & Sleytr, U. B. S-layer proteins. J. Bacteriol. 182, 859–868 (2000).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Gerbino, E., Carasi, P., Mobili, P., Serradell, M. A. & Gomez-Zavaglia, A. Role of S-layer proteins in bacteria. World J. Microbiol. Biotechnol. 31, 1877–1887 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. 11.

    Mignot, T., Mesnage, S., Couture-Tosi, E., Mock, M. & Fouet, A. Developmental switch of S-layer protein synthesis in Bacillus anthracis. Mol. Microbiol. 43, 1615–1627 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12.

    Mesnage, S., Tosi-Couture, E., Mock, M., Gounon, P. & Fouet, A. Molecular characterization of the Bacillus anthracis main S-layer component: evidence that it is the major cell-associated antigen. Mol. Microbiol. 23, 1147–1155 (1997).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  13. 13.

    Kern, V. J., Kern, J. W., Theriot, J. A., Schneewind, O. & Missiakas, D. Surface-layer (S-layer) proteins Sap and EA1 govern the binding of the S-layer-associated protein BslO at the cell septa of Bacillus anthracis. J. Bacteriol. 194, 3833–3840 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Baranova, E. et al. SbsB structure and lattice reconstruction unveil Ca2+ triggered S-layer assembly. Nature 487, 119–122 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  15. 15.

    Kern, J. et al. Structure of surface layer homology (SLH) domains from Bacillus anthracis surface array protein. J. Biol. Chem. 286, 26042–26049 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Sychantha, D. et al. Molecular basis for the attachment of S-layer proteins to the cell wall of Bacillus anthracis. Biochemistry 57, 1949–1953 (2018).

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Candela, T., Mignot, T., Hagnerelle, X., Haustant, M. & Fouet, A. Genetic analysis of Bacillus anthracis Sap S-layer protein crystallization domain. Microbiology 151, 1485–1490 (2005).

    CAS  PubMed  Article  Google Scholar 

  18. 18.

    Pavkov, T. et al. The structure and binding behavior of the bacterial cell surface layer protein SbsC. Structure 16, 1226–1237 (2008).

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Teixeira, L. M. et al. Entropically driven self-assembly of Lysinibacillus sphaericus S-layer proteins analyzed under various environmental conditions. Macromol. Biosci. 10, 147–155 (2010).

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Rad, B. et al. Ion-specific control of the self-assembly dynamics of a nanostructured protein lattice. ACS Nano 9, 180–190 (2015).

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Kirk, J. A. et al. New class of precision antimicrobials redefines role of Clostridium difficile S-layer in virulence and viability. Sci. Transl. Med. 9, eaah6813 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  22. 22.

    Wang, X. Y. et al. A S-layer protein of Bacillus anthracis as a building block for functional protein arrays by in vitro self-assembly. Small 11, 5826–5832 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23.

    Domanska, K. et al. Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic β2-microglobulin variant. Proc. Natl Acad. Sci. USA 108, 1314–1319 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24.

    Conrath, K. E. et al. β-lactamase inhibitors derived from single-domain antibody fragments elicited in the Camelidae. Antimicrob. Agents Chemother. 45, 2807–2812 (2001).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    Pardon, E. et al. A general protocol for the generation of nanobodies for structural biology. Nat. Protoc. 9, 674–693 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010).

    CAS  Article  Google Scholar 

  27. 27.

    Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D 67, 293–302 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    Sheldrick, G. M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479–485 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. 29.

    Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M. & Paciorek, W. Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D 59, 2023–2030 (2003).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    Abrahams, J. P. & Leslie, A. G. W. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996).

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

    Article  Google Scholar 

  32. 32.

    Cowtan, K. D. & Main, P. Phase combination and cross validation in iterated density-modification calculations. Acta Crystallogr. D 52, 43–48 (1996).

    CAS  PubMed  Article  Google Scholar 

  33. 33.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  PubMed  Article  Google Scholar 

  34. 34.

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).

    CAS  PubMed  Article  Google Scholar 

  35. 35.

    BUSTER v.2.10.3 (Global Phasing Ltd, 2017).

  36. 36.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007).

    CAS  Article  Google Scholar 

  37. 37.

    Holm, L. & Sander, C. Dali: a network tool for protein structure comparison. Trends Biochem. Sci. 20, 478–480 (1995).

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Jones, D. T., Taylor, W. R. & Thornton, J. M. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8, 275–282 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Kumar, S., Stecher, G. & Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Petoukhov, M. V. et al. New developments in the ATSAS program package for small-angle scattering data analysis. J. Appl. Cryst. 45, 342–350 (2012).

    CAS  Article  Google Scholar 

  42. 42.

    Svergun, D. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Cryst. 25, 495–503 (1992).

    CAS  Article  Google Scholar 

  43. 43.

    Svergun, D., Barberato, C. & Koch, M. H. J. CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Cryst. 28, 768–773 (1995).

    CAS  Article  Google Scholar 

  44. 44.

    Kozin, M. B. & Svergun, D. I. Automated matching of high- and low-resolution structural models. J. Appl. Cryst. 34, 33–41 (2001).

    CAS  Article  Google Scholar 

  45. 45.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. 48.

    Gipson, B., Zeng, X., Zhang, Z. Y. & Stahlberg, H. 2dx—user-friendly image processing for 2D crystals. J. Struct. Biol. 157, 64–72 (2007).

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Leppla, S. H. Production and purification of anthrax toxin. Methods Enzymol. 165, 103–116 (1988).

    CAS  PubMed  Article  Google Scholar 

  50. 50.

    Zwietering, M. H., de Koos, J. T., Hasenack, B. E., de Witt, J. C. & van’t Riet, K. Modeling of bacterial growth as a function of temperature. Appl. Environ. Microbiol. 57, 1094–1101 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank P. Wattiau (CODA–CERVA Brussels) for providing the B. anthracis 34F2 strain as well as P. Goossens (Institut Pasteur, Paris) for providing the S-layer knockout mutants RBA91 and SM91; P. Borghgraef for assistance with SEM acquisition; BEI Resources, NIAID, NIH for providing us with anti-PA monoclonal antibodies; A. E. Pirro Lundqvist for assistance with selection and identification of Nbs; R. K. Singh for assistance with SAXS data analysis; and the beamline staff at I03, Diamond Light Source, UK, for support with the data collection under proposal MX12718-10. This research was supported by VIB and FWO Flanders through project grant number G028113N.

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Contributions

A.F. and H.R. conceived the project and wrote the manuscript; H.D.G. performed gene assembly; A.F. performed cloning, protein production, functional and biophysical analysis, bacterial work as well as identification of Nbs; E.P. and J.S. supervised Ilama immunization and identification of Nbs; W.J. assisted in protein production; A.F. and H.R performed structural studies; S.E.V.d.V. performed and analysed TEM experiments, supervised by A.F. and H.R.; A.F. performed all microscopy experiments, with assistance of A.G. for fluorescent microscopy; F.V.H. performed mouse experiments with the assistance of A.F. and supervised by M.L. and H.R.

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Correspondence to Antonella Fioravanti or Han Remaut.

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Competing interests

A priority application on compounds used to inhibit bacterial S-layer protein assembly has been filed by VIB and Vrije Universiteit Brussel at the European Patent Office listing A.F. and H.R. as inventors. The other authors declare no competing interests.

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Supplementary Information

Supplementary Tables 1–3, legend for Supplementary Video 1, Supplementary Figs. 1–9, Supplementary Table 1 and full length blots.

Reporting Summary

Supplementary Video 1

Timelapse of B. anthracis growth in the presence or absence of NbsSAI.

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Fioravanti, A., Van Hauwermeiren, F., Van der Verren, S.E. et al. Structure of S-layer protein Sap reveals a mechanism for therapeutic intervention in anthrax. Nat Microbiol 4, 1805–1814 (2019). https://doi.org/10.1038/s41564-019-0499-1

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