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

Nature Microbiology volume 4, pages 1805–1814 (2019)Cite this article

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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.

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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.

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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.

Author information

Author notes

  1. Filip Van Hauwermeiren & Mohamed Lamkanfi

    Present address: Janssen Immunosciences, Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium

Authors and Affiliations

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

    Antonella Fioravanti, Sander E. Van der Verren, Wim Jonckheere, Henri De Greve & Han Remaut

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

    Antonella Fioravanti, Sander E. Van der Verren, Wim Jonckheere, Els Pardon, Jan Steyaert, Henri De Greve & Han Remaut

  3. Center for Inflammation Research, Vlaams Instituut voor Biotechnologie, Ghent, Belgium

    Filip Van Hauwermeiren & Mohamed Lamkanfi

  4. Department of Internal Medicine, Ghent University, Ghent, Belgium

    Filip Van Hauwermeiren & Mohamed Lamkanfi

  5. VIB Bio Imaging Core, UGent-VIB, Gent, Belgium

    Amanda Goncalves

  6. Structural Biology Research Center, VIB, Brussels, Belgium

    Els Pardon & Jan Steyaert

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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.

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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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