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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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.
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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