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Surface Architecture of Endospores of the Bacillus Cereus/Anthracis/Thuringiensis Family at the Subnanometer Scale

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Surface Architecture of Endospores of the Bacillus Cereus/Anthracis/Thuringiensis Family at the Subnanometer Scale Surface architecture of endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale Lekshmi Kailasa, Cassandra Terrya,1, Nicholas Abbotta, Robert Taylora, Nic Mullinb, Svetomir B. Tzokova, Sarah J. Todda, B. A. Wallacec, Jamie K. Hobbsb, Anne Moira, and Per A. Bullougha,2 aKrebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom; bDepartment of Physics and Astronomy, University of Sheffield, Sheffield S3 7RH, United Kingdom; and cDepartment of Crystallography, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom Edited by Richard M. Losick, Harvard University, Cambridge, MA, and approved August 5, 2011 (received for review June 14, 2011) Bacteria of the Bacillus cereus family form highly resistant spores, carbohydrate (9). Proteins of the exosporium are likely to have which in the case of the pathogen B. anthracis act as the agents of intimate interactions with the environment and are also potential infection. The outermost layer, the exosporium, enveloping spores candidates for vaccines and ligands for spore detection (10, 11). of the B. cereus family as well as a number of Clostridia, plays roles in Two exosporium glycoproteins, BclA and BclB, have been spore adhesion, dissemination, targeting, and germination control. identified in B. anthracis (8, 12, 13). These contain a central tri- We have analyzed two naturally crystalline layers associated with meric collagen-like domain, a processed N-terminal domain an- the exosporium, one representing the “basal” layer to which the choring the protein to the basal layer, and a C-terminal head outermost spore layer (“hairy nap”) is attached, and the other likely domain whose structure has been elucidated to atomic resolution representing a subsurface (“parasporal”) layer. We have used elec- in the case of BclA (14). BclA filaments are the major components tron cryomicroscopy at a resolution of 0.8–0.6 nm and circular di- of the hairy nap (8, 13). ExsFA/BxpB and its homolog ExsFB are chroism spectroscopic measurements to reveal a highly α-helical required for attachment of BclA to the basal layer (15, 16). BclA, structure for both layers. The helices are assembled into 2D arrays ExsFA, and ExsY form high-molecular weight complexes, ExsY of “cups” or “crowns.” High-resolution atomic force microscopy of being required for the complete assembly of the exosporium (17– the outermost layer showed that the open ends of these cups face 19). The exosporium is likely to contain a number of other struc- the external environment and the highly immunogenic collagen-like tural proteins (many reviewed in ref. 4) as well as loosely bound fibrils of the hairy nap (BclA) are attached to this surface. Based on proteins such as immune inhibitor A (20) and arginase (21). our findings, we present a molecular model for the spore surface and The exosporium is likely to play multiple roles in the interaction propose how this surface can act as a semipermeable barrier and of the spore with its environment. For example, in B. anthracis, a matrix for binding of molecules involved in defense, germination BclA–integrin interaction promotes spore uptake by macrophages control, and other interactions of the spore with the environment. in vitro (22). The exosporium contributes protection against macrophage-mediated damage (21), whereas enzymes in the electron crystallography | cryoelectron microscopy | two-dimensional crystal exosporium (23) moderate responses to germinants. However, although the exosporium may have a role in natural anthrax acterial endospores are uniquely resistant survival structures infections, a BclA-containing exosporium is not required for an- Bthat result from a complex differentiation process. The thrax infection in animal models (24). The adherent properties dormancy, resistance (1), and widespread dispersal of bacterial exhibited by the exosporium (25) point to a role in attachment endospores mean that they are ubiquitous in the environment. to surfaces; this has important implications for development of Spores of a few species can be very effective infectious agents, decontamination protocols. remaining dormant in the environment until the opportunity for A substantial part of the basal layer of the exosporium from infection arises, in addition to being resistant to host defenses members of the B. cereus family takes the form of a natural 2D after infection. Formation of spores involves a tightly controlled crystal (7). Ball et al. have isolated fragments of this layer (type II sequence of steps of gene expression and cellular morphological crystals) from B. cereus, B. anthracis, and B. thuringiensis, de- change. Although these steps have been well-studied (2), the scribing the 3D structure to ∼30 Å resolution (3). The basic structure of the fully assembled spore remains poorly understood architecture of the crystalline basal layer, including unit cell at the molecular scale. Coupled with previous genetic and dimensions, is identical in all three species and the following morphological studies, our recent work on electron crystallog- description applies to all of them. The layer forms a network of raphy of spore proteins suggests that spores may present an at- “crown”-like structures, which were cautiously proposed to be tractive model system for exploring the molecular mechanisms trimeric although the density showed strong indications of higher driving the formation of complex supramolecular structures (3). symmetry. The open ends of the crowns are linked together to Spores contain a relatively dehydrated cellular core that is membrane-bound and surrounded by a peptidoglycan cortex and an outer protective coat containing multiple protein layers (4–6). Author contributions: L.K., C.T., B.A.W., A.M., and P.A.B. designed research; L.K., C.T., Some species, including, but by no means limited to, pathogens N.A., R.T., N.M., S.B.T., S.J.T., B.A.W., and P.A.B. performed research; L.K., C.T., N.A., R.T., N.M., S.B.T., B.A.W., J.K.H., A.M., and P.A.B. analyzed data; and L.K., B.A.W., such as Bacillus cereus and Clostridium botulinum (food-borne A.M., and P.A.B. wrote the paper. pathogens), B. thuringiensis (an insect pathogen), and B. anthracis The authors declare no conflict of interest. (animal and human pathogen), possess a further outermost layer This article is a PNAS Direct Submission. called the “exosporium” (7), a loosely fitting shell surrounding the 1Present address: Medical Research Council Prion Unit and Department of Neurodegen- coat. The volume between the spore coat and exosporium, the erative Disease, University College London Institute of Neurology, Queen Square, Lon- “interspace,” contains proteins but has not been well-studied. In don WC1N 3BG, United Kingdom. B. cereus and the closely related B. anthracis and B. thuringiensis, 2To whom correspondence should be addressed. E-mail: p.bullough@sheffield.ac.uk. “ ” fi the exosporium is made up of a basal layer supporting a la- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mentous “hairy nap” (8) and is composed of proteins, lipid, and 1073/pnas.1109419108/-/DCSupplemental. 16014e16019 | PNAS | September 20, 2011 | vol. 108 | no. 38 www.pnas.org/cgi/doi/10.1073/pnas.1109419108 Downloaded by guest on September 24, 2021 form tunnels connecting one surface of the basal layer with the maps calculated from crystals embedded in a glucose/uranyl other (figure 5 in ref. 3). In some spores, there is evidence of formate mixture, we have been able to superimpose the 6-Å map another crystalline layer, a “parasporal” layer (type I crystals), onto a projection of the negatively stained protein assembly, located within the interspace of the spore. This layer does not whose approximate envelope is indicated by the filled gray circles appear to envelop the spore completely; it follows the same ar- (Fig. 1A). This allowed us to identify which threefold symmetry chitectural theme as the basal layer, but the crowns appear axis corresponds to the central symmetry axis of the trimeric smaller and more closely packed (3). The parasporal layer may crown. The envelope of the trimeric crown encloses a number of represent a different conformation of the same complex found in very dense circular features, which have the appearance of pro- the basal layer, and/or it may be an intermediate assembly state jected α-helices; we have circled the six densest features in an on the way to forming a mature exosporium basal layer. arbitrary cluster in magenta with their trimeric partners in blue. Our aim is to move on to the next stage in visualizing the detailed molecular architecture of the exosporium, to map Basal Layer Crystals: Projection Structure at 8 Å Resolution. Electron identified proteins onto the structure, and to understand how micrographs of exosporium fragments from B. cereus 10876 em- these various proteins determine function and architecture. A bedded in ice were recorded and 16 of them were processed. A few key questions are: (i) Which surface revealed in the B. cereus representative Fourier transform computed after unbending is family basal layer structure of Ball et al. (3) corresponds to the shown in Fig. S1B. Fourier phases were consistent with p6 plane ii group symmetry (Table S3). Table S4 shows that the mean phase spore surface exposed to the environment? ( ) Where and how is fi the hairy nap attached to this outer surface? (iii) Where are the error is signi cantly less than that expected for random phases “core” structural proteins located in the exosporium? These are (45°) to a resolution of 8 Å, and we have truncated all subsequent calculations to this resolution. Temperature factors applied to likely to be the proteins resistant to all washing regimes and fi 2 making up the crystalline lattice. (iv) Where are the more easily individual lms ranged from 546 to 1,000 Å .
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