bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906404; this version posted January 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Architecture and self-assembly of the Clostridium sporogenes/botulinum spore 2 surface illustrate a general protective strategy across spore formers 3 4 Thamarai K. Janganan1,2,6, Nic Mullin1,3, Ainhoa Dafis-Sagarmendi1,2, Jason Brunt4,5, 1,2 4 1,2 1,2 5 Svetomir B. Tzokov , Sandra Stringer , Anne Moir , Roy Chaudhuri , Robert P. 6 Fagan1,2, Jamie K. Hobbs1,3 and Per A. Bullough1,2,7. 7 8 1Krebs Institute, University of Sheffield, 2Dept. of Molecular Biology and Biotechnology, 9 University of Sheffield, Sheffield S10 2TN, UK, 1, 3Dept. of Physics and Astronomy, 10 University of Sheffield, Sheffield, S3 7RH, UK, 4Quadram Institute, Norwich Research 11 Park, Norwich, NR4 7UA, UK, 5current address: Dept. of Chemical Engineering and 12 Biotechnology, University of Cambridge, Philippa Fawcett Drive, Cambridge CB3 0AS, UK 13 6current address: School of Life Sciences, Park Square, University of Bedfordshire, Luton, 14 LU1 3JU, UK. 15 16 7To whom correspondence should be addressed. E-mail: [email protected] 17 Tel. (+44) 114 2224245 18 Fax (+44) 114 2222800 19 20 21 22 23 24 25 26 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906404; this version posted January 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Abstract 2 Spores, the infectious agents of many Firmicutes, are remarkably resilient cell forms. Even 3 distant relatives have similar spore architectures incorporating protective proteinaceous 4 envelopes. We reveal in nanometer detail how the outer envelope (exosporium) in 5 Clostridium sporogenes (surrogate for C. botulinum group I), and in other Clostridial 6 relatives, forms a hexagonally symmetric molecular filter. A cysteine-rich protein, CsxA, 7 when expressed in E. coli, self-assembles into a highly thermally stable structure identical 8 to native exosporium. Like exosporium, CsxA arrays require harsh reducing conditions for 9 disassembly. We conclude that in vivo, CsxA self-organises into a highly resilient, 10 disulphide cross-linked array decorated with additional protein appendages enveloping the 11 forespore. This pattern is remarkably similar in Bacillus spores, despite lack of protein 12 homology. In both cases, intracellular disulphide formation is favoured by the high lattice 13 symmetry. We propose that cysteine-rich proteins identified in distantly related spore 14 formers may adopt a similar strategy for intracellular assembly of robust protective 15 structures. 16 17 Introduction 18 Spores formed by bacteria of the genera Clostridium and Bacillus provide a uniquely 19 effective means of surviving environmental stress (1); they act as the infectious agent in 20 pathogens such as Bacillus anthracis, Clostridium botulinum and Clostridium difficile. In 21 the anaerobic Clostridia they are essential to survival in air. Despite the early evolutionary 22 divergence of the genera Clostridium and Bacillus, the overall process of spore formation 23 is strikingly similar, and a large number of the genes responsible for regulation and 24 morphogenesis in sporulation are conserved. However, proteins making up the spore 25 outer layers are much less conserved (2). These layers include a complex protein coat, 26 and in some species, such as the pathogens B. anthracis and C. botulinum (but not B. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906404; this version posted January 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 subtilis) a distinct and deformable outermost exosporium enveloping the spore. The outer 2 protein layers confer much of the spore’s resistance to chemical and enzymatic insult (1). 3 The genetic control and the role of key morphogenetic proteins in B. subtilis spore outer 4 layer assembly are well studied (3), but far less so in other species, particularly the 5 Clostridia. 6 7 The exosporium defines the interface between the spore and its environment. Where the 8 spore acts as an infectious agent, it is the first point of contact between the spore and the 9 host. In B. anthracis it has roles in modulating spore germination, adhesion, protection, 10 (reviewed in (4)), host cell uptake (5) and immune inhibition (6). The physical and structural 11 properties of the exosporium have been best studied in the B. cereus/anthracis group, 12 where it comprises a thin, continuous and hexagonally crystalline proteinaceous layer (7) 13 (known as the basal layer) whose lattice is formed by cysteine-rich proteins ExsY and 14 CotY (8). Its external face is decorated by a hairy nap composed of BclA, which has an 15 internal collagen-like repeat (CLR) domain (9) that is associated with the basal layer 16 through the ExsFA/BxpB protein (10). 17 18 In the Clostridia much less is known, with the exception of the medically important 19 Clostridium difficile, where several proteins important in spore coat and exosporium 20 assembly have been identified (11-13). Now reclassified as Clostridioides difficile, this 21 species is rather distant from the main group of Clostridia however (14-16). Clostridium 22 botulinum has an exosporium but its composition and assembly are poorly understood. 23 This species is significant as a potential bioterror agent; its toxin is responsible for 24 botulism, a severe neuroparalytic disease that affects humans, mammals and birds (17). 25 For the highly pathogenic proteolytic strains of Group I C. botulinum, the closely related 26 Clostridium sporogenes is a useful non-pathogenic experimental surrogate (17, 18). This 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906404; this version posted January 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 makes C. sporogenes an attractive target for probing Clostridial spore structure and 2 function. C. sporogenes exosporium is morphologically similar to that of the B. cereus 3 group and has been proposed to have a hexagonally symmetric crystalline basal layer (19) 4 and a hairy nap (20), but the detailed molecular architecture of the exosporium has not 5 been explored. Proteins extracted from purified C. sporogenes exosporium (20) include, 6 amongst others, a Clostridial-specific cysteine-rich protein, CsxA, that was detected in very 7 high molecular weight material, together with a BclA-like protein; the latter is a possible 8 contributor to the hairy nap, by analogy with B. cereus. 9 10 We now reveal the three dimensional molecular structure of a Clostridial exosporium, 11 using electron crystallography and atomic force microscopy. The novel cysteine-rich CsxA 12 protein has been identified as the defining structural component of the basal layer array. 13 This provides the first detailed view of the structure of the spore envelope of C. botulinum 14 Group I strains, and as CsxA homologues are encoded more widely, it will provide insights 15 for future spore coat and exosporium research in the genus Clostridium. We show that 16 recombinant CsxA can self-assemble into crystalline arrays identical in structure to the 17 exosporium. Thus, we show that apparently unrelated cysteine-rich proteins from different 18 spore-forming species can self-assemble to form remarkably similar and robust structures. 19 We propose that diverse cysteine-rich proteins identified in the genomes of a broad range 20 of spore formers may adopt a similar strategy of molecular tiling to build up spore 21 structures. 22 23 24 25 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906404; this version posted January 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Results and Discussion 2 The exosporium of C. sporogenes, and other Clostridia, is formed from a hexagonally 3 symmetric two dimensional lattice enveloping the spore 4 Electron microscopy shows the exosporium enveloping the electron dense spore core; it 5 is generally more extended at one pole (Fig. 1A). In all electron transparent areas, the 6 exosporium appears as a thin two-dimensional crystalline layer, mostly associated with a 7 ‘hairy nap’ and various other appendages (Fig. 1B), (20). Fourier amplitudes and phases 8 were averaged from 5 high magnification images of negatively stained exosporium. Unit 9 cell parameters are a = b = 110 ± 5 Å and γ = 120 ± 3°; phases are consistent with p6 10 symmetry. The projection map (Fig. S1A) reveals a densely stained core surrounded by a 11 ring of 6 stain-excluding densities (black circle), separated by deeply stained pits (black 12 rectangle). Each ring is connected to two adjacent rings by a trimeric linker (Fig. S1A; 13 arrow). We also determined projection maps from exosporium of C. acetobutylicum, C. 14 tyrobutyricum, C. puniceum and C. pasteurianum (Fig. S2). These all display a density 15 distribution nearly identical to that of C. sporogenes (Fig. S1A). 16 17 Three-dimensional (3D) reconstruction of Clostridium sporogenes exosporium reveals a 18 semi-permeable protein lattice 19 61 images of negatively stained exosporium from intact spores were recorded and 20 processed in 10 separate tilt series of ±55°. 3D merging statistics are given in Table S1. In 21 3D (Fig. 2), the basic repeating unit is revealed as a cog-like ring with sixfold symmetry 22 linked to adjacent rings through a small threefold symmetric bridge (arrow in Fig.