Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains

Thamarai K Janganan,Nic Mullin,Svetomir B Tzokov,Sandra Stringer,Robert P Fagan,Jamie K Hobbs,Anne Moir,Per A Bullough

doi:10.1016/j.fm.2016.06.003

Thamarai K Janganan, Nic Mullin + Show 6 more

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https://doi.org/10.1016/j.fm.2016.06.003

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Abstract

Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75–100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores.

Highlights

Endospores are produced by Bacillus and Clostridium spp; their extreme resistance properties contribute to their persistence and dissemination in the environment
C. sporogenes NCIMB 701792 (NCDO 1792), the subject of this study, has been included in a microarray study of genome relatedness within C. botulinum Group I and C. sporogenes strains (Carter and Peck, 2015), and we have found the exosporium of this C. sporogenes strain amenable to proteomic and structural analysis
C. sporogenes spore surface features revealed by Electron microscopy (EM)

Summary

Introduction

Endospores are produced by Bacillus and Clostridium spp; their extreme resistance properties contribute to their persistence and dissemination in the environment. The only exosporium layer studied in detail is that of Bacillus anthracis spores and the spores of the closely related Bacillus cereus and Bacillus thuringiensis, where it contains an outer hairy nap and a paracrystalline basal layer with hexagonal arrays (Stewart, 2015). The BclA glycoprotein of B. anthracis, which contains a collagen-like repeat (CLR) domain, is the major contributor to the hairy nap layer of the exosporium (Sylvestre et al, 2003). This protein is likely to be involved in adherence and entry into the host (Xue et al, 2011) and influences spore surface properties (Lequette et al, 2011). The cysteine-rich ExsY protein is essential for the formation of exosporium (Boydston et al, 2006; Johnson et al, 2006)

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