Role Played by Exosporium Glycoproteins in the Surface Properties of Bacillus cereus Spores and in Their Adhesion to Stainless Steel

Abstract

Bacillus cereus spores are surrounded by a loose-fitting layer called the exosporium, whose distal part is mainly formed from glycoproteins. The role played by the exosporium glycoproteins of B. cereus ATCC 14579 (BclA and ExsH) was investigated by considering hydrophobicity and charge, as well as the properties of spore adhesion to stainless steel. The absence of BclA increased both the isoelectric point (IEP) and hydrophobicity of whole spores while simultaneously reducing the interaction between spores and stainless steel. However, neither the hydrophobicity nor the charge associated with BclA could explain the differences in the adhesion properties. Conversely, ExsH, another exosporium glycoprotein, did not play a significant role in spore surface properties. The monosaccharide analysis of B. cereus ATCC 14579 showed different glycosylation patterns on ExsH and BclA. Moreover, two specific glycosyl residues, namely, 2-O-methyl-rhamnose (2-Me-Rha) and 2,4-O-methyl-rhamnose (2,4-Me-Rha), were attached to BclA, in addition to the glycosyl residues already reported in B. anthracis.The food-borne pathogen Bacillus cereus has been extensively isolated in the form of spores from various environments, including food contact surfaces (26,33). Spores of the B. cereus group (closely related species, such as B. cereus, B. anthracis, or B. thuringiensis) are characterized by the presence of an outer layer called the exosporium, which surrounds the spore and is suspected of playing a major role in the interface phenomena. Indeed, the exosporium has been shown to play a role in spore adhesion to abiotic surfaces (14) and to professional phagocytic cells (25), in the escape of spores from macrophages (27), and in spore germination (16). As revealed by electron microscopy, the exosporium is made of an external hair-like nap on top of a paracrystalline basal layer (18). The hair-like nap is mainly composed of the collagen-like glycoprotein BclA in B. anthracis (34). The BclA protein contains three domains: the N-terminal domain (NTD) of 44 amino acids (aa) is involved in targeting and anchoring the protein in the exosporium (38); the C-terminal domain (CTD) is composed of multiple ␤-strands (28); and the collagen-like region (CLR), with a triple-helix conformation (34), is composed of GXX repeats, mostly GPT, whose threonine residues provide putative glycosylation sites (8). The filament length is proportional to the number of GXX repeats (35). The presence of BclA at the surfaces of B. anthracis spores has been shown to have an impact on the overall hydrophobicity (6) and charge (7) of the spores. Furthermore, the presence of BclA affects the germination of B. anthracis spores and is involved in specific interaction with immune cells (4, 24) and surfactants (28). Genes of other collagen-like proteins are also present in the B. anthracis genome (22, 39), as well as in other Bacillus species, such as exsH, which encodes a protein with an NTD of 145 aa with putative helical secondary structures, or exsJ (40). However, their loc...

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

“…In contrast to the results obtained with B. anthracis, a study with a strain of B. cereus (ATCC 14579) indicated an increase in spore hydrophobicity in bclA deletion mutants, and this coincided with a reduction in spore binding to stainless steel (99). However, overall spore hydrophobicity does not adequately explain the differences in stainless steel adhesion properties.…”

“…The exosporium layer and the BclA glycoprotein, in spores possessing it, contribute to the overall hydrophobicity of the spore (80,84,99). The hydrophobic character contributes to the binding properties of the spore in the environment.…”

“…Studies have attributed roles for the exosporium (114,115) or BclA (78,99) in binding to stainless steel surfaces. In contrast to the results obtained with B. anthracis, a study with a strain of B. cereus (ATCC 14579) indicated an increase in spore hydrophobicity in bclA deletion mutants, and this coincided with a reduction in spore binding to stainless steel (99).…”

“…ExsH was identified as a collagen-like spore surface glycoprotein of B. cereus and B. thuringiensis (94,98,99). B. cereus ExsH has a C-terminal BclB C-terminal domain (exospore_TM), defined as a C-terminal region in a number of proteins that have extensive collagen-like triple helix repeat regions.…”

Single cell profiling of surface carbohydrates on Bacillus cereus

“…Compared with the vegetative cells, the new synthetic protective layers on spore surfaces during sporulation may lead to the change of carbohydrate compositions when detecting surface carbohydrates using AFM force mapping. Among these newly formed surface structures of spores, the outermost exosporium, a transparent loose-fitting membrane which is mainly composed of highly glycosylated glycoprotein BclA, known to envelop spores of B. cereus [49,50]. Our estimated carbohydrate composition value (GlcNAc :Glu/Man) is in excellent agreement with a previously reported value of 1 : 3 (molar ratio) measured from the B. cereus T-strain spore (same strain as this study) exosporium [13], in which only the outer exosporium was extracted and analysed.…”