Glycosylation of BclA Glycoprotein from Bacillus cereus and Bacillus anthracis Exosporium Is Domain-specific

Abstract

The spores of the Bacillus cereus group (B. cereus, Bacillus anthracis, and Bacillus thuringiensis) are surrounded by a paracrystalline flexible yet resistant layer called exosporium that plays a major role in spore adhesion and virulence. The major constituent of its hairlike surface, the trimerized glycoprotein BclA, is attached to the basal layer through an N-terminal domain. It is then followed by a repetitive collagen-like neck bearing a globular head (C-terminal domain) that promotes glycoprotein trimerization. The collagen-like region of B. anthracis is known to be densely substituted by unusual O-glycans that may be used for developing species-specific diagnostics of B. anthracis spores and thus targeted therapeutic interventions. In the present study, we have explored the species and domain specificity of BclA glycosylation within the B. cereus group. First, we have established that the collagen-like regions of both B. anthracis and B. cereus are similarly substituted by short O-glycans that bear the species-specific deoxyhexose residues anthrose and the newly observed cereose, respectively. Second we have discovered that the C-terminal globular domains of BclA from both species are substituted by polysaccharide-like O-linked glycans whose structures are also species-specific. The presence of large carbohydrate polymers covering the surface of Bacillus spores may have a profound impact on the way that spores regulate their interactions with biotic and abiotic surfaces and represents potential new diagnostic targets.All Bacillus spores share a common architecture that consists of a set of concentric layers with a nucleotide-containing inner core surrounded by a peptidoglycan cortex and a spore coat. Species may differ according to the presence or not of an additional loosely fitting envelope enclosing individual spores. Indeed, mature spores from the Bacillus cereus group, which includes B. cereus, Bacillus anthracis, and Bacillus thuringiensis, are surrounded by a protein-rich, flexible envelope called the exosporium, whereas other species such as Bacillus subtilis and Bacillus licheniformis are not (1-3). Exosporia appear as a semipermeable barrier and are thought to exhibit a wide range of functions, including resistance to chemical and enzymatic treatments (4), enhancement of spore adhesion to biotic (5) and abiotic surfaces (6, 7), and germination (8). Some of the properties of the exosporium may also exert an influence on the infection process of pathogenic B. anthracis strains by protecting spores from macrophage-induced degradation (4, 9) and by targeting them toward phagocytic cells (5). As revealed by electron microscopy, the exosporium is made up of an external hairlike nap that sits on top of a paracrystalline basal layer (10, 11). The supporting basal layer of B. anthracis was shown to contain about 20 different proteins (12, 13) that form hexagonal subunits to which the hairlike structures are attached (11,14,15). Filaments are mostly composed of trimers of the major BclA glycoprotein (for ...

Contributions of crust proteins to spore surface properties in Bacillus subtilis

“…The discoveries of the sugars anthrose and cereose on the surface of B. anthracis and B. cereus spores, respectively, implies that the carbohydrate profiles of spores can be highly species‐specific (Daubenspeck et al , ; Maes et al , ; Li et al , ). These properties are especially important for understanding pathogenic spore forming bacteria and could significantly influence strategies deployed for their elimination.…”

Galactosylation of the Secondary Cell Wall Polysaccharide of Bacillus anthracis and Its Contribution to Anthrax Pathogenesis

“…Similar to UDP-GlcNAc 2-epimerase, the genome of B. anthracis also includes two genes, galE1 and galE2, for UDP-Glc 4-epimerase, an enzyme that catalyzes the reversible interconversion of UDP-Glc and UDP-Gal (19). One of these two genes, galE2, is expressed only during B. anthracis sporulation, providing UDP-Gal and UDP-GalNAc substrates for the glycosylation of BclA, an exosporium protein in the outer spore nap that is produced by all members of the B. cereus sensu lato group (21,(43)(44)(45). In contrast, galE1 is only expressed during vegetative growth and provides UDP-Gal for the galactosylation of the SCWP in B. anthracis.…”

“…B. anthracis strain Sterne 34F2 lacking galE2, a gene that is only expressed during sporulation, cannot synthesize GalNAc (19). Unlike the spores of wild-type B. anthracis, spores of galE2 mutant bacilli elaborate BclA (Bacillus collagen-like protein of anthracis), the main glycoprotein of the exosporium, harboring GlcNAc, not GalNAc, as the anchoring moiety for two different oligosaccharides, 3-O-Me-Rha(␣1-2)Rha(␣1-3)GalNAc and Ant(␤1-3)Rha(␣1-3)Rha(␣1-2)Rha(␣1-3)GalNAc (19)(20)(21)(22).…”

Structure of the Bacillus anthracis dTDP-L-rhamnose-biosynthetic enzyme dTDP-4-dehydrorhamnose 3,5-epimerase (RfbC)

“…In B. anthracis, l-rhamnose is not found in the cell wall of the vegetative cells, but is a major component of the spore exosporium layer (Fox et al, 2003). In the closely related Bacillus cereus, l-rhamnose is the second most abundant sugar, constituting 6.4% of the exosporium (Daubenspeck et al, 2004;Stewart, 2015;Maes et al, 2016;Matz et al, 1970). l-Rhamnose-deficient strains of B. anthracis exhibit reduced host adherence, but this deficiency does not translate into reduced virulence (Chitlaru et al, 2011;Bozue et al, 2005).…”