Identification of a Second Collagen-Like Glycoprotein Produced by
            <i>Bacillus anthracis</i>
            and Demonstration of Associated Spore-Specific Sugars

Waller, Lashanda N.; Stump, Michael J.; Fox, Kevin M.; Harley, William; Fox, Alvin; Stewart, George C.; Shahgholi, Mona

doi:10.1128/jb.187.13.4592-4597.2005

Cited by 47 publications

(47 citation statements)

References 38 publications

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“…The anthrax spore contains a second collagen-like glycoprotein containing a GXT tandem repeat domain (46). Since sporespecific sugars (rhamnose, 3-O-methyl rhamnose, and galactosamine) were shown to be a component of BclA of B. anthracis (13,15,31,47) and are also found in this newly discovered protein, it was named BclB (46).…”

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The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

et al. 2007

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Anthrax is a highly fatal disease caused by the gram-positive, endospore-forming, rod-shaped bacterium Bacillus anthracis. Spores, rather than vegetative bacterial cells, are the source of anthrax infections. Spores of B. anthracis are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. Filaments of the hair-like nap are made up largely of a single collagen-like glycoprotein called BclA. A second glycoprotein, BclB, has been identified in the exosporium layer. The specific location of this glycoprotein within the exosporium layer and its role in the biology of the spore are unknown. We created a mutant strain of B. anthracis ⌬Sterne that carries a deletion of the bclB gene. The mutant was found to possess structural defects in the exosporium layer of the spore (visualized by electron microscopy, immunofluorescence, and flow cytometry) resulting in an exosporium that is more fragile than that of a wild-type spore and is easily lost. Immunofluorescence studies also indicated that the mutant strain produced spores with increased levels of the BclA glycoprotein accessible to the antibodies on the surface. The resistance properties of the mutant spores were unchanged from those of the wild-type spores. A bclB mutation did not affect spore germination or kinetics of spore survival within macrophages. BclB plays a key role in the formation and maintenance of the exosporium structure in B. anthracis.Bacillus anthracis is a gram-positive, rod-shaped bacterium that causes anthrax, principally in ruminants, and is a major concern as both a zoonotic human pathogen and an agent of bioterrorism (reviewed in reference 25). Anthrax is acquired following contact with B. anthracis spores, which are the infectious form of this organism. The exosporium of B. anthracis and the other members of the Bacillus cereus group of sporeforming bacteria is the most external protein layer enclosing the spore. It consists of an inner basal layer and outer nap region having a hair-like appearance (4,11,20,24,29). The filaments of the hair-like nap are apparently formed by a single collagen-like glycoprotein called BclA (38-42), whereas the basal layer is composed of a number of different proteins in tight and loose associations (35,39). In the past few years, the protein constituents of the exosporium have begun to be elucidated, but there have been few studies of the role of these proteins in the function of the exosporium (2,34,35,38,44).The first spore surface glycoprotein discovered in B. anthracis was referred to as BclA (for Bacillus collagen-like protein anthracis) and is the most prominent protein component of the exosporium (41,42). BclA is the immunodominant antigen located in the filaments of the nap (38). BclA contains an internal tandem repeat region (consisting primarily of GPT repeats containing most of the glycosylation sites) and N -and C-terminal regions (41). These repeats are the primary anchor point for rhamnose oligosaccharides wit...

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The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

et al. 2007

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“…Most likely, BxpB anchors BclA to the exosporium basal layer, probably through covalent interactions between the two proteins (36). The exosporium is clearly a complex of these proteins and involves additional proteins, including CotY, CotE, ExsY, ExsA, ExsK, and ExsM (1,5,11,15,24,27,28,31,43). CotY and ExsY are notable for playing a role in the early-stage assembly of the exosporium (5,23).…”

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“…BclA and associated rhamnose residues play a major role in the interactions between the spore and the host macrophages and tissues (6,7,17,26). A second collagenlike glycoprotein found in the B. anthracis exosporium is BclB (40,41,43). This protein plays an important role in exosporium assembly; bclB mutant spores have a fragile exosporium, suggesting an exosporium assembly defect (41).…”

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Localization and Assembly of the Novel Exosporium Protein BetA of Bacillus anthracis

et al. 2011

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The exosporium of Bacillus anthracis is comprised of two distinct layers: a basal layer and a hair-like nap that covers the basal layer. The hair-like nap contains the glycoproteins BclA and, most likely, BclB. BclA and BclB are directed to assemble into the exosporium by motifs in their N-terminal domains. Here, we identify a previously uncharacterized putative gene encoding this motif, which we have named betA (Bacillus exosporium-targeted protein of B. anthracis). Like bclA, betA encodes a putative collagenlike repeat region. betA is present in several genomes of exosporium-producing Bacillus species but, so far, not in any others. Using fluorescence microscopic localization of a BetA-enhanced green fluorescent protein (eGFP) fusion protein and immunofluorescence microscopy with antiBetA antibodies, we showed that BetA resides in the exosporium basal layer, likely underneath BclA. BetA assembles at the spore surface at around hour 5 of sporulation and under the control of BxpB, similar to the control of deposition of BclA. We suggest a model in which BclA and BetA are incorporated into the exosporium by a mechanism that depends on their similar N termini. These data suggest that BetA is a member of a growing family of exosporium proteins that assemble under the control of targeting sequences in their N termini.

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“…These spores have been shown to be enclosed by an exosporangial layer which is composed of a number of different proteins (13,14) and which includes an outermost hair-like nap layer. The filaments of the nap layer are primarily composed of a highly immunogenic collagenlike protein, BclA (15)(16)(17), as well as a second exosporangial protein, BclB (18). Both BclA and BclB have been well characterized and shown to be glycosylated with an O-linked pentasaccharide that contains the novel terminal sugar 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-D-glucopyranose, which is also referred to as anthrose (16).…”

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Identification and Characterization of Glycoproteins on the Spore Surface of Clostridium difficile

et al. 2014

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In this study, we identify a major spore surface protein, BclA, and provide evidence that this protein is glycosylated. Following extraction of the spore surface, solubilized proteins were separated by one-dimensional PAGE and stained with glycostain to reveal a reactive high-molecular-mass region of approximately 600 kDa. Tandem mass spectrometry analysis of in-gel digests showed this band to contain peptides corresponding to a putative exosporangial glycoprotein (BclA3) and identified a number of glycopeptides modified with multiple N-acetyl hexosamine moieties and, in some cases, capped with novel glycans. In addition, we demonstrate that the glycosyltransferase gene sgtA (gene CD3350 in strain 630 and CDR3194 in strain R20291), which is located immediately upstream of the bclA3 homolog, is involved in the glycosylation of the spore surface, and is cotranscribed with bclA3. The presence of anti-␤-O-GlcNAc-reactive material was demonstrated on the surface of spores by immunofluorescence and in surface extracts by Western blotting, although each strain produced a distinct pattern of reactivity. Reactivity of the spore surface with the anti-␤-O-GlcNAc antibody was abolished in the 630 and R20291 glycosyltransferase mutant strains, while complementation with a wild-type copy of the gene restored the ␤-O-GlcNAc reactivity. Phenotypic testing of R20291 glycosyltransferase mutant spores revealed no significant change in sensitivity to ethanol or lysozyme. However, a change in the resistance to heat of R20291 glycosyltransferase mutant spores compared to R20291 spores was observed, as was the ability to adhere to and be internalized by macrophages.

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Identification of a Second Collagen-Like Glycoprotein Produced by Bacillus anthracis and Demonstration of Associated Spore-Specific Sugars

Cited by 47 publications

References 38 publications

The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

Localization and Assembly of the Novel Exosporium Protein BetA of Bacillus anthracis

Identification and Characterization of Glycoproteins on the Spore Surface of Clostridium difficile

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