The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

Abstract

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...

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

“…Steichen et al (56) demonstrated that during spore germination and outgrowth, the emerging vegetative cell escapes from its exosporium shell through the displaced cap, suggesting that the cap is designed to pop off the exosporium to permit the emergence of the outgrowing cell. Mutants of B. anthracis lacking the BclB collagen-like protein have an exosporium that exhibits damage and loss of the exosporium at one pole (59). Furthermore, the distribution of exosporium proteins, such as CotY and BxpB, is altered in this mutant, suggesting that BclB is required for correct exosporium assembly.…”

“…BclB assembly into spores is prevented in bxpB-null mutant spores, suggesting that the BxpB basal layer protein is directly or indirectly required for BclB assembly. Spores from a ⌬bclB mutant strain exhibited ruptures of the exosporium layer, usually at one pole of the elliptical spore (59). As a consequence of this exosporium instability, a population of exosporium-free spores is present in a sample of bclB-null spores.…”

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

“…Protein samples for Western blots were collected in two ways. For samples isolated from synchronous cultures at set time points, 1 ml of sporulating culture was harvested by centrifugation and resuspended in 100 l of 8 M urea-containing SDS sample buffer (38,41) and immediately boiled. For blots containing extracts obtained from purified spores, 10 7 spores were harvested by centrifugation (10,000 ϫ g) and the pellets resuspended in 100 l SDS sample buffer with 8 M urea and boiled for 5 min.…”

“…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).…”

“…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).…”