Identification of the Immunodominant Protein and Other Proteins of theBacillus anthracisExosporium

Abstract

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose-fitting, balloon-like layer called the exosporium. Although the exosporium serves as the source of surface antigens and a primary permeability barrier of the spore, its molecular structure and function are not well characterized. In this study, we identified five major proteins in purified B. anthracis (Sterne strain) exosporia. One protein was the recently identified collagen-like glycoprotein BclA, which appears to be a structural component of the exosporium hair-like nap. Using a large panel of unique antispore monoclonal antibodies, we demonstrated that BclA is the immunodominant antigen on the B. anthracis spore surface. We also showed that the BclA protein and not a carbohydrate constituent directs the dominant immune response. In addition, the length of the central (GXX) n repeat region of BclA appears to be strain specific. Two other unique proteins, BxpA and BxpB, were identified. BxpA is unusually rich in Gln and Pro residues and contains several different tandem repeats, which also exhibit strain-specific variation. In addition, BxpA was found to be cleaved approximately in half. BxpB appears to be glycosylated or associated with glycosylated material and is encoded by a gene that (along with bclA) may be part of an exosporium genomic island. The other two proteins identified were alanine racemase and superoxide dismutase, both of which were reported to be associated with the surface of other Bacillus spores. Possible functions of the newly identified proteins are discussed.The genus Bacillus includes a diverse collection of grampositive, rod-shaped, aerobic bacteria that form an endospore (or spore) upon deprivation of an essential nutrient (10, 30). In this process, an asymmetric septation of the starved vegetative cell produces a large and a small genome-containing compartment called the mother cell and forespore, respectively. The mother cell then engulfs the forespore, thereby surrounding it with two opposing cell membranes. A thick layer of modified peptidoglycan called the cortex is synthesized between the two membranes, and proteins synthesized in the mother cell form multiple layers of a spore coat that covers the cortex. While the coat forms the outermost detectable layer for spores of some species (e.g., Bacillus subtilis), in others (e.g., Bacillus anthracis), the spore is enclosed by an additional layer called the exosporium, a prominent, loose-fitting, balloon-like layer also synthesized by the mother cell (12,17). After a final stage of maturation, the mother cell lyses to release the mature spore, which is dormant and capable of persisting in the soil for many years until it encounters a germination signal.Most Bacillus species are not pathogenic to humans. The most notable exception is B. anthracis, the causative agent of anthrax (25). In light of the recent use of B. anthracis spores as a terrorist weapon in the United States and the development of these spores as a weapon of mass destruct...

Structure and specificity of lamprey monoclonal antibodies

“…6). Fourteen of 212 VLR-B transfectants (6.6%) were found to secrete VLR-B antibodies against the C-terminal domain of the major exosporium protein BclA (BclA-CTD) (11,12), a major epitope recognized by primary VLR-B antibodies made in the lamprey response. We selected the eight recombinant antibodies that recognized BclA-CTD at the highest levels above background and one weakly binding clone, VLR5, for more comprehensive analysis (Fig.…”

Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model

“…BclA, being the most immunogenic structure of the exosporium, has been shown to be immunogenic in other species (Steichen et al, 2003(Steichen et al, , 1903(Steichen et al, -1910. Likewise, immunization with SLSV in other species resulted in detectable anti-BclA titres (Hahn et al, 2006, 4569-4571, ChunQiang et al, 2008.…”

The integrin Mac-1 (CR3) mediates internalization and directsBacillus anthracisspores into professional phagocytes

“…BclA forms the hair-like nap of the exosporium and is the most abundant and immunogenic protein on the spore surface (18,19). As its name implies, BclA is structurally similar to mammalian collagen (20,21).…”

“…Collagen-like proteins are rare in prokaryotic organisms, and those described seem to be located on microbial surfaces and are often involved in adherence (22,23). Because BclA forms the outermost component of the spore (18,24), we hypothesized that it would play a role in spore-host interactions. To study this possibility, the effect of the ⌬bclA mutation on cellular adherence and uptake was determined.…”