Construction, crystal structure and application of a recombinant protein that lacks the collagen‐like region of BclA from Bacillus anthracis spores

Abstract

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called BclA, which comprises a central collagen-like region (CLR) and a globular C-terminal domain. Here, the entire CLR coding sequence of BclA was removed, and the resulting protein (tBclA) produced in Escherichia coli. The crystallographic structure of tBclA was determined to 1.35 A resolution, and consists of an all-beta structure with a TNF-like jelly fold topology (12 beta-strands which form 2 beta-sheets of five strands each) consistent with previous studies on wild-type BclA. These globular domains are tightly packed into trimeric structures (surface shape complementarity; S (c) = 0.83), demonstrating that formation of the core structure of BclA is independent of the anchoring collagen-like region. A polyclonal antibody raised against tBclA recognized B. anthracis spores directly, and showed little cross-reactivity (<10%) with the spores of the closely related species Bacillus cereus and Bacillus thuringiensis, when compared to two other polyclonal antibodies raised against B. anthracis spore extracts and inactivated spores. The tBclA protein was used to purify a pool of specific antibodies from bovine colostrum whey samples from cows inoculated with the Sterne strain anthrax vaccine, which also showed reactivity with B. anthracis spores. Together, these results demonstrate that tBclA provides a safer and more effective way to the production and purification of antibodies with high binding affinity for B. anthracis spores. Biotechnol. Bioeng. 2008;99: 774-782. (c) 2007 Wiley Periodicals, Inc.

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

“…The number of glycine-containing triplet amino acid repeats in BclA varies among strains (17 to 91 triplet repeats), and this variation is responsible for the different lengths of the hairlike nap observed on spores of different B. anthracis strains (32,33). The 134-residue carboxy-terminal domain is organized into an all-␤ structure with a jelly roll topology (77,78). It is structurally similar (but dissimilar in sequence) to the globular domain of the human C1q complement protein, which belongs to the tumor necrosis factor-like family of proteins.…”

“…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).…”

BclA and toxin antigens augment each other to protect NMRI mice from lethal Bacillus anthracis challenge

“…Analysis of the blood of animals which have survived challenge gave the indication of a possible sterile infection as inferred from lack of PA antibodies [43]. To our knowledge the level of protection achieved in this study following immunisation with BclAD1D3 exceeds that previously reported for BclA in protein or DNA vaccine form [14,20,40,44].…”

“…The sequence for murine CIITA (Mn01492) was acquired from GeneCopoeia (Rockville, MD) and the sequence of mIPS-1 was used as given for pUNO1-mIPS1 (GenBank: NM 144888.2). Antigens used comprised of rPA83 (2208 bp) [35], LFD1PAD4 (1300 bp) [13] and BclAD1D3 (480 bp) [20]. Vaccines were prepared and applied as described previously [35].…”

“…One such component is the Bacillus collagen like protein of anthracis (BclA) which forms hair-like structures projecting from the spore surface and represents a major spore immunogen [17,18] results in a smaller peptide which is easier to incorporate into a multicomponent vaccine [19,20]. In this study we determined the immunogenicity of a CLR-deficient version of BclA called rBclAD1D3 when administered as a DNA-vaccine in two different vectors.…”