Brian M. Thompson

Targeting of the BclA and BclB proteins to the Bacillus anthracis spore surface

The exosporium is the outermost layer of the Bacillus anthracis spore. The predominant protein on the exosporium surface is BclA, a collagen‐like glycoprotein. BclA is incorporated on the spore surface late in the B. anthracis sporulation pathway. A second collagen‐like protein, BclB, has been shown to be surface‐exposed on B. anthracis spores. We have identified sequences near the N‐terminus of the BclA and BclB glycoproteins responsible for the incorporation of these proteins into the exosporium layer of the spore and used these targeting domains to incorporate reporter fluorescent proteins onto the spore surface. The BclA and BclB proteins are expressed in the mother cell cytoplasm and become spore‐associated in a two‐step process involving first association of the protein with the spore surface followed by attachment of the protein in a process that involves a proteolytic cleavage event. Protein domains associated with each of these events have been identified. This novel targeting system can be exploited to incorporate foreign proteins into the exosporium of inactivated, spores resulting in the surface display of recombinant immunogens for use as a potential vaccine delivery system.

The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

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 DeltaSterne 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.

The co-dependence of BxpB/ExsFA and BclA for proper incorporation into the exosporium of Bacillus anthracis

The outermost layer of the Bacillus anthracis spore consists of an exosporium comprised of two distinct layers, an outer hair‐like nap layer and an internal basal layer. The hair‐like nap is primarily comprised of the glycosylated collagen‐like protein BclA. BclA is found in a trimeric form in close association with many other exosporium proteins in high‐molecular weight complexes. We previously had characterized an N‐terminal sequence of BclA that is sufficient for incorporation into the exosporium. Here we utilized site‐directed mutagenesis to identify BclA residues critical to two steps in this process, positioning of the protein at the site of the developing exosporium basal layer and stable incorporation which includes a proteolytic cleavage of BclA after residue 19. The BxpB (ExsFA) protein is known to be important for proper incorporation of BclA onto the exosporium. BxpB and BclA were found to be expressed at the same time in sporulating cells of B. anthracis and immediately colocalize to high‐molecular weight complexes. The BxpB protein was found to be in close proximity to the BclA NTD. BxpB and BclA are co‐dependent for exosporium incorporation, with the BclA NTD being sufficient to deliver BxpB to the exosporium.

Current Physical and SDS Extraction Methods Do Not Efficiently Remove Exosporium Proteins from Bacillus anthracis spores

Biochemical studies of the outermost spore layers of the Bacillus cereus family are hindered by difficulties in efficient dispersal of the external spore layers and difficulties in dissociating protein complexes that comprise the exosporium layer. Detergent and physical methods have been utilized to disrupt the exosporium layer. Herein we compare commonly used SDS extraction buffers used to extract spore proteins and demonstrate the incomplete extractability of the exosporium layer by these methods. Sonication and bead beating methods for exosporium layer removal were also examined. A combination of genetic and physical methods is the most effective for isolating proteins found in the spore exosporium.

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

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 anti-BetA 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.

Assembly of the BclB glycoprotein into the exosporium and evidence for its role in the formation of the exosporium “cap” structure in Bacillus anthracis

The outermost layer of the Bacillus anthracis spore consists of an exosporium comprised of an outer hair‐like nap layer and an internal basal layer. A major component of the hair‐like nap is the glycosylated collagen‐like protein BclA. A second collagen‐like protein, BclB, is also present in the exosporium. BclB possesses an N‐terminal sequence that targets it to the exosporium and is similar in sequence to a cognate targeting region in BclA. BclB lacks, however, sequence similarity to the region of BclA thought to mediate attachment to the basal layer via covalent interactions with the basal layer protein BxpB. Here we demonstrate that BxpB is critical for correct localization of BclB during spore formation and that the N‐terminal domains of the BclA and BclB proteins compete for BxpB‐controlled assembly sites. We found that BclB is located principally in a region of the exosporium that excludes a short arc on one side of the exosporium (the so‐called bottle‐cap region). We also found that in bclB mutant spores, the distribution of exosporium proteins CotY and BxpB is altered, suggesting that BclB has roles in exosporium assembly. In bclB mutant spores, the distance between the exosporium and the coat, the interspace, is reduced.

A genetic approach for the identification of exosporium assembly determinants of Bacillus anthracis.

The exosporium is the outermost layer of spores of the zoonotic pathogen Bacillus anthracis. The composition of the exosporium and its functions are only partly understood. Because this outer spore layer is refractive to traditional biochemical analysis, a genetic approach is needed in order to define the proteins which comprise this important spore layer and its assembly pathway. We have created a novel genetic screening system for the identification and isolation of mutants with defects in exosporium assembly during B. anthracis spore maturation. The system is based on the targeting sequence of the BclA exosporium nap layer glycoprotein and a fluorescent reporter. By utilizing this screening system and gene inactivation with Tn916, several novel putative exosporium-associated determinants were identified. A sampling of the mutants obtained was further characterized, confirming their exosporium defect and validating the utility of this screen to identify novel spore determinants in the genome of this pathogen.