anthracisstrains (23). SM2. We also demonstrate that cleavage of the BclA NTD is necessary for efficient attachment to the basal layer and that the site of cleavage is usually somewhat flexible, at least in certain mutant NTDs. Finally, we propose a mechanism for BclA attachment and discuss the possibility that analogous mechanisms are involved in the attachment of many different collagen-like proteins ofB. anthracisand closely relatedBacillusspecies. Bacillus anthracis, a Gram-positive, rod-shaped, aerobic bacterium, is the causative agent of anthrax (17). When vegetative cells ofB. anthracisare starved for certain essential nutrients, they form dormant spores that can survive in harsh soil environments for many years (12,19). Spore formation starts with asymmetric septation that divides the starved vegetative cell into two genome-containing compartments, a mother cell compartment and a smaller forespore compartment. The mother cell then engulfs the forespore and surrounds it with three protective layers: a cortex composed of peptidoglycan, a closely apposed proteinaceous coat, and a loosely fitting exosporium (11). After a spore maturation stage, the mother cell lyses and releases the mature spore. IRL-2500 When spores encounter an aqueous environment made up of nutrients, they can germinate and grow as vegetative cells (18). Anthrax is typically caused by contact with spores (17). The outermost layer ofB. anthracisspores, the exosporium, has been studied intensively in recent years because it is usually both the first point of contact with the immune system of an infected host and the target of new IRL-2500 detectors for brokers of bioterrorism (21,28,32). The exosporium ofB. anthracisand closely related pathogenic species, such asBacillus cereusandBacillus thuringiensis, is usually a prominent structure consisting of a paracrystalline basal layer and an external hair-like nap (1,9). The filaments of the nap are formed by trimers of the collagen-like glycoprotein BclA (2,29). Recent studies suggest that BclA plays a major role in pathogenesis by directing spores to professional phagocytic cells, a critical step in disease progression (4,21). The basal layer is composed of approximately 20 different proteins (23,25,26), several of which have been shown to Rabbit Polyclonal to TAF1 play key roles in exosporium assembly (3,13,27). One of these proteins is usually IRL-2500 BxpB (also called ExsFA) (25,30,34), which is required for the attachment of approximately 98% of spore-bound BclA to the basal layer (26,30). Residual BclA attachment requires the basal layer protein ExsFB, a paralog of BxpB (30). BclA contains three distinct domains: a IRL-2500 38-residue amino-terminal domain name (NTD), a central collagen-like region made up of a strain-specific number of XXG (mostly PTG) repeats, and a 134-residue carboxyl-terminal domain name (CTD) (25,29,31). The CTD apparently functions as the major nucleation site for trimerization of BclA (24), and CTD trimers form the globular distal ends of the filaments in the nap (2). The highly extended collagen-like region is extensively glycosylated (5), and its length determines the depth of the nap (2,31). The NTD is the site of attachment of BclA to the basal layer, and deletion of the NTD prevents this attachment (2). The NTD is normally proteolytically processed to remove the first 19 amino acids, and it is this mature form of BclA that is attached to the basal layer (25,29). In an earlier report, we suggested that NTD processing of BclA is required for basal-layer attachment, perhaps through a direct covalent linkage to BxpB (26). Recently, Thompson and Stewart identified conserved 11-residue sequences in the NTDs of BclA and the minorB. anthraciscollagen-like glycoprotein BclB and showed that these sequences are IRL-2500 involved in the incorporation of BclA and BclB into the exosporium. These investigators used a truncated BclA NTD that lacked residues 2 through 19 but included the conserved 11-amino-acid sequence to target enhanced green fluorescent protein (EGFP) to the surface of the developing forespore (33). Thompson and Stewart also reported that cleavage of the BclA NTD occurred after its association with the forespore and suggested that this cleavage was involved indirectly in the attachment process. Actual cleavage sites were not determined in these studies, however. We have performed related studies of the attachment of BclA to the exosporium that provide a more detailed and somewhat different view of this process. In our studies, which are reported here, we identified short segments, or submotifs, of the BclA NTD that can be arranged in different combinations to produce 10-amino-acid motifs sufficient for tight attachment of BclA, and probably most proteins, to the exosporium basal layer. Additionally, we present direct evidence showing that BclA NTD cleavage is required for efficient attachment to the basal layer and that selection of the cleavage site can be somewhat flexible. Finally, we discuss a.