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

Abstract

SummaryThe 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.

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

“…Therefore, my colleagues and I have proposed a model whereby BxpB is delivered to the spore by virtue of forming a complex with BclA and possibly, to a lesser extent, with other exosporium-targeting motif sequences (58). In keeping with this, we observed a reduction in the amount of BxpB extractable from spores of a BclA-negative strain, a finding similar to that observed by others (65,66,68). However, this model is disputed by Rodenburg et al (39), based on their analysis of B. anthracis spore structure by high-resolution electron microscopy.…”

“…An N-terminal targeting sequence of BclA and certain other collagen-like proteins is responsible for positioning these proteins at the site of the developing exosporium (57,58,(65)(66)(67). No such localization domain is present in the BxpB sequence.…”

“…Assembly begins with the incorporation of an as yet undefined set of proteins (but likely including CotY and ExsY) which provides the scaffold for assembly of the basal layer. After scaffold assembly has initiated, the assembly of the BclA-and BxpB-containing complexes occurs at this mother cell central spore pole and follows scaffold assembly around the spore (57,66). When positioning of BclA-containing complexes has occurred over approximately 75% of the spore, proteolytic cleavage of the BclA N terminus after amino acid 19 occurs, beginning at the cap end of the exosporium, and the Nterminal peptide is released into the mother cell cytoplasm (57).…”

Simple detection of Bacillus anthracis spores by precipitation method with goat antibody anti anthrosa

“…Study result previously showed that to obtain protein samples for immunoprecipitation, BclA-eGFP fusion-expressing sporulating cultures in Tiger broth at T 5 were harvested by centrifugation, and the cells lysed by bead-beating using 0.1 milli meter glass beads (Biospec Products). 28 Other studies show that there are similarities endospores surface antigen between Bacillus cereus and Bacillus anthracis. Result of analysis showe that anthrose is monosaccharide, who compiled tetrasaccharide on endospre of Bacillus anthracis.…”

“…Sporulation was essentially complete (>95%) after 72 hours. 28 Some spore proteins play roles in assembly of both the coat and exosporium. CotE of Bacillus anthracis directs assembly of at least one coat protein other than itself.…”