Non‐uniform assembly of the Bacillus anthracis exosporium and a bottle cap model for spore germination and outgrowth

Abstract

SummarySpores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is formed by a collagen-like glycoprotein called BclA, while the basal layer contains many different proteins, one of which is a sporespecific alanine racemase (Alr). In this study, we employed fluorescence microscopy and a fluorescently labelled anti-Alr monoclonal antibody (mAb) to examine the distribution of Alr within the exosporium. Binding of the mAb occurred over approximately three-quarters of the exosporium but not in a cap-like region at one end of the spore, indicating the absence or inaccessibility of Alr in this region. We also determined that the cap-like region, or cap, corresponds to the first part of the exosporium assembled within the mother cell during sporulation and the only part of the exosporium assembled in a DexsY mutant strain of B. anthracis. Our results provide the first direct evidence that exosporium assembly is a non-uniform process and suggest that exosporium formation is discontinuous. Finally, we demonstrated that during spore germination and outgrowth, the outgrowing cell always escapes from its exosporium shell by popping through the cap, suggesting that the cap is designed to facilitate the emergence of the outgrowing cell.

Surface architecture of endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale

“…ExsFA and BclA are clearly not essential to hold the lattice together, because exosporium from ΔexsFA and ΔbclA, mutants maintains the crystal lattice (8,15). We speculate that ExsY and CotY could be good candidates for major components of the lattice because a ΔexsY strain of B. cereus makes only a small terminal cap of exosporium and a ΔexsY ΔcotY strain is completely devoid of exosporium (17,18,31). It is possible that the main bulk of the exosporium contains ExsY whereas the cap contains CotY, which is 85% identical to ExsY (17).…”

“…5 summarizes the main conclusions of this paper, although it should be noted that in different strains the basal layer thickness may vary depending on whether the layer has a single tier or is made up of multiple layers stuck together: (i) The exosporium forms a semipermeable barrier enclosing the spore and breached by passages as narrow as ∼20 Å in diameter and small enough to exclude typical proteolytic enzymes but large enough to allow entry of spore germinants. The barrier may not be identically structured around the whole spore surface because a specialized "cap" structure directs the escape of the germinating cell enclosed within the exosporium (31). (ii) The exosporium is made up of interlinked cups opening to the external environment of the spore.…”

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

“…In a study of an exsY mutant of B. anthracis, Turnbough and coworkers discovered that the exosporium layer is not a uniform structure but is comprised of two distinct entities of differing compositions (42,56). The "bottle cap" part of the exosporium is synthesized first, originating at the mother cell central pole of the spore.…”

Biochemical characterization of Alanine racemase- a spore protein produced by Bacillus anthracis

“…The present study focused on biochemical characterization of alanine racemase, an immunodominant antigen constituting 75% of the exosporium (8). Further studies are currently in progress to evaluate the protective efficacy of antibodies generated against this protein alone and in conjunction with PA in response to B. anthracis spore challenge.…”

“…Understanding these mechanisms requires enzyme-substrate complex analysis to determine if it requires a PLP cofactor. Therefore, the present study was conducted to characterize alanine racemase (YP_026523), a spore-specific enzyme produced by B. anthracis (8).…”