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

Abstract

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

“…The protein compositions of the cap and noncap regions of the exosporium differ. CotY is a cap-specific protein, whereas BclB, ExsY, and Alr are found in the noncap portion of the exosporium (56)(57)(58). BclA is found on the exosporium surface in both cap and noncap sites, whereas BxpB is found in larger amounts, but not exclusively, in the noncap portion of the exosporium (58).…”

“…(2) This is followed closely by outer basal layer assembly, which includes BxpB and the BclA nap layer. When the BxpB layer is then approximately 75% complete, cleavage of the BclA N terminus after amino acid 19 (arrow 3) begins at the mother cell central spore pole, and this wave of cleavage follows the assembly around the spore periphery (57).…”

“…sis of the exosporium initiates at the mother cell central spore pole, the region of the exosporium that will become the bottle cap (35,56,57,62). Exosporium assembly progresses by continued deposition of proteins at the leading edge of this nascent exosporium, terminating at the opposite spore pole.…”

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

“…Two exosporium glycoproteins, BclA and BclB, have been identified in B. anthracis (8,12,13). These contain a central trimeric collagen-like domain, a processed N-terminal domain anchoring the protein to the basal layer, and a C-terminal head domain whose structure has been elucidated to atomic resolution in the case of BclA (14).…”

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

“…12 Various ways have been conducted to identify the spores of Bacillus anthracis, among others, with poly chain reaction (PCR), 2,14,15,16 spectrometry, flow cytometry and transmission electron microscopy. 17,18 Methods developed to detect anthrax spores contained in an envelope that is by engineering mikrospectroscopy. 19 Amperometric technique was also used for detecting Bacillus anthracis.…”