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Abstract
The glycoprotein BclA is an important constituent of the exosporium of Bacillusanthracis spores. This glycoprotein is substituted with an oligosaccharide composed ofa-L-rhamnoside substituted with the previously unknown terminal saccharide, 2-Omethyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-D-glucopyranose, also referred to as anthrose. Anthrose has not been found in spores of B. Cereus and B. thuringiensis, making it a potential species-specific marker for B. anthracis. In order to study the antigenicity of anthrose, efficient syntheses of an anthrose-containing trisaccharide and a series of structurally related analogues were developed. The analogues lacked either the methyl ether at C-2 or contaminated modified C-4 amino functionalities of anthrose. The synthetic compounds were equipped with an aminopropyl spacer to facilitate conjugation to the carrier proteins mariculture Keyhole Limpet Hemocyanin (mcKLH) and bovine serum albumin (BSA). Serum antibodies of rabbits immunized with live or irradiated spores of B. anthracis Sterne 34F2 were able to recognize the synthetic trisaccharide-mcKLH conjugate. The specificity of the interaction was confirmed by competitive inhibition with the free and BSA-conjugated trisaccharides. Inhibition using the trisaccharide analogues demonstrated that the isovaleric acid moiety of anthrose is an important structural motif for antibodyrecognition. These data demonstrate that 1) anthrose is a specific antigenic determinantof the B. antrhacis Sterne spore; 2) this antigen is presented to the immune system ofrabbits receiving the anthrax live-spore vaccine; 3) synthetic analogues of theoligosaccharide retain the antigenic structure; and 4) the antigenic region is localized tospecific terminal groups of the oligosaccharide. Collectively these data provide animportant proof-of-concept step in the synthesis and development of spore-specificreagents for detection and targeting of non-protein structures in B. anthracis.