Synthesis and antigenic analysis of the BclA glycoprotein oligosaccharide from the Bacillus anthracis exosporium.
The glycoprotein BclA is an important constituent of the exosporium of Bacillus anthracis spores. This glycoprotein is substituted with an oligosaccharide composed of a beta-L-rhamnoside substituted with the previously unknown terminal saccharide, 2-O-methyl-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 contained 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 34F(2) 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 antibody recognition. These data demonstrate that 1) anthrose is a specific antigenic determinant of the B. anthracis Sterne spore; 2) this antigen is presented to the immune system of rabbits receiving the anthrax live-spore vaccine; 3) synthetic analogues of the oligosaccharide retain the antigenic structure; and 4) the antigenic region is localized to specific terminal groups of the oligosaccharide. Collectively these data provide an important proof-of-concept step in the synthesis and development of spore-specific reagents for detection and targeting of non-protein structures in B. anthracis.
Mehta
Alok S
AS
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA.
Saile
Elke
E
Zhong
Wei
W
Buskas
Therese
T
Carlson
Russell
R
Kannenberg
Elmar
E
Reed
Yvonne
Y
Quinn
Conrad P
CP
Boons
Geert-Jan
GJ
D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D001408:Bacillus anthracis / Q000737:chemistry*; D002240:Carbohydrate Sequence; D005260:Female; D008562:Membrane Glycoproteins / Q000138:chemical synthesis* / Q000276:immunology*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000942:Antigens, Bacterial; C470593:BclA protein, Bacillus anthracis; D008562:Membrane Glycoproteins