Centers for Disease Control and Prevention, Atlanta, GA, United States of America, Complex Carbohydrate Research Center, University of Georgia, Athens, GA, United States of America, Indian Institute of Engineering Science and Technology, Shibpur, West Bengal, India, Robert Koch-Institute, Center for Biological Threats and Special Pathogens, Berlin, Germany
Bacillus anthracis (Ba) and human infection-associated Bacillus cereus (Bc) strains Bc G9241 and Bc 03BB87 have secondary cell wall polysaccharides (SCWPs) comprising an aminoglycosyl trisaccharide repeat: →4)-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→6)-α-d-GlcpNAc-(1→, substituted at GlcNAc residues with both α- and β-Galp. In Bc G9241 and Bc 03BB87, an additional α-Galp is attached to O-3 of ManNAc. Using NMR spectroscopy, mass spectrometry and immunochemical methods, we compared these structures to SCWPs from Bc biovar anthracis strains isolated from great apes displaying "anthrax-like" symptoms in Cameroon (Bc CA) and Côte d'Ivoire (Bc CI). The SCWPs of Bc CA/CI contained the identical HexNAc trisaccharide backbone and Gal modifications found in Ba, together with the α-Gal-(1→3) substitution observed previously at ManNAc residues only in Bc G9241/03BB87. Interestingly, the great ape derived strains displayed a unique α-Gal-(1→3)-α-Gal-(1→3) disaccharide substitution at some ManNAc residues, a modification not found in any previously examined Ba or Bc strain. Immuno-analysis with specific polyclonal anti-Ba SCWP antiserum demonstrated a reactivity hierarchy: high reactivity with SCWPs from Ba 7702 and Ba Sterne 34F2, and Bc G9241 and Bc 03BB87; intermediate reactivity with SCWPs from Bc CI/CA; and low reactivity with the SCWPs from structurally distinct Ba CDC684 (a unique strain producing an SCWP lacking all Gal substitutions) and non-infection-associated Bc ATCC10987 and Bc 14579 SCWPs. Ba-specific monoclonal antibody EAII-6G6-2-3 demonstrated a 10-20 fold reduced reactivity to Bc G9241 and Bc 03BB87 SCWPs compared to Ba 7702/34F2, and low/undetectable reactivity to SCWPs from Bc CI, Bc CA, Ba CDC684, and non-infection-associated Bc strains. Our data indicate that the HexNAc motif is conserved among infection-associated Ba and Bc isolates (regardless of human or great ape origin), and that the number, positions and structures of Gal substitutions confer unique antigenic properties. The conservation of this structural motif could open a new diagnostic route in detection of pathogenic Bc strains.
table 2, fig.2, B. cereus G9241
13C NMR, 1H NMR, methylation, NMR-2D, GC-MS, sugar analysis, biological assays, immunochemical methods, HF treatment, reduction with NaBD4
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6
4,4,6,4,4,2 Ac 176.20 20.99
4,4,6,4,4,3 aDGalp 99.40 69.76 70.19 69.76 71.70 61.6
4,4,6,4,4,4 bDGalp 103.48 71.70 73.30 69.33 76.21 61.8
4,4,6,4,4 aDGlcpN 99.19 53.43 75.72 77.06 71.47 67.4
4,4,6,4,2 Ac 175.97 22.93
4,4,6,4 bDManpN 100.04 54.51 73.41 76.20 75.35 61.20
4,4,6,2 Ac 174.46 22.93
4,4,6,3 aDGalp 97.91 69.54 70.19 69.76 71.70 61.4
4,4,6 bDGlcpN 101.54 55.15 75.98 77.31 75.99 60.7
4,4,2 Ac 176.20 20.99
4,4,3 aDGalp 99.62 69.76 70.19 69.76 71.70 61.6
4,4,4 bDGalp 103.48 71.70 73.30 69.33 76.21 61.8
4,4 aDGlcpN 96.61 52.79 75.56 77.70 69.98 68.0
4,2 Ac 175.97 22.93
4,3 aDGalp 101.56 68.68 70.19 69.97 72.13 62.46
4 bDManpN 99.40 54.51 80.28 77.27 75.56 60.7
2 Ac 174.46 22.93
3 aDGalp 98.33 69.54 70.19 69.76 71.70 61.4
bDGlcpN 101.54 55.15 75.98 77.31 75.99 60.7
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6
4,4,6,4,4,2 Ac - 2.08
4,4,6,4,4,3 aDGalp 5.55 3.76 3.72 3.99 3.83 3.74-3.83
4,4,6,4,4,4 bDGalp 4.44 3.54 3.64 3.94 3.66 3.75-3.82
4,4,6,4,4 aDGlcpN 5.21 4.08 4.01 4.03 3.96 4.13-4.14
4,4,6,4,2 Ac - 2.03
4,4,6,4 bDManpN 4.90 4.50 4.05-4.10 3.67-3.73 3.51 3.82-3.91
4,4,6,2 Ac - 2.08
4,4,6,3 aDGalp 5.68 3.80 3.71 3.98 3.81 3.72-3.72
4,4,6 bDGlcpN 4.65 3.90-3.91 3.90 4.10 3.52 3.74-3.92
4,4,2 Ac - 2.08
4,4,3 aDGalp 5.52 3.76 3.71 3.98 3.86 3.74-3.83
4,4,4 bDGalp 4.44 3.54 3.64 3.94 3.66 3.75-3.82
4,4 aDGlcpN 5.79 4.15 3.99 4.07 3.86 4.14-4.14
4,2 Ac - 2.03
4,3 aDGalp 5.05 3.77 3.65 3.93 4.15-4.20 3.73-3.73
4 bDManpN 4.91 4.65 4.24 4.10 3.52 3.89-3.91
2 Ac - 2.08
3 aDGalp 5.63 3.80 3.72 3.99 3.81 3.72-3.72
bDGlcpN 4.65 3.90-3.91 3.90 4.10 3.52 3.74-3.92
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6
4,4,6,4,4,2 Ac 20.99/2.08
4,4,6,4,4,3 aDGalp 99.40/5.55 69.76/3.76 70.19/3.72 69.76/3.99 71.70/3.83 61.6/3.74-3.83
4,4,6,4,4,4 bDGalp 103.48/4.44 71.70/3.54 73.30/3.64 69.33/3.94 76.21/3.66 61.8/3.75-3.82
4,4,6,4,4 aDGlcpN 99.19/5.21 53.43/4.08 75.72/4.01 77.06/4.03 71.47/3.96 67.4/4.13-4.14
4,4,6,4,2 Ac 22.93/2.03
4,4,6,4 bDManpN 100.04/4.90 54.51/4.50 73.41/4.05-4.10 76.20/3.67-3.73 75.35/3.51 61.20/3.82-3.91
4,4,6,2 Ac 22.93/2.08
4,4,6,3 aDGalp 97.91/5.68 69.54/3.80 70.19/3.71 69.76/3.98 71.70/3.81 61.4/3.72-3.72
4,4,6 bDGlcpN 101.54/4.65 55.15/3.90-3.91 75.98/3.90 77.31/4.10 75.99/3.52 60.7/3.74-3.92
4,4,2 Ac 20.99/2.08
4,4,3 aDGalp 99.62/5.52 69.76/3.76 70.19/3.71 69.76/3.98 71.70/3.86 61.6/3.74-3.83
4,4,4 bDGalp 103.48/4.44 71.70/3.54 73.30/3.64 69.33/3.94 76.21/3.66 61.8/3.75-3.82
4,4 aDGlcpN 96.61/5.79 52.79/4.15 75.56/3.99 77.70/4.07 69.98/3.86 68.0/4.14-4.14
4,2 Ac 22.93/2.03
4,3 aDGalp 101.56/5.05 68.68/3.77 70.19/3.65 69.97/3.93 72.13/4.15-4.20 62.46/3.73-3.73
4 bDManpN 99.40/4.91 54.51/4.65 80.28/4.24 77.27/4.10 75.56/3.52 60.7/3.89-3.91
2 Ac 22.93/2.08
3 aDGalp 98.33/5.63 69.54/3.80 70.19/3.72 69.76/3.99 71.70/3.81 61.4/3.72-3.72
bDGlcpN 101.54/4.65 55.15/3.90-3.91 75.98/3.90 77.31/4.10 75.99/3.52 60.7/3.74-3.92
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