Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, →4)-β-D-ManpNAc-(1→4)-β-D-GlcpNAc-(1→6)-α-D-GlcpNAc-(1→. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-β-D-ManpNAc-(1→4)-[3-O-acetyl]-β-D-GlcpNAc-(1→6)-α-D-GlcpNH(2)-(1→.

structure, polysaccharide, cell wall, Bacillus anthracis, pyruvylation

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,6,4,6	xSPyr	176.67	102.97	25.79
4,6,4,2	Ac	176	?
4,6,4	bDManpN	101.08	54.70	70.16	75.12	68.01	65.32
4,6,2	Ac	174.30	21.72
4,6,3	Ac	175.58	23.21
4,6	bDGlcpN
4	aDGlcpN	101.30	56.15	74.12	70.68	72.6	69.3
	Subst


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,6,4,6	xSPyr	-	-	1.45
4,6,4,2	Ac	-	2.07
4,6,4	bDManpN	4.87	4.53	3.95	3.58	3.37	3.72-3.99
4,6,2	Ac	-	2.09
4,6,3	Ac	-	1.98
4,6	bDGlcpN	4.67	3.88	5.08	3.92	3.56	3.73-3.87
4	aDGlcpN	5.27	2.8	3.56	3.39	3.79	3.83-4.10
	Subst


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,6,4,6	xSPyr			25.79/1.45
4,6,4,2	Ac		?/2.07
4,6,4	bDManpN	101.08/4.87	54.70/4.53	70.16/3.95	75.12/3.58	68.01/3.37	65.32/3.72-3.99
4,6,2	Ac		21.72/2.09
4,6,3	Ac		23.21/1.98
4,6	bDGlcpN	NMR TSV error 2: unequal length of 13C and 1H datasets
4	aDGlcpN	101.30/5.27	56.15/2.8	74.12/3.56	70.68/3.39	72.6/3.79	69.3/3.83-4.10
	Subst

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

R1 = cell wall polysaccharide (ID 27153)
[1*]O[C@H]1O[C@H](CO[C@@H]2O[C@H](CO)[C@@H](O[C@@H]3O[C@@H]4CO[C@@](C)(C(=O)O)O[C@H]4[C@H](O)[C@@H]3NC(C)=O)[C@H](OC(C)=O)[C@H]2NC(C)=O)[C@@H](O)[C@H](O)[C@H]1N

696.636 g/mol (C₂₇H₄₂RN₃O₁₈, R = underdetermined substituent(s), not counted in mol weight)