Whole-cell high-resolution magic angle spinning (HR-MAS) NMR was employed to survey the surface polysaccharides of a group of clinical and environmental isolates of Clostridium difficile. Results indicated that a highly conserved surface polysaccharide profile among all strains studied. Multiple additional peaks in the anomeric region were also observed which prompted further investigation. Structural characterization of the isolated surface polysaccharides from two strains confirmed the presence of the conserved water soluble polysaccharide originally described by Ganeshapillai et al. which was composed of a hexaglycosyl phosphate repeat consisting of [→6)-β-D-Glcp-(1-3)-β-D-GalpNAc-(1-4)-α-D-Glcp-(1-4)-[β-D-Glcp(1-3]-β-D-GalpNAc-(1-3)-α-D-Manp-(1-P→]. In addition, analysis of phenol soluble polysaccharides revealed a similarly conserved lipoteichoic acid (LTA) which could be detected on whole cells by HR-MAS NMR. Conventional NMR and mass spectrometry analysis indicated that the structure of this LTA consisted of the repeat unit [→6)-α-D-GlcpNAc-(1-3)-[→P-6]-α-D-GlcpNAc-(1-2)-D-GroA] where GroA is glyceric acid. The repeating units were linked by a phosphodiester bridge between C-6 of the two GlcNAc residues (6-P-6). A minor component consisted of GlcpN-(1-3) instead of GlcpNAc-(1-3) in the repeat unit. Through a 6-6 phosphodiester bridge this polymer was linked to →6)-β-D-Glcp-(1-6)-β-D-Glcp-(1-6)-β-D-Glcp-(1-1)-Gro, with glycerol (Gro) substituted by fatty acids. This is the first report of the utility of HR-MAS NMR in the examination of surface carbohydrates of Gram positive bacteria and identification of a novel LTA structure from Clostridium difficile.

capsular polysaccharide, lipoteichoic acid, Clostridium difficile, lipocarbohydrate, High-resolution magic angle spinning (HRMAS) NMR

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
1,6,6,6,0,6,0,1	lDGroA	175.1	77.2	63.7
1,6,6,6,0,6,0,2	Ac	175.3	23.3
1,6,6,6,0,6,0,3,2	Ac	175.3	23.3
1,6,6,6,0,6,0,3	aDGlcpN	99.0	54.7	71.7	70.1	72.5	61.2
1,6,6,6,0,6,0	aDGlcpN	97.6	53.1	78.0	71.3	72.5	64.9
1,6,6,6,0,6	P
1,6,6,6,0	Subst
1,6,6,6	P
1,6,6	bDGlcp	104.1	74.2	76.6	70.5	75.7	65.0
1,6	bDGlcp	104.1	74.2	76.6	70.4	76.0	69.9
1	bDGlcp	103.7	74.2	76.6	70.6	76.0	69.9
	xDGro	72.0	?	63.4


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
1,6,6,6,0,6,0,1	lDGroA	-	4.41	3.92-3.96
1,6,6,6,0,6,0,2	Ac	-	2.08
1,6,6,6,0,6,0,3,2	Ac	-	2.08
1,6,6,6,0,6,0,3	aDGlcpN	5.35	3.91	3.66	3.63	3.71	3.84-3.84
1,6,6,6,0,6,0	aDGlcpN	4.98	4.10	3.95	3.83	3.90	4.12-4.17
1,6,6,6,0,6	P
1,6,6,6,0	Subst
1,6,6,6	P
1,6,6	bDGlcp	4.52	3.34	3.50	3.44	3.58	4.08-4.19
1,6	bDGlcp	4.52	3.32	3.50	3.50	3.64	3.86-4.21
1	bDGlcp	4.49	3.32	3.50	3.46	3.64	3.86-4.21
	xDGro	3.77-3.92	?	3.61-3.68


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
1,6,6,6,0,6,0,1	lDGroA		77.2/4.41	63.7/3.92-3.96
1,6,6,6,0,6,0,2	Ac		23.3/2.08
1,6,6,6,0,6,0,3,2	Ac		23.3/2.08
1,6,6,6,0,6,0,3	aDGlcpN	99.0/5.35	54.7/3.91	71.7/3.66	70.1/3.63	72.5/3.71	61.2/3.84-3.84
1,6,6,6,0,6,0	aDGlcpN	97.6/4.98	53.1/4.10	78.0/3.95	71.3/3.83	72.5/3.90	64.9/4.12-4.17
1,6,6,6,0,6	P
1,6,6,6,0	Subst
1,6,6,6	P
1,6,6	bDGlcp	104.1/4.52	74.2/3.34	76.6/3.50	70.5/3.44	75.7/3.58	65.0/4.08-4.19
1,6	bDGlcp	104.1/4.52	74.2/3.32	76.6/3.50	70.4/3.50	76.0/3.64	69.9/3.86-4.21
1	bDGlcp	103.7/4.49	74.2/3.32	76.6/3.50	70.6/3.46	76.0/3.64	69.9/3.86-4.21
	xDGro	72.0/3.77-3.92	?/?	63.4/3.61-3.68

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

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

R1 = repeating unit LTA (ID 28606, ID 28607)
CC(=O)N[C@H]1[C@@H](O[C@H]2[C@H](O)[C@@H](COP(=O)(O)O[1*]OP(=O)(O)OC[C@H]3O[C@@H](OC[C@H]4O[C@@H](OC[C@H]5O[C@@H](OC[C@H](O)CO)[C@H](O)[C@@H](O)[C@@H]5O)[C@H](O)[C@@H](O)[C@@H]4O)[C@H](O)[C@@H](O)[C@@H]3O)O[C@H](O[C@H](CO)C(=O)O)[C@@H]2NC(C)=O)O[C@H](CO)[C@@H](O)[C@@H]1O

1248.924 g/mol (C₄₀H₇₀RN₂O₃₈P₂, R = underdetermined substituent(s), not counted in mol weight)