Clostridium difficile is a Gram-positive bacterium that is known to be a cause of enteric diseases in humans. It is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. Recently, large outbreaks of C. difficile-associated diarrhea have been reported internationally, and there have been reports of increases in severe disease, mortality and relapse rates. At the moment, there is no vaccine against C. difficile, and the medical prevention of C. difficile infection is mostly based on the prophylactic use of antibiotics; however, this has led to an increase in the incidence of the disease. Here, we describe the chemical structure of C. difficile cell-surface polysaccharides. The polysaccharides of three C. difficile strains were structurally analyzed; ribotype 027 (North American pulsotype 1) strain was observed to express two polysaccharides, one was composed of a branched pentaglycosyl phosphate repeating unit: [→4)-α-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap-(1→3]-α-D-Glcp-(1→2)-α-D-Glcp-(1→P] and the other was composed of a hexaglycosyl phosphate repeating unit: [→6)-β-D-Glcp-(1→3)-β-D-GalpNAc-(1→4)-α-D-Glcp-(1→4)-[β-D-Glcp-(1→]-β-D-GalpNAc-(1→3)-α-D-Manp-(1→P]. The latter polysaccharide was also observed to be produced by strains MOH900 and MOH718. The results described here represent the first literature report describing the covalent chemical structures of C. difficile cell-surface polysaccharides, of which PS-II appears to be a regular C. difficile antigen. These C. difficile teichoic-acid-like polysaccharides will be tested as immunogens in vaccine preparations in a rat and horse model.

Structural characterization, Clostridium difficile, Teichoic-acid polysaccharide

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
0,2,4,3	aLRhap	101.4	71.2	70.9	78.9	68.6	17.8
0,2,4	bDGlcp	102.4	75.2	83.0	69.0	77.1	62.2
0,2,3	aLRhap	101.9	71.1	71.0	73.0	69.4	17.5
0,2	aDGlcp	98.0	73.6	77.5	73.6	72.4	?
0	aDGlcp	93.5	77.3	72.1	70.1	73.8	?
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
0,2,4,3	aLRhap	5.17	4.09	3.97	4.07	4.12	1.33
0,2,4	bDGlcp	4.53	3.38	3.62	3.46	3.45	3.80-3.95
0,2,3	aLRhap	5.23	4.07	3.85	3.46	4.44	1.27
0,2	aDGlcp	5.13	3.70	4.01	3.86	4.06	?
0	aDGlcp	5.75	3.68	3.89	3.53	3.91	?
	P


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
0,2,4,3	aLRhap	101.4/5.17	71.2/4.09	70.9/3.97	78.9/4.07	68.6/4.12	17.8/1.33
0,2,4	bDGlcp	102.4/4.53	75.2/3.38	83.0/3.62	69.0/3.46	77.1/3.45	62.2/3.80-3.95
0,2,3	aLRhap	101.9/5.23	71.1/4.07	71.0/3.85	73.0/3.46	69.4/4.44	17.5/1.27
0,2	aDGlcp	98.0/5.13	73.6/3.70	77.5/4.01	73.6/3.86	72.4/4.06	?/?
0	aDGlcp	93.5/5.75	77.3/3.68	72.1/3.89	70.1/3.53	73.8/3.91	?/?
	P

The spectrum also has 2 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](C)O[C@@H](O[C@@H]2[C@@H](O)[C@H](O[C@@H]3[C@@H](CO)O[C@H](O[C@@H]4[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]4OP(*)(=O)O)[C@H](O)[C@H]3O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](O)[C@H]3O)O[C@H](CO)[C@H]2O)[C@H](O)[C@@H]1O

858.686 g/mol (C₃₀H₅₁R₂O₂₆P, R = next and previous repeats, not counted in mol weight)