The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic acid and legionaminic acid, respectively. A minor high molecular weight polysaccharide component was recognized as the fructose homopolymer levan, and a glucosylated lipoteichoic acid (LTA) was identified in a micellar fraction. The polysaccharides were conjugated to the CRM197 carrier protein, and the resulting glycoconjugates were used to immunize rabbits. Rabbit immune sera were evaluated for their ability to kill Tx16 in opsonophagocytic assays and in a mouse passive protection infection model. Although antibodies raised against levan failed to mediate opsonophagocytic killing, the other glycoconjugates induced effective opsonic antibodies, with the altruronic acid-containing polysaccharide antisera showing the greatest opsonophagocytic assay activity. Antibodies directed against either novel heteroglycan or the LTA reduced bacterial load in mouse liver or kidney tissue. To assess antigen prevalence, we screened a diverse collection of blood isolates (n = 101) with antibodies to the polysaccharides. LTA was detected on the surface of 80% of the strains, and antigens recognized by antibodies to the two major heteroglycans were co-expressed on 63% of these clinical isolates. Collectively, these results represent the first steps toward identifying components of a glycoconjugate vaccine to prevent E. faecium infection.

antigen, teichoic acid, vaccine, uronic acids, carbohydrate structure, glycoconjugate vaccine, Enterococcus faecium, sialic acids

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
0,2	aDGlcp	99.0	72.7	74.2	70.8	73.0	61.7
0	x?Gro	65.9-66.4	76.5	65.9-66.4
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
0,2	aDGlcp	5.19	3.54	3.77	3.42	3.94	3.78-3.89
0	x?Gro	4.05	4.14	4.05
	P


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
0,2	aDGlcp	99.0/5.19	72.7/3.54	74.2/3.77	70.8/3.42	73.0/3.94	61.7/3.78-3.89
0	x?Gro	65.9-66.4/4.05	76.5/4.14	65.9-66.4/4.05
	P

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*OCC(COP(*)(=O)O)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O

316.199 g/mol (C₉H₁₇R₂O₁₀P, R = next and previous repeats, not counted in mol weight)