All enterococci produce a complex polysaccharide called the enterococcal polysaccharide antigen (EPA). This polymer is required for normal cell growth and division and for resistance to cephalosporins and plays a critical role in host-pathogen interaction. The EPA contributes to host colonization and is essential for virulence, conferring resistance to phagocytosis during the infection. Recent studies revealed that the "decorations" of the EPA polymer, encoded by genetic loci that are variable between isolates, underpin the biological activity of this surface polysaccharide. In this work, we investigated the structure of the EPA polymer produced by the high-risk enterococcal clonal complex Enterococcus faecalis V583. We analyzed purified EPA from the wild-type strain and a mutant lacking decorations and elucidated the structure of the EPA backbone and decorations. We showed that the rhamnan backbone of EPA is composed of a hexasaccharide repeat unit of C2- and C3-linked rhamnan chains, partially substituted in the C3 position by α-glucose (α-Glc) and in the C2 position by β-N-acetylglucosamine (β-GlcNAc). The so-called "EPA decorations" consist of phosphopolysaccharide chains corresponding to teichoic acids covalently bound to the rhamnan backbone. The elucidation of the complete EPA structure allowed us to propose a biosynthetic pathway, a first essential step toward the design of antimicrobials targeting the synthesis of this virulence factor.IMPORTANCE Enterococci are opportunistic pathogens responsible for hospital- and community-acquired infections. All enterococci produce a surface polysaccharide called EPA (enterococcal polysaccharide antigen) required for biofilm formation, antibiotic resistance, and pathogenesis. Despite the critical role of EPA in cell growth and division and as a major virulence factor, no information is available on its structure. Here, we report the complete structure of the EPA polymer produced by the model strain E. faecalis V583. We describe the structure of the EPA backbone, made of a rhamnan hexasaccharide substituted by Glc and GlcNAc residues, and show that teichoic acids are covalently bound to this rhamnan chain, forming the so-called "EPA decorations" essential for host colonization and pathogenesis. This report represents a key step in efforts to identify the structural properties of EPA that are essential for its biological activity and to identify novel targets to develop preventive and therapeutic approaches against enterococci.

teichoic acids, cell wall polysaccharide, rhamnan, Enterococcus faecalis, E.faecalis, enterococcal polysaccharide antigen

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
2,2,3,3,2,3	aDGlcp	95.95	74.29	74.08	72.93	70.80	61.72
2,2,3,3,2	aLRhap	102.25	75.35	75.10	71.72	70.65	18.01
2,2,3,3	aLRhap	101.84	79.47	71.48	73.11	70.74	18.04
2,2,3,2,2	Ac
2,2,3,2	bDGlcpN	104.05	57.21	71.70	73.47	76.85	61.66
2,2,3	aLRhap	101.82	79.33	76.80	73.45	70.65	18.10
2,2	aLRhap	103.24	71.13	78.70	72.93	70.61	18.11
2	aLRhap	102.16	79.45	71.27	73.52	70.50	18.00
	aLRhap	102.07	79.37	71.37	73.49	70.50	18.00


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
2,2,3,3,2,3	aDGlcp	5.095	3.608	3.772	3.919	3.463	3.748-3.782
2,2,3,3,2	aLRhap	5.173	4.347	3.953	3.621	3.738	1.280
2,2,3,3	aLRhap	5.235	4.011	3.861	3.545	3.734	1.333
2,2,3,2,2	Ac
2,2,3,2	bDGlcpN	4.582	3.702	3.628	3.468	3.425	3.766-3.872
2,2,3	aLRhap	5.171	4.210	3.904	3.484	3.728	1.282
2,2	aLRhap	4.966	4.159	3.843	3.558	3.779	1.282
2	aLRhap	5.109	4.106	3.901	3.471	3.740-3.780	1.280
	aLRhap	5.159	4.067	3.944	3.513	3.740-3.780	1.280


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
2,2,3,3,2,3	aDGlcp	95.95/5.095	74.29/3.608	74.08/3.772	72.93/3.919	70.80/3.463	61.72/3.748-3.782
2,2,3,3,2	aLRhap	102.25/5.173	75.35/4.347	75.10/3.953	71.72/3.621	70.65/3.738	18.01/1.280
2,2,3,3	aLRhap	101.84/5.235	79.47/4.011	71.48/3.861	73.11/3.545	70.74/3.734	18.04/1.333
2,2,3,2,2	Ac
2,2,3,2	bDGlcpN	104.05/4.582	57.21/3.702	71.70/3.628	73.47/3.468	76.85/3.425	61.66/3.766-3.872
2,2,3	aLRhap	101.82/5.171	79.33/4.210	76.80/3.904	73.45/3.484	70.65/3.728	18.10/1.282
2,2	aLRhap	103.24/4.966	71.13/4.159	78.70/3.843	72.93/3.558	70.61/3.779	18.11/1.282
2	aLRhap	102.16/5.109	79.45/4.106	71.27/3.901	73.52/3.471	70.50/3.740-3.780	18.00/1.280
	aLRhap	102.07/5.159	79.37/4.067	71.37/3.944	73.49/3.513	70.50/3.740-3.780	18.00/1.280

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](O[C@H]2[C@H](O[C@@H]3[C@@H](O)[C@H](C)O[C@@H](O[C@@H]4[C@@H](O)[C@H](C)O[C@@H](O[C@H]5[C@H](O[C@H]6[C@H](*)O[C@@H](C)[C@H](O)[C@H]6O)O[C@@H](C)[C@H](O)[C@H]5O)[C@@H]4O)[C@@H]3O[C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3NC(C)=O)O[C@@H](C)[C@H](O)[C@H]2O)O[C@@H](C)[C@H](O)[C@H]1O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O

1242.187 g/mol (C₅₀H₈₃R₂NO₃₄, R = next and previous repeats, not counted in mol weight)