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
3,3	l?Lac	180.33	77.49	19.23
3,5	Ac	174.85	21.49
3	bDGalf	109.74	80.52	85.67	81.94	72.78	70.21
	bDGlcp	104.02	74.44	83.21	69.24	76.90	61.94


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3	l?Lac	-	4.101	1.381
3,5	Ac	-	2.167
3	bDGalf	5.297	4.330	3.778	4.408	5.391	3.908-4.103
	bDGlcp	4.498	3.398	3.612	3.433	3.479	3.731-3.914


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3	l?Lac		77.49/4.101	19.23/1.381
3,5	Ac		21.49/2.167
3	bDGalf	109.74/5.297	80.52/4.330	85.67/3.778	81.94/4.408	72.78/5.391	70.21/3.908-4.103
	bDGlcp	104.02/4.498	74.44/3.398	83.21/3.612	69.24/3.433	76.90/3.479	61.94/3.731-3.914

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*OC[C@@H](OC(C)=O)[C@@H]1O[C@@H](O[C@@H]2[C@@H](O)[C@H](*)O[C@H](CO)[C@H]2O)[C@H](O)[C@H]1OC(C)C(=O)O

438.382 g/mol (C₁₇H₂₆R₂O₁₃, R = next and previous repeats, not counted in mol weight)