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
5,2	Ac
5,3	aLRhap	103.66	71.39	71.19	73.13	70.39	17.85
5	bDGalpN	102.83	52.76	79.99	68.43	74.63	64.97
	x?Rib-ol	67.79	72.61	71.82	71.76	71.89


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
5,2	Ac
5,3	aLRhap	4.887	3.840	3.804	3.432	3.825	1.284
5	bDGalpN	4.591	4.044	3.789	4.072	3.877	4.046-4.046
	x?Rib-ol	3.983-4.053	3.766	3.903	3.969	3.856-3.997


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
5,2	Ac
5,3	aLRhap	103.66/4.887	71.39/3.840	71.19/3.804	73.13/3.432	70.39/3.825	17.85/1.284
5	bDGalpN	102.83/4.591	52.76/4.044	79.99/3.789	68.43/4.072	74.63/3.877	64.97/4.046-4.046
	x?Rib-ol	67.79/3.983-4.053	72.61/3.766	71.82/3.903	71.76/3.969	71.89/3.856-3.997

There are 2 chemically distinct structures. Please, select:
-6)[aLRhap(1-3),Ac(1-2)]bDGalpN(1-5)xDRib-ol(1- -6)[aLRhap(1-3),Ac(1-2)]bDGalpN(1-5)xLRib-ol(1-

SVGMWSMILES3D molIonize

 

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

483.467 g/mol (C₁₉H₃₃R₂NO₁₃, R = next and previous repeats, not counted in mol weight)