about cell envelope polysaccharides as a target of protective antibodies against E. faecalis. Hancock, Gilmore and Thurlow [12,13,14,15] described a capsule in E. faecalis synthesized by the cps locus. This capsule mediates resistance to killing by serum and neutrophils or macrophages, augments bacterial persistence in vivo, and impedes C3b deposition on the bacterial surface [4,14]. However, to date, no definite chemical structure of the capsular polysaccharide has been published. In the current study, we have revisited the cell wall carbohydrates of E. faecalis and investigated their role as antigens in the CPS-serotyping system by Hufnagel and colleagues. Using highly purified polysaccharides, we were able to show that opsonic antibodies are directed against only two of these antigens: In acapsular strains, LTA is the major opsonic epitope and in encapsulated strains opsonic antibodies bind to a novel diheter- oglycan, the putative capsular polysaccharide of E. faecalis in CPS- C and CPS-D strains. Results CPS-A and CPS-B strains but not CPS-C and CPS-D strains are opsonized by LTA-specific antibodies We reported previously that E. faecalis strains belonging to the CPS-A serotype are opsonized by antibodies specific to a teichoic- acid like polysaccharide, which was later shown to be structurally identical to LTA [3,4]. More than half of the E. faecalis strains, however, belong to serotypes CPS B – D and are not opsonized by this antiserum [4,21]. To further explore the role of antibodies against LTA in the serodiversity of E. faecalis strains, we vaccinated a rabbit with LTA from E. faecalis 12030 (CPS-A), which was extracted and purified using non-degrading condi- tions. In a western blot analysis with whole cell lysates of the vaccine strain this antiserum was monospecific to LTA (data not shown). In the opsonophagocytic killing assay, anti-LTA antibodies mediated killing not only of CPS-A strains, but also of E. faecalis strains that belong to the CPS-B serotype. In contrast, CPS-C and CPS-D strains were not opsonized (Fig. 1B). Next, we wanted to explore if CPS-C and CPS-D strains may express a structurally distinct LTA molecule that is not recognized by antibodies that are raised against LTA from a CPS-A strain. We purified LTA from three E. faecalis strains that belonged to the serotypes CPS-B – CPS-D and measured binding by ELISA (Fig. 1A). Anti-LTA bound equally well, however, to LTA derived from CPS-A (E. faecalis 12030), CPS-B (12107), CPS-C (FA2-2) and CPS-D (type 5) strains, suggesting that antigenic variability of LTA is irrelevant for the lack opsonic activity of LTA antiserum against CPS-C and CPS-D strains (Fig. 1, for specifications of E. faecalis strains see table 1). Purification of a novel capsular polysaccharide in CPS-C and CPS-D strains Since CPS-C and CPD-D strains are not killed by anti-LTA antibodies, we hypothesized that LTA in these strains is masked by a polysaccharide capsule, an assumption also supported by agglutination experiments by Thurlow and coworkers [15]. To investigate this hypothesis, we released cell wall associated carbohydrates in CPS-C (E. faecalis FA2-2 and E. faecalis type 2) and CPS-D (E. faecalis type 5) strains by enzymatic digestion of peptidoglycan and separated the extracted material by gel- permeation chromatography. Carbohydrate eluting at void volume consisted of LTA as determined by 1H NMR analysis. A large second peak eluting around a Kav of 0.45 was further purified by anion-exchange chromatography. The majority of the material eluted from Q-Sepharose around 300 mM NaCl and Opsonic Antibodies against E. faecalis contained rhamnose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine, as determined by sugar analysis. The compositional analysis of this polysaccharide was consistent with the previously described rhamnopolysaccharide [10,11,12,16,22]. A smaller, adjacent peak eluted at 450 mM NaCl and contained only glucose and galactose. This material migrated as a single, broad band around 100 kDa on SDS-PAGE electrophoresis and stained positive with periodic acid-Schiff (PAS) but not with Commassie blue (Fig. S1). Preliminary analysis by 1H-NMR spectroscopy revealed that this material contained a novel diheteroglycan and impurities of lipoteichoic acid, which were removed by a final purification step using gel-permeation chromatography on a Toyopearl column HW-40S. This final preparation contained ,3% protein and ,1% phosphorus and was used for consecutive chemical and biological analysis. Structural analysis of capsular polysaccharide from E. faecalis CPS-C and CPS-D strains reveals a novel diheteroglycan Compositional analysis identified the presence of D-Glc. The 1H NMR spectrum of the diheteroglycan isolated from E. faecalis type 2 (CPS-C, Fig. 2A) showed two anomeric signals at d 5.297 (residue A, {3JH1,H2 ,2 Hz}), and at d 4.491 (residue B, {3JH1,H2 = 7.8 Hz}), which were assigned to H-1 of b-Galf and b-Glcp, respectively. In addition, a broad signal at d 5.379 was identified, which was assigned to proton H-5 of b-Galf due to the substitution of position C-5 by an O-acetyl group (d 2.164). Furthermore, the doublet at d 1.366 was recognized as methyl group belonging to lactic acid (LA) residue [23,24,25]. All 1H and 13C chemical shifts of the capsular polysaccharides were established from 1H,1H correlation and total correlation as well as 1H,13C heteronuclear multiple quantum coherence NMR experiments. Low-field shifted signals of carbon atoms demon- strated substitutions at C-6 and C-3 of b-Galf (d 69.63 and d 84.85, respectively) and substitution at C-3 of b-Glcp (d 82.58) The chemical shifts are summarized in table S1. The sequence of the residues in the repeating unit of the capsular polysaccharides was established by rotating frame nuclear Overhauser effect (NOE) spectroscopy (ROESY, Fig. 2B) and heteronuclear multiple bond correlation (HMBC, Fig. S2) experiments. Strong interresidual NOE contacts were observed between H-1 A/H-3 B (d 5.297/3.622), as well as between H-1 B/H-6a A (d 4.491/3.897), and H-1 B/H-6b A (d 4.491/4.101). Additional weak NOE contacts were found between H-5 A and LA methyl group (d 5.379/1.336), and H-5 A and O-acetyl methyl group (d 5.379/2.164). The HMBC data confirmed the sequence of the constituents assigned from ROESY data. The following interresidual proton-carbon correlations were observed: H-1 A/C-3 B (d 5.297/82.58), C-1 A/H-3 B (d 109.20/3.622), C-1 B/H-6a A (d 103.44/3.897), as well as H-3 A/C-2 LA (d 3.747/77.88), and C-3 A/H-2 LA (d 84.85/3.966). The chemical structure of the isolated polysaccharide is shown on the Fig. 2C. 1H NMR spectroscopy of diheteroglycans isolated from E. faecalis FA2-2 (CPS-C) and type 5 (CPS-D) revealed a polysaccharide that they only differed from the E. faecalis type 2 (CPS-C) diheteroglycan by their lack of O-acetylation of b-Galf (for structural analysis of diheteroglycan from type 5 see Fig. S3, S4 and table S2). In subsequent experiments we isolated O- acetylated and O-deacetylated diheteroglycan from E. faecalis type 2 using the same culture conditions. Thus, it cannot be excluded that the chosen purification scheme may result in the loss of the labile O-acetyl group during the isolation of the polysaccharide. PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17839