Chemical characterization of E. faecalis diheteroglycan Protein and phosphorus content of purified diheteroglycan as quantified using standard assays [36,38]. The polysaccharide was further characterized by SDS-PAGE in gradient gels (4/12% w/v, Invitrogen), followed by staining for proteins with Coomassie blue and with the PAS reaction for carbohydrates [39]. Compositional analyses were performed as described previously [3,40,41]. Polyacrylamide gel electrophoresis Cell wall carbohydrates were released from E. faecalis FA2-2 or HG101 by treatment with mutanolysin, lysozyme and nuclease and proteinase K treatment as described above. Next, 25% ethanol was added and precipitated material was discarded. More ethanol was added to a final concentration of 75% for the precipitation of carbohydrates. Purified diheteroglycan and cell wall extracts were analyzed by electrophoresis through 3% polyacrylamide (33:1) in Tris-borate buffer (0.2 M Tris-base/ 0.2 M boric acid/20 mM EDTA, pH 8.3), and detected using Stains-All (3,39-dimethyl-9-methyl-4,5,4959-dibenzothiacarbocya- nine) according to the method of Hancock et al. [12]. NMR spectroscopy Samples were exchanged three times with 99.90% 2H2O, lyophilized, and redissolved in 99.99% 2H2O. All one-dimensional and two-dimensional spectra were recorded at 27uC with a Bruker DRX Avance 600 MHz spectrometer as described previously [3]. Chemical shifts were reported relative to internal acetone (dH 2.225; dC 31.45). ELISA studies ELISA experiments were performed by standard methods as described previously [3]. In brief, microtiter plates were coated with the carbohydrate antigen specified in the respective experiment (10 mg/ml in 0.04 M phosphate buffer, pH 7.0) and incubated for 18 h at 4uC. Washing steps were performed with PBS containing 0.05% Tween 20. Plates were blocked with 3% skim milk in PBS-0.02% sodium azide at 37uC for 2 h. A goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma) diluted 1:1,000 was used as secondary antibody, and p-nitrophenyl phosphate was used as a substrate (Sigma). After 60 min of incubation at 37uC, the absorbance was measured at 405 nm. Opsonophagocytic killing assay An opsonophagocytic killing assay was used with modifications as previously described [3,42]. In brief, E. faecalis strains were grown to logarithmic phase (OD 600 nm 0.4) in TSB and diluted in RPMI plus 15% heat-inactivated fetal calf serum. Baby rabbit serum (Cedarlane Laboratories, Hornby, Ontario, Canada) absorbed with the target bacterial strain served as a source of complement. White blood cells (WBC) were isolated from healthy volunteers by sedimentation with heparin-dextrane [42]. Rabbit immune serum was heat-inactivated at 56uC for 30 min before use and diluted to the concentration indicated for the individual experiments. 2.56106 white blood cells, 2.56106 CFU of E. faecalis, 0.00125–2.5% rabbit antiserum (as indicated in the individual experiment), and 1.7% complement in a total volume of 400 ml were incubated in tubes and rotated end over end at 37uC for 90 min. Negative controls included tubes from which leukocytes, complement or serum were omitted. The opsonic activity of the serum was calculated as follows: {1 - (CFU immune serum at 90 min/CFU of control without WBC at 90 min)} x 100. For studies on inhibition of opsonophagocytic killing, rabbit antiserum was incubated at a concentration of 1:800 (final Opsonic Antibodies against E. faecalis concentration) with various concentrations of inhibitor for 60 min at 4uC. After incubation, the opsonophagocytic killing assay was continued with the absorbed antiserum as described above. Inhibition assays were performed at serum dilutions yielding ,70 – 80% killing of the inoculum without the addition of the inhibitor (for individual dilutions see text). The percentage of inhibition of opsonophagocytic killing was compared to controls without inhibitor. Mouse bacteremia model To test protective efficacy of rabbit immune serum, we employed a modified mouse bacteremia model developed previously in our laboratory [42,43,44]. Six- to eight-week old female BALB-C mice (7–8 mice per group) were injected i.p. with 200 ml of heat-inactivated rabbit immune serum 48 h and 24 h before and 4 h after bacterial challenge and infected intravenously as indicated in the individual experiments. After 48 h, mice were sacrificed, blood, kidneys and livers were harvested under sterile conditions and bacterial counts enumerated by culture of serially diluted samples. The lower limit of detection of the assay was 16101 CFU. Ethics statement All animal experiments were performed in compliance with the German animal protection law (TierSchG). The mice were housed and handled in accordance with good animal practice as defined by FELASA and the national animal welfare body GV-SOLAS. The animal welfare committees of the University of Freiburg (Regierungspra¨sidium Freiburg Az 35/9185.81/G-07/15) ap- proved all animal experiments. The institutional review board of the University of Freiburg approved the study protocol and written informed consent was obtained from all study participants. Statistcal analysis Statistical significance for two-way comparisons was determined by an unpaired t test. Analysis of variance (ANOVA) for multigroup comparisons was used on log-transformed data, and the Tukey’s multiple-comparison test was used for posthoc analysis for pairwise comparisons. Statistical results were calculated using the Prism 3 software package. Supporting Information Table S1 1H and 13C NMR data of the capsular polysaccharide from E. faecalis strain type 2. Spectra were recorded of a solution in 2H2O at 600 MHz and 27uC relative to internal acetone (dH 2.225; dC 31.45). (DOC) Table S2 1H and 13C NMR chemical shifts [d] of diheterogly- can isolated from of E. faecalis type 5. Spectra were recorded of a solution in 2H2O at 600 MHz and 27uC relative to internal acetone (dH 2.225; dC 31.45). (DOC) Figure S1 SDS PAGE electrophoresis of purified diheteroglycan from E. faecalis type 2. Lane 1 protein molecular mass marker, lane 2 Coomassie stain, lane 3 PAS stain. (TIFF) Figure S2 Section of the HMBC spectrum of diheteroglycan isolated from E. faecalis type 2. The interresidual connectivities are underlined. (PDF) PLoS ONE | www.plosone.org 9 March 2011 | Volume 6 | Issue 3 | e17839