cps locus and was identified as a 130 kDa glycan. The com- position of the material isolated from strain FA2-2 (CPS-C) was determined to be glucose, galactose, glycerol and phosphate at a ratio of 4:1:1:2 [12]. Serum absorptions experiments, acrylamide gel elecrophoresis and structural data presented here suggest that both polysaccharide antigens – diheteroglycan and capsular polysaccharide produced by the cps locus - are identical. Our studies demonstrate that opsonic antibodies directed against diheteroglycan bind to capsule-bearing CPS-C strains, but not to its isogenic acapsular mutant. Also, both antigens co-migrate in native PAGE and contain glucose and galactose, albeit at different ratios. We can only speculate about the reason for this discrepancy in monosaccharide composition, but impurities in the capsular polysaccharide described by Hancock may explain the compositional differences. As mentioned above, the diheterogly- can partially co-elutes during the chromatography process with LTA and only an additional purification step following ion exchange chromatography yielded pure material in our study. Hence, impurities of LTA could explain why Hancock and Gilmore found more glucose, glycerol and phosphate in their preparations of capsular polysaccharide. Of note, high-pH anion- exchange chromatography–pulsed-amperometric detection em- ployed for compositional analysis in Hancock’s study is not suitable to detect LTA in mixtures of complex carbohydrates [12,15]. To test if capsule-specific antibodies can protect against E. faecalis infection, we passively immunized mice with either anti- diheteroglycan or anti-LTA rabbit serum and challenged them with E. faecalis CPS-C and CPS-D strains intravenously. Bacterial counts were 1.4–3.4 logs lower in kidneys and livers of mice immunized with anti-diheteroglycan for the three strains tested. The variable protective efficacy against the tested E. faecalis strains is somewhat surprising, but may be explained by differences of in vivo expression or the degree of O-acetylation of diheteroglycan between strains. In broader terms, this level of reduction of bacterial load by passive protection is comparable if not superior to vaccine efficacy achieved for various vaccine antigens evaluated in Staphylococcus aureus [29,30,31,32]. It is difficult to predict, if this level of vaccine efficacy in mice will translate into protection in the human host. In contrast to other Gram-positive pathogens like Streptococcus pneumoniae or group-B streptococci, which are virulent pathogens in humans and mice, mice clear enterococcal bacteremia or peritonitis spontaneously unless a very high inoculum (.108–109 bacteria per mouse) is given. Hence, in our animal model we may have overwhelmed adaptive immunity with bacterial loads much higher than encountered during human infection, a problem that also riddles vaccine research for other nosocomial pathogens of low virulence (e.g. Staphylococcus epidermi- dis, Candida spp.). Nevertheless, we expect that conjugation of the diheteroglycan capsule to a protein carrier will enhance immuno- genicity of the vaccine and elicit better protection in vivo. Active and passive vaccination strategies with a diheteroglycan conjugate vaccine could potentially be employed in patients that are at high risk of invasive enterococcal infections such as patients hemato- logic malignancies, neutropenia, or liver-transplant recipients [33,34,35]. In summary, the results presented in this study provide evidence that two carbohydrates of the cell envelope are targets of opsonic antibodies in typeable strain of E. faecalis. Active vaccination with LTA and diheteroglycan or a passive immuno- therapy approach using recombinant human monoclonal anti- bodies could therefore target non-encapsulated and capsule- expressing strains and may provide protection against a majority of E. faecalis strains. Opsonic Antibodies against E. faecalis Materials and Methods Bacterial strains and culture Bacterial strains and the respective serotype according to Hufnagel et al. are specified in table 1. Unless otherwise indicated, bacterial cells were grown from starter cultures in Columbia broth (Becton Dickinson, Sparks, MD, USA) supplemented with 1% glucose at 37uC until they reached an optical density at 600 nm of 0.6 to 0.8 and harvested by centrifugation. Antisera Rabbit antisera against whole bacterial cells of E. faecalis type 2 and type 5 have been described previously [4]. Anti-LTA rabbit antiserum was prepared using LTA purified from E. faecalis 12030 by butanol extraction and hydrophobic interaction chromatogra- phy [3]. Antiserum against diheteroglycan and LTA was raised by s.c. immunization of one female New Zealand white rabbit for each antigen with 100 mg of antigen suspended in complete Freund’s adjuvant followed by the same dose in incomplete Freund’s adjuvant seven days later and intravenous booster doses of 10 mg every three days in the consecutive week. Antiserum was obtained five weeks after the beginning of the immunization. Preparation and characterization of capsular polysaccharide Diheteroglycan of E. faecalis was isolated by methods similar to those described previously [3,22]. Briefly, E. faecalis strains type 2, FA2-2 and type 5 were cultivated as described above and harvested by centrifugation. The bacteria were washed in PBS and cell walls were digested by addition of mutanolysin and lysozyme (each at 100 mg/ml, Sigma Chemicals, St. Louis, MO, USA in PBS supplemented with 5 mM MgCl2, 1 mM CaCl2 and 0.05% NaN3) at 37uC for 18 h. Insoluble material was removed by centrifugation, and the supernatant was treated with nucleases (DNase I and RNase A, 100 mg/ml) at 37uC for 4 h followed by addition of proteinase K (100 mg/ml, all Sigma Chemicals) at 56uC for 18 h. The supernatant was precipitated by the addition of ethanol (80% final volume), collected by centrifugation, dialyzed against deionized H2O, and lyophilized. For size exclusion chromatography, the material was redissolved in 0.01 M ammo- nium carbonate buffer (pH 8.0) and applied to a column (1.6690 cm) of Sephacryl S-400 (GE Healthcare, Uppsala, Sweden). Fractions eluting at around a Kav of 0.45 were combined, dialyzed and lyophilized. The material was resus- pended in 20 mM NaHCO3, pH 8.4 and applied to an anion- exchange column (Sepharose Q FF, GE Healthcare). Bound antigen was eluted from the column by a linear gradient of 0–1 M NaCl and fractions were assayed for hexose content by the Dubois assay and for phosphorus using the Lowry method [36,37]. Hexose-positive and phosphorus-negative fractions eluting at 450 mM NaCl were combined, dialyzed and lyophilized. As a final purification step, gel-permeation chromatography was performed on a 1.5675 cm Toyopearl HW-40 column (Tosoh Corporation, Tokyo, Japan). Preparation of LTA LTA was prepared by butanol extraction and hydrophobic interaction chromatography as described previously [3]. LTA preparations were evaluated for purity by the Bradford assay, SDS-PAGE and western blot analysis with the respective antiserum to whole bacterial cells (see above). Structural identity of LTA was confirmed by NMR spectroscopy as described recently [3]. PLoS ONE | www.plosone.org 8 March 2011 | Volume 6 | Issue 3 | e17839