Opsonic Antibodies against E. faecalis Figure 2. Structural characterization of diheterglycan by nuclear magnetic resonance (NMR) analysis. (A) The 1H NMR spectrum of diheteroglycan isolated from E. faecalis type 2. The spectrum was recorded at 600 MHz and 27uC. The letters refer to the carbohydrate residues as shown in chemical structure (Fig. 2C), and the numbers refer to the protons in the respective residues; LA, lactic acid. (B) Sections of the ROESY spectrum of E. faecalis diheteroglycan. The interresidual NOE contacts are underlined. (C) Chemical structure of the repeating unit of E. faecalis capsular diheteroglycan. * Acetic acid remain of the final gel-permeation chromatography step. doi:10.1371/journal.pone.0017839.g002 At a serum dilution of 1:40, diheteroglycan-specific antibodies were opsonic to all E. faecalis strains of the CPS-C and CPS-D serotype evaluated (i.e. killing .70%). At higher serum dilutions, opsonic killing activity of $50% was observed only for three out of five heterologous E faecalis strains, indicating overall moderate titers of opsonic antibodies induced by vaccination with diheter- oglycan (Fig. 4). Diheteroglycan shares antigenic and structural similarities with the gene product of the cps locus Hancock and Gilmore previously described a gene locus involved in the biosynthesis of a putative capsular polysaccharide of E. faecalis strains of the CPS-C and CPS-D serotype [12,14,15]. The product of the cps-locus is a capsular polysaccharide composed of glucose, galactose, glycerol, and phosphate [12,15]. To examine antigenic similarity of diheteroglycan and the capsular polysaccharide produced by the cps locus, we absorbed the antiserum raised against diheterglycan derived from E. faecalis type 2 (CPS-C) with an acapsular cpsI mutant in E. faecalis FA2-2 and the isogenic wild type strain (CPS-C) [12]. Absorption of diheteroglycan-specific rabbit antiserum with the wild-type E. faecalis but not with the acapsular mutant abolished opsonic killing of the E. faecalis FA2-2 (Fig. 5A). For comparison of the biosynthetic product of the cps-locus with diheteroglycan we released cell wall carbohydrates from E. faecalis FA2-2 and its isogenic cpsI mutant and applied it along with purified polysaccharide to acrylamide gel electrophoresis (Fig. 5B). Electrophoretic mobility of diheteroglycan was identical to a high molecular weight band around 100 kDa that was present in E. faecalis FA2-2 wild type strain but not in the cpsI mutant (Fig. 5B). Antibodies to diheteroglycan protect against bacteremia in mice Having demonstrated that diheteroglycan-specific antibodies are opsonic to E. faecalis CPS-C and CPS-D strains, we assessed in vivo protection by passive immunization in a mouse bacteremia model. To this end, BALB-C mice received rabbit antibodies against diheteroglycan or against LTA (as a control) 48 h and 24 h before and 4 h after i.v. challenge. A total of three E. faecalis strains were evaluated (E. faecalis type 2 and FA2-2 (CPS-C), type 5 [CPS-D]). Anti-LTA was chosen as control because natural LTA-specific antibodies were present in the diheteroglycan antiserum and because identical vaccination protocols including complete Freunds adjuvant were used for production of both antisera. Forty-eight hours after infection, bacterial counts from the blood, kidneys and liver were enumerated. At this time point, mice of both immunization groups had cleared the bacteremia, but – depending on the E. faecalis challenge strain - bacterial counts in the kidney and liver were reduced 1.4 to 3.4 logs in mice immunized with diheteroglycan-specific antibodies compared to mice that had received rabbit anti-LTA (Fig. 6). Discussion Defining serotypes and corresponding structures of the cell envelope that constitute the basis of serospecificity is a critical step in vaccine development. Using formaldehyde-killed bacteria for immunization, in 1992, Maekawa identified 21 serotypes of E. faecalis by cross-agglutination and absorption studies [26]. More recently, Hufnagel defined a simplified serotyping system based largely on carbohydrate antigens of E. faecalis [4]. Figure 3. Specificity of antibodies raised by vaccination with whole bacterial cells of E. faecalis. (A) Binding of rabbit IgG raised against whole bacterial cells of E. faecalis type 2 to diheteroglycan and LTA purified from the same strain. The coating antigen and serum dilutions are indicated in the legend. (B) Absorption of opsonic activity against E. faecalis type 2 (CPS-C) and type 5 (CPS-D) by cell envelope carbohydrate antigens. Rabbit antiserum raised against whole bacterial cells of the respective target strain was used. For the inhibition opsonophagocytic killing assay, rabbit antiserum was preincubated for 60 min with 10 mg/ml of either LTA or diheteroglycan purified from the homologous E. faecalis strain and used at a final dilution of 1:800 in the assay. Bars are means and error bars the SEM four determinations. doi:10.1371/journal.pone.0017839.g003 PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e17839