Figure 4. Opsonophagocytic killing of E. faecalis CPS-serotype C and D strains by rabbit antiserum after immunization with diheteroglycan purified from E. faecalis type 2 (CPS-C). Serum dilutions were used as indicated in the legend. Bars represent the mean of four determinations and the error bar the SEM. doi:10.1371/journal.pone.0017839.g004 Prototype sera for Hufnagel’s study were generated by vaccination with purified polysaccharide or heat-killed, protein- ase K-digested whole bacteria, and strains were typed according to cross-reactivity measured by ELISA and the opsonophagocytic killing assay. Using this methodology, 60% of a total of 29 clinical E. faecalis isolates could be assigned to one of four serotypes, CPS-A to CPS-D [4]. However, serospecificity of opsonic antibodies was not unequivocal between CPS-A and CPS-B strains or between CPS-C and CPS-D strains [4]. McBride and Gilmore examined the diversity of capsule expression in E. faecalis on a genetic basis in 106 strains of diverse origin and found that approximately half of the strains lacked genes of the cps locus that are essential for capsule production [21]. Opsonic Antibodies against E. faecalis Our group has demonstrated previously that the teichoic acid- like cell envelope polysaccharide that is the target of opsonic antibodies in CPS-A strains is structurally identical to LTA [3]. Using the highly purified LTA obtained from our previous study we reassessed the cross-reactivity of LTA-specific antibodies. In a previous publication by us, we demonstrated that opsonic antibodies against CPS-A and CPS-B serotypes are not cross- reative [4]. In our current study antibodies raised against purified LTA reacted with both CPS-A strains (E. faecalis 12030) and CPS- B strains (E. faecalis 12107, OG1RF, type 1). The discrepancy of our current and previous study may be related to the different antigen preparations used for production of rabbit antisera. For a previous investigation we utilized a partially deacylated and dealanylated LTA molecule that may have lost antigenic properties during the purification process [3,4]. Our finding that CPS-A and CPS-B only represent a single serotype is supported by genetic characterization of the cps-locus: CPS-A and CPS-B strains both contain only the first two cps-genes (cpsA and cpsB) but lack remaining the genes of this locus (cpsC – cpsK) which are essential for capsule production [4,15]. Also, LTA-specific antibodies agglutinate CPS-A and CPS-B but not CPS-C and CPS-D strains, indicating that LTA is surface exposed in the former serotypes [15]. In contrast to acapsular CPS-A and CPS-B strains, our and Thurlow’s data indicate that LTA of CPS-C and CPS-D strains is not available on the bacterial surface to bind specific antibodies for opsonization via the classical pathway, probably because a polysaccharide capsule masks this antigen [14]. With evidence suggesting the presence of an antiphagocytic capsule in E. faecalis, we next purified cell envelope polysaccharides from the CPS-C strain E. faecalis type 2 and identified a novel diheteroglycan. The isolated diheteroglyan represented only a small propor- tion of carbohydrates released by enzymatic digestion of peptidoglycan. Most of the material obtained by this mode of extraction was a rhamnopolysaccharide with similar composition as described previously [8,9,10,11,12,22]. The diheterglycan Figure 5. Relationship of diheteroglycan to the biosynthetic product of the cps locus. (A) Loss of opsonic activity after absorption of rabbit antiserum raised against E. faecalis type 2 (CPS-C) diheteroglycan with whole bacteria. Before the assay the serum was absorbed with either E. faecalis FA2-2 (CPS-C) wild type or its isogenic, acapsular cpsI mutant (E. faecalis HG101) for 60 min. Absorbed serum was used at a final dilution of 1:40 in the assay. Bars represent the mean of four determinations and the error bar the SEM. (B) Native PAGE of purified diheteroglycan from E. faecalis type 2 (lane 1), cell wall lysates of E. faecalis FA2-2 wild type (CPS-C, lane 2) and the isogenic cpsI mutant (HG101, lane 3). Cell envelope carbohydrates were released by digestion of peptidoglycan by lysozyme and mutanolysin and acrylamide gels were stained with Stains-All according to the method of Hancock et al. [12]. doi:10.1371/journal.pone.0017839.g005 PLoS ONE | www.plosone.org 6 March 2011 | Volume 6 | Issue 3 | e17839