Opsonic Antibodies against E. faecalis Figure 6. Passive protection by rabbit antiserum in a mouse bacteremia model. Six to eight weeks old female Balb-C mice were passively immunized by i.p. injection of 200 ml of heat-inactivated rabbit antiserum raised against diheterglycan (anti-DHG) from E. faecalis type 2 or LTA purified from E. faecalis 12030 (anti-LTA) 24 and 12 h before and 4 h after infection. Fourty-eight h after i.v. injection of bacteria via the tail vein, mice were sacrificed and bacterial counts quantified. Mice were infected with E. faecalis FA2-2 (3.06109 cfu per mouse), E. faecalis type 2 (2.06109) and E. faecalis type 5 (2.56109) as indicated in the graph. Bars represent geometric means. Seven to eight mice per group were used. The lower limit of detection was 10 CFU/ml. Groups of mice were compared using the T-test of log-transformed CFU counts. doi:10.1371/journal.pone.0017839.g006 described here eluted in close association with the rhamnopo- lysaccharide and LTA during the chromatographic separation process, making it challenging to isolate this carbohydrate in high purity. Capsule extracts from two additional E. faecalis strains (FA2-2 [CPS-C] and type 5 [CPS-D]) contained a polysaccharide with an identical repeating unit of R6)- b-Galf- (1R3)- b-D-Glcp-(1R. In E. faecalis type 2 we recovered this polysaccharide with and without O-acetylation in position C-5 of Galf, while the polysaccharide isolated from E. faecalis type 5 and FA2-2 lacked O-acetylation. Since O-acetyl substituents are pH labile and hydrolyzed under mild basic conditions, the O- deacetylated diheteroglycan may represent an artifact of the conditions we have chosen for column chromatography. Alternatively, we cannot exclude that the degree of O-acetylation varies due to minor differences in culture conditions between the batches used for purification. O-acetyl groups may influence the biochemical properties of carbohydrates (e.g. solubility in water). They also can be part of antigenic epitopes of bacterial polysaccharides recognized by opsonic antibodies [27] but may also mask them [28]. Pazur et al. described a diheteroglycan of very similar composition in E. faecalis [5,6]. The proposed structure of Pazur’s diheteroglycan is a backbone consisting of a trisaccharide repeating unit of R4)-b-Glc-(1R6)-b-Glc-(1R4)-b-Gal-(1R sub- stituted with lactosyl and cellobiosyl side chains attached by b- (1R4) linkages to alternate glucose residues of the backbone [5]. Since this structure was determined before the availability of NMR spectrometry for more definitive carbohydrate analysis, it is tempting to speculate that this molecule is in fact identical with the carbohydrate identified by us. The composition of our diheteroglycan also bears resemblance to the capsular polysac- charide described by Hancock and Gilmore [12,15] although these authors never reported complete structural information for their antigen. Their capsular polysaccharide is synthesized by the PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e17839