Serodiversity of Opsonic Antibodies against Enterococcus faecalis —Glycans of the Cell Wall Revisited Christian Theilacker1, Zbigniew Kaczyn´ ski2, Andrea Kropec1, Irina Sava1, Libin Ye1, Anna Bychowska2, Otto Holst3, Johannes Huebner1* 1 Center for Infectious Disease and Travel Medicine and Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany, 2 Faculty of Chemistry, University of Gdansk, Gdansk, Poland, 3 Division of Structural Biochemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany Abstract In a typing system based on opsonic antibodies against carbohydrate antigens of the cell envelope, 60% of Enterococcus faecalis strains can be assigned to one of four serotypes (CPS-A to CPS-D). The structural basis for enterococcal serotypes, however, is still incompletely understood. Here we demonstrate that antibodies raised against lipoteichoic acid (LTA) from a CPS-A strain are opsonic to both CPS-A and CPS-B strains. LTA-specific antibodies also bind to LTA of CPS-C and CPS-D strains, but fail to opsonize them. From CPS-C and CPS-D strains resistant to opsonization by anti-LTA, we purified a novel diheteroglycan with a repeating unit of R6)-b-Galf-(1R3)- b-D-Glcp-(1R with O-acetylation in position 5 and lactic acid substitution at position 3 of the Galf residue. The purified diheteroglycan, but not LTA absorbed opsonic antibodies from whole cell antiserum against E. faecalis type 2 (a CPS-C strain) and type 5 (CPS-D). Rabbit antiserum raised against purified diheteroglycan opsonized CPS-C and CPS-D strains and passive protection with diheteroglycan-specific antiserum reduced bacterial counts by 1.4 – 3.4 logs in mice infected with E. faecalis strains of the CPS-C and CPS-D serotype. Diheteroglycan- specific opsonic antibodies were absorbed by whole bacterial cells of E. faecalis FA2-2 (CPS-C) but not by its isogenic acapsular cpsI-mutant and on native PAGE purified diheteroglycan co-migrated with the gene product of the cps-locus, suggesting that it is synthesized by this locus. In summary, two polysaccharide antigens, LTA and a novel diheteroglycan, are targets of opsonic antibodies against typeable E. faecalis strains. These cell-wall associated polymers are promising candidates for active and passive vaccination and add to our armamentarium to fight this important nosocomial pathogen. Citation: Theilacker C, Kaczyn´ ski Z, Kropec A, Sava I, Ye L, et al. (2011) Serodiversity of Opsonic Antibodies against Enterococcus faecalis —Glycans of the Cell Wall Revisited. PLoS ONE 6(3): e17839. doi:10.1371/journal.pone.0017839 Editor: Shan Lu, University of Massachusetts Medical Center, United States of America Received October 14, 2010; Accepted February 15, 2011; Published March 18, 2011 Copyright: 2011 Theilacker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: ZK was supported by Polish Ministry of Research and Higher Education under grant no. N N301 030134. No other current external funding sources supported this this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: johannes.huebner@uniklinik-freiburg.de Introduction The classification system for streptococci was developed by Rebecca Lancefield at the beginning of the 20th century [1]. Enterococci were assigned to the serogroup D in the Lancefield typing system, and the group-specific antigen was subsequently identified as lipoteichoic acid (LTA) by Wicken and colleagues [2]. About fifty years later, our group demonstrated that antibodies that opsonize E. faecalis strain 12030 bind to the group antigen LTA [3]. In a recent serotyping system based on carbohydrate- specific antibodies, 60% of E. faecalis strains were typeable and assigned to four serotypes, designated CPS-A to CPS-D [4]. However, the structural equivalents of the type-specific antigens in this serotyping system are still unknown. This is surprising because several major carbohydrate structures of the enterococcal cell wall were described by Pazur, Bleiweis, and Krause in a number of landmark studies almost forty years ago [5,6,7,8]. These authors identified two major glycans from cell wall extracts of E. faecalis: a rhamnopolysaccharide (also called tetraheteroglycan or cell wall polysaccharide) and a diheteroglycan. The rhamnopolysaccharide was first described by Elliott et al. as the type antigen of Streptococcus faecalis in 1960 [9]. Bleiweis and Krause characterized the type antigen in more detail and reported that it is a complex carbohydrate containing rhamnose, glucose, glucosamine, and galactosamine as well as ribitol and phosphorus [8]. Rhamnopo- lysaccharides of similar composition were also described by Pazur and Karakawa [6] and in two more recent studies [10,11]. In the early 1970ies, Pazur et al also isolated a polysaccharide containing glucose and galactose from E. faecalis and named it diheterglycan [5,6]. A number of more recent studies have investigated genetic loci involved in the biosynthesis of polysaccharides of the enterococcal cell envelope, but chemical structures of the respective polysaccharides and their potential as vaccine antigens have not been explored [11,12,13,14,15,16,17,18]. Enterococcus is currently ranked third among Gram-positive pathogens to cause hospital-associated infections in the US [19] and was the second most common pathogen isolated from ICU patients worldwide in a point-prevalence study conducted in 2007, causing 10% of nosocomial infections on the ICU [20]. With limited options in antimicrobial chemotherapy available, a renewed interest in alternative treatment and prevention strategies such as active and passive immunization has evolved. Capsular polysaccharides have been highly successful vaccine antigens for vaccines against various bacterial pathogens, but little is known PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e17839