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Characterization of the Linkage between the Type III Capsular Polysaccharide and the Bacterial Cell Wall of Group BStreptococcus *

Open AccessPublished:March 17, 2000DOI:https://doi.org/10.1074/jbc.275.11.7497
      The capsular polysaccharide of group BStreptococcus is a key virulence factor and an important target for protective immune responses. Until now, the nature of the attachment between the capsular polysaccharide and the bacterial cell has been poorly defined. We isolated insoluble cell wall fragments from lysates of type III group B Streptococcus and showed that the complexes contained both capsular polysaccharide and group B carbohydrate covalently bound to peptidoglycan. Treatment with the endo-N-acetylmuramidase mutanolysin released soluble complexes of capsular polysaccharide linked to group B carbohydrate by peptidoglycan fragments. Capsular polysaccharide could be enzymatically cleaved from group B carbohydrate by treatment of the soluble complexes with β-N-acetylglucosaminidase, which catalyzes hydrolysis of the β-d-GlcNAc(1→4)β-d-MurNAc subunit produced by mutanolysin digestion of peptidoglycan. Evidence from gas chromatography/mass spectrometry and 31P NMR analysis of the separated polysaccharides supports a model of the group BStreptococcus cell surface in which the group B carbohydrate and the capsular polysaccharide are independently linked to the glycan backbone of cell wall peptidoglycan; group B carbohydrate is linked to N-acetylmuramic acid, and capsular polysaccharide is linked via a phosphodiester bond and an oligosaccharide linker to N-acetylglucosamine.
      GBS
      group BStreptococcus
      GC/MS
      gas chromatography/mass spectrometry
      MurNAc
      N-acetylmuramic acid
      ELISA
      enzyme-linked immunosorbent assay
      FPLC
      fast protein liquid chromatography
      Group B Streptococcus(GBS)1 is a common cause of serious infections in neonates, including bacteremia, pneumonia, and meningitis. Almost all GBS strains isolated from neonates with invasive infection are encapsulated with one of several capsular polysaccharides (
      • Baker C.J.
      • Barrett F.F.
      ,
      • Wenger J.D.
      • Hightower A.W.
      • Facklam R.R.
      • Gaventa S.
      • Broome C.V.
      ,
      • Harrison L.H.
      • Elliott J.A.
      • Dwyer D.M.
      • Libonati J.P.
      • Ferrieri P.
      • Billman L.
      • Schuchat A.
      ). Studies in experimental animals have provided evidence that the capsular polysaccharide serves an important virulence function in GBS infection. Acapsular mutants derived from type III GBS strains by transposon mutagenesis are attenuated in their ability to cause lethal infection in neonatal rats (
      • Rubens C.E.
      • Wessels M.R.
      • Heggen L.M.
      • Kasper D.L.
      ,
      • Rubens C.E.
      • Heggen L.M.
      • Haft R.F.
      • Wessels M.R.
      ). Studies of opsonophagocytic killing of type III GBS in vitro demonstrated a direct correlation between the amount of capsule produced by a strain and its level of resistance to complement-mediated phagocytic killing by human blood leukocytes (
      • Marques M.B.
      • Kasper D.L.
      • Pangburn M.K.
      • Wessels M.R.
      ). Other studies suggest not only that the capsule is important in virulence but also that the amount of capsule produced under different circumstances may vary, perhaps as a means to enhance adaptation of the organism to various ecological niches within the human host (
      • Hakansson S.
      • Granlund-Edstedt M.
      • Sellin M.
      • Holm S.E.
      ). Despite the importance of the capsular polysaccharide in the pathogenesis of GBS infection, relatively little is known about the biochemistry of capsule biosynthesis or the nature of the linkage of the capsular polysaccharide to the bacterial cell surface.
      Nine GBS capsular types have been identified serologically, and the repeating unit structure of each has been defined (
      • Jennings H.J.
      • Katzenellenbogen E.
      • Lugowski C.
      • Kasper D.L.
      ,
      • Jennings H.J.
      • Rosell K.-G.
      • Katzenellenbogen E.
      • Kasper D.L.
      ,
      • Wessels M.R.
      • Pozsgay V.
      • Kasper D.L.
      • Jennings H.J.
      ,
      • DiFabio J.L.
      • Michon F.
      • Brisson J.-R.
      • Jennings H.J.
      • Wessels M.R.
      • Benedi V.-J.
      • Kasper D.L.
      ,
      • Wessels M.R.
      • DiFabio J.L.
      • Benedi V.-J.
      • Kasper D.L.
      • Michon F.
      • Brisson J.-R.
      • Jelinkova J.
      • Jennings H.J.
      ,
      • Kogan G.
      • Uhrin D.
      • Brisson J.-R.
      • Paoletti L.C.
      • Kasper D.L.
      • von Hunolstein C.
      • Orefici G.
      • Jennings H.J.
      ,
      • Kogan G.
      • Brisson J.-R.
      • Kasper D.L.
      • von Hunolstein C.
      • Orefici G.
      • Jennings H.J.
      ,
      • Kogan G.
      • Uhrin D.
      • Brisson J.-R.
      • Paoletti L.C.
      • Blodgett A.E.
      • Kasper D.L.
      • Jennings H.J.
      ). The type III capsular polysaccharide is one of the three major capsular types associated with invasive neonatal infection and the most common serotype in GBS meningitis (
      • Wenger J.D.
      • Hightower A.W.
      • Facklam R.R.
      • Gaventa S.
      • Broome C.V.
      ,
      • Dillon Jr., H.C.
      • Khare S.
      • Gray B.M.
      ). The repeating unit structure of GBS type III polysaccharide is illustrated in Fig.1. Although previous investigations have suggested the capsular polysaccharide is linked to peptidoglycan in the cell wall (
      • deCueninck B.J.
      • Shockman G.D.
      • Swenson R.M.
      ,
      • Yeung M.K.
      • Mattingly S.J.
      ), the nature of the attachment of the capsular polysaccharide to the GBS cell wall has not been clearly defined. In this study, we present evidence to show that the type III GBS capsular polysaccharide is covalently linked via a phosphodiester bond and a linker oligosaccharide to N-acetylglucosamine residues of the disaccharide repeating unit of cell wall peptidoglycan, while the group B antigen is linked to N-acetylmuramic acid.
      Figure thumbnail gr1
      Figure 1Panel A, structure of the pentasaccharide repeating unit of type III GBS capsular polysaccharide (
      • Wessels M.R.
      • Pozsgay V.
      • Kasper D.L.
      • Jennings H.J.
      ). The large arrows indicate the cleavage sites of endo-β-galactosidase. Panel B, proposed tetraantenary structure of group B carbohydrate (
      • Michon F.
      • Chalifour R.
      • Feldman R.
      • Wessels M.
      • Kasper D.L.
      • Gamian A.
      • Pozsgay V.
      • Jennings H.J.
      ). A–Drepresent the major component oligosaccharides, and Prepresents phosphate. The sequences of the four oligosaccharides are as follows. A, α-l-Rhap-(1→3)-α-d-Galp-(1→3)-β-d-GlcpNAc-(1→4)-α-l-Rhap-(1→2)-[α-l-Rhap-(1→3)-α-d-Galp-(1→3)-β-d-GlcpNAc-(1→4)-]-α-l-Rhap-(1→2)-α-l-Rhap-(1→1“)-d-glucitol(3”→1)-α-l-Rhap. B, α-l-Rhap-(1→2)-[α-l-Rhap-(1→3)-α-d-Galp-(1→3)-β-d-GlcpNAc-(1→4)-]-α-l-Rhap-(1→2)-α-l-Rhap-(1→1“)-d-glucitol(3”→1)-α-l-Rhap. C, α-l-Rhap-(1→2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→1“)-d-glucitol(3”→1)-α-l-Rhap-.D, α-l-Rhap-(1→3)-α-d-Galp-(1→3)-β-d-GlcpNAc-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-l-Rhap-(1→4)-d-GlcNAc. The group B carbohydrate also contains additional minor component oligosaccharides.

      DISCUSSION

      The capsular polysaccharide is a major surface structure of GBS isolates associated with human infection. However, as with other Gram-positive bacterial species, the nature of the attachment between the GBS capsular polysaccharide and the bacterial cell is incompletely understood. In Gram-negative bacteria, the capsular polysaccharide is linked via a lipid moiety to the bacterial outer membrane (
      • Gotschlich E.C.
      • Fraser B.A.
      • Nishimura O.
      • Robbins J.B.
      • Liu T.-Y.
      ,
      • Kuo J.S.-C.
      • Doelling V.W.
      • Graveline J.F.
      • McCoy D.
      ). By contrast, evidence from studies of S. pneumoniae,Staphylococcus aureus, and GBS suggest that the capsular polysaccharide of each of these Gram-positive pathogens is covalently attached to the bacterial cell wall (
      • deCueninck B.J.
      • Shockman G.D.
      • Swenson R.M.
      ,
      • Yeung M.K.
      • Mattingly S.J.
      ,
      • Sorenson U.B.S.
      • Henrichsen J.
      • Chen H.-C.
      • Szu S.C.
      ,
      • Karakawa W.W.
      • Fournier J.M.
      • Vann W.R.
      • Arbeit R.
      • Schneerson R.
      • Robbins J.B.
      ). Treatment of the bacterial cells with enzymes that cleave peptidoglycan releases the capsular polysaccharide from the cell, suggesting a direct or indirect linkage of capsule to cell wall peptidoglycan. deCueninck et al. (
      • deCueninck B.J.
      • Shockman G.D.
      • Swenson R.M.
      ) found that mutanolysin treatment of type III GBS cell walls yielded soluble complexes that contained constituents of peptidoglycan, group B carbohydrate, and capsular polysaccharide. The complexes could be cleaved by base treatment to release capsular polysaccharide free of peptidoglycan. In similar experiments, Yeung and Mattingly (
      • Yeung M.K.
      • Mattingly S.J.
      ) analyzed capsular polysaccharide released by base treatment and detected alanine, lysine, and glutamic acid in addition to the constituent sugars of the type III polysaccharide. On the basis of this evidence, they suggested that the capsular polysaccharide was attached to the peptide cross-bridges of GBS peptidoglycan.
      In the present study, we confirmed the hypothesis suggested by earlier reports that both the capsular polysaccharide and the group B carbohydrate are covalently bound to the cell wall. Insoluble cell wall complexes isolated from type III GBS were found to contain immunoreactive type III polysaccharide and to contain sugars characteristic of both type III capsular polysaccharide and group B carbohydrate in addition to the expected sugars and amino acids of GBS peptidoglycan. Treatment of the insoluble cell wall complexes with the endo-N-acetylmuramidase mutanolysin released soluble complexes that contained both capsular polysaccharide and group B carbohydrate. Covalent attachment of the two polysaccharides was suggested by their coelution on both ion exchange and gel filtration chromatography and by their comigration in electrophoresis. Separation of the capsular polysaccharide and group B carbohydrate was achieved by treatment with β-N-acetylglucosaminidase, an enzyme that cleaves the glycosidic bond of the β-d-GlcNAc(1→4)β-d-MurNAc disaccharide that is the product of complete mutanolysin digestion of the glycan backbone of GBS peptidoglycan. Analysis of the separated polysaccharides revealed that the group B carbohydrate was linked to muramic acid directly or via a cross-linking peptide, while the capsular polysaccharide was linked via a phosphodiester bond and a linking oligosaccharide to N-acetylglucosamine.
      In general, β-N-acetylglucosaminidase cleaves the glycosidic bond of a terminal residue ofN-acetylglucosamine. However, in the present study, enzyme treatment of the capsular polysaccharide-group B carbohydrate complex produced products of M r ∼10,000 to >100,000, a result that implies cleavage of internal linkages within the complex. Several pieces of evidence suggest the site of action of the enzyme is the β-d-GlcNAc(1→4)β-d-MurNAc bond in polysaccharide-substituted disaccharide fragments of peptidoglycan produced by mutanolysin digestion. 1) Sequential treatment of purified GBS peptidoglycan with mutanolysin followed by β-N-acetylglucosaminidase resulted in the release of freeN-acetylglucosamine (data not shown), a result that confirms susceptibility of the β-d-GlcNAc(1→4)β-d-MurNAc bond to β-N-acetylglucosaminidase (the released freeN-acetylglucosamine presumably represents residues that were unsubstituted in the native state or residues from which the capsular polysaccharide was cleaved during purification of the peptidoglycan). 2) The capsular polysaccharide was released from insoluble cell wall complexes by β-N-acetylglucosaminidase only after digestion of the peptidoglycan with mutanolysin, which exposesN-acetylglucosamine residues. 3) β-N-Acetylglucosaminidase digestion of the complexes separated capsular polysaccharide from group B carbohydrate, an observation that implies the enzyme acts on a moiety that links the two polysaccharides. 4) Muramic acid remained with the group B carbohydrate after β-N-acetylglucosaminidase treatment, while reducing terminal N-acetylglucosamine residues were detected in the capsular polysaccharide fraction. Thus, evidence from several different experimental approaches indicates that β-N-acetylglucosaminidase cleaves the glycosidic linkage to muramic acid of (polysaccharide-substituted)N-acetylglucosamine residues exposed by mutanolysin digestion of peptidoglycan.
      Our results do not support the earlier suggestion that the capsular polysaccharide is attached to the peptide cross-bridges of peptidoglycan (
      • Yeung M.K.
      • Mattingly S.J.
      ). Rather, the data indicate that the capsule is linked to N-acetylglucosamine residues of the glycan backbone. Potentially available sites on N-acetylglucosamine residues of the GBS peptidoglycan are at C-3 and C-6. Substitution at C-6 may be more compatible with the observed sensitivity in our studies of the GBS capsular polysaccharide-peptidoglycan fragment complex to digestion with β-N-acetylglucosaminidase. We speculate that substitution at C-6 is more likely to permit β-N-acetylglucosaminidase to act on a (substituted) β-linked N-acetylglucosamine residue, since a substituent at the 6-position is separated from the pyranose ring by an additional C–C bond compared with a substituent at the 3- or 4-position. The additional C–C bond separating C-6 from the pyranose ring not only places the substituent group at a greater distance from the ring structure but also probably confers greater flexibility to the portion of the molecule bearing the substituent. These effects may permit more efficient utilization by β-N-acetylglucosaminidase of a β-linked N-acetylglucosamine substituted at C-6 than one substituted at C-3. In a separate experiment, we found that the β-N-acetylglucosaminidase preparation used in our studies cleaved 4-methylumbelliferyl-7-(6-sulfo-2-acetamido-2-deoxy-β-d-glucopyranoside), albeit more slowly than the standard fluorogenic substrate 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide (data not shown), indicating that the enzyme can cleave β-linkedN-acetylglucosamine residues bearing a substituent group at C-6.
      Others have reported evidence of attachment of accessory polysaccharides to the glycan portion of peptidoglycan in several Gram-positive organisms. Examples include teichoic acid ofBacillus subtilis, teichuronic acid of Micrococcus luteus, and arabinogalactan of mycobacteria (
      • Munson R.S.
      • Glaser L.
      ,
      • Gassner G.T.
      • Dickie J.P.
      • Hamerski D.A.
      • Magnuson J.K.
      • Anderson J.S.
      ,
      • McNeil M.
      • Daffe M.
      • Brennan P.J.
      ). Where it has been examined, linkage of these polymers appears to be to C-6 of theN-acetylmuramic acid residues in contrast to the linkage of the GBS capsular polysaccharide to N-acetylglucosamine residues. In the case of GBS, N-acetylmuramic acid residues may be unavailable or energetically less favorable for attachment of the capsular polysaccharide because the group B carbohydrate is linked at this site. These results indicate that the capsular polysaccharide is distinctive among GBS accessory polysaccharides not only in its central role in virulence but also in its mode of attachment to the bacterial cell. It remains to be determined whether linkage of the capsular polysaccharide to N-acetylglucosamine residues of peptidoglycan is a feature unique to GBS or a general property of encapsulated Gram-positive bacteria.
      Results of these studies provide direct evidence that both the capsular polysaccharide and group B carbohydrate are covalently bound to peptidoglycan of the GBS cell wall. They indicate further that the two polysaccharides are attached independently and at separate sites. The general features of this model of the GBS cell surface are likely to apply generally to other encapsulated Gram-positive bacteria. These data represent strong evidence that the mechanism and site of attachment of capsular polysaccharides to the Gram-positive cell surface is fundamentally different from that in Gram-negative bacteria and from the linkage of other accessory cell wall polysaccharides in Gram-positives. Characterization of the site of attachment and of the nature of the linkage between the capsular polysaccharide and the GBS cell wall provides new insight into the basic structure of this important pathogen and may suggest potential targets for novel antimicrobial drug design for this and other encapsulated Gram-positive bacteria.

      Acknowledgments

      We thank Lawrence Paoletti for providing endo-β-galactosidase; Claudia Gravekamp, Barbara G. Reinap, and Yansong Chen for technical assistance; and Harold J. Jennings, Michael McNeil, and John S. Anderson for helpful discussions.

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