A polysaccharide from the human commensal Bacteroides fragilis protects against CNS demyelinating disease.

The intestinal microbiome may have a critical roll in susceptibility or resistance to immune-mediated diseases. Alterations of the gut microflora after oral antibiotic treatment can regulate encephalomyelitis (EAE), an animal model for human multiple sclerosis (MS). We now show that a zwitterionic capsular polysaccharide A (PSA) of Bacteroides fragilis can protect against central nervous system demyelinating disease. Oral administration with purified PSA protected mice against EAE prophylactic and therapeutically. PSA treatment enhanced CD103 expressing dendritic cells (DCs) that accumulated in the cervical lymph nodes. Exposure of naïve DCs to PSA induced the conversion of naïve CD4(+) T cells into interleukin (IL)-10-producing FoxP3(+)Treg cells. Protection against EAE was completely abrogated in IL-10-deficient mice. Our results show an important role for a molecule from human commensal bacteria in protecting against EAE and suggest the possibility for protection in MS.

Extensive surface diversity of a commensal microorganism by multiple DNA inversions.

The dynamic interactions between a host and its intestinal microflora that lead to commensalism are unclear. Bacteria that colonize the intestinal tract do so despite the development of a specific immune response by the host. The mechanisms used by commensal organisms to circumvent this immune response have yet to be established. Here we demonstrate that the human colonic microorganism, Bacteroides fragilis, is able to modulate its surface antigenicity by producing at least eight distinct capsular polysaccharides-a number greater than any previously reported for a bacterium-and is able to regulate their expression in an on-off manner by the reversible inversion of DNA segments containing the promoters for their expression. This means of generating surface diversity allows the organism to exhibit a wide array of distinct surface polysaccharide combinations, and may have broad implications for how the predominant human colonic microorganisms, the Bacteroides species, maintain an ecological niche in the intestinal tract.

Novel engagement of CD14 and multiple toll-like receptors by group B streptococci.

Group B streptococcus (GBS) imposes a major health threat to newborn infants. Little is known about the molecular basis of GBS-induced sepsis. Both heat-inactivated whole GBS bacteria and a heat-labile soluble factor released by GBS during growth (GBS-F) induce nuclear translocation of NF-kappaB, the secretion of TNF-alpha, and the formation of NO in mouse macrophages. Macrophages from mice with a targeted disruption of MyD88 failed to secrete TNF-alpha in response to both heat-inactivated whole bacteria and GBS-F, suggesting that Toll-like receptors (TLRs) are involved in different aspects of GBS recognition. Immune cell activation by whole bacteria differed profoundly from that by secreted GBS-F. Whole GBS activated macrophages independently of TLR2 and TLR6, whereas a response to the secreted GBS-F was not observed in macrophages from TLR2-deficient animals. In addition to TLR2, TLR6 and CD14 expression were essential for GBS-F responses, whereas TLR1 and TLR4 or MD-2 did not appear to be involved. Heat lability distinguished GBS-F from peptidoglycan and lipoproteins. GBS mutants deficient in capsular polysaccharide or beta-hemolysin had GBS-F activity comparable to that of wild-type streptococci. We suggest that CD14 and TLR2 and TLR6 function as coreceptors for secreted microbial products derived from GBS and that cell wall components of GBS are recognized by TLRs distinct from TLR1, 2, 4, or 6.

Effects of alum adjuvant or a booster dose on immunogenicity during clinical trials of group B streptococcal type III conjugate vaccines.

Phase 1 and 2 clinical trials of group B streptococcal (GBS) capsular polysaccharide (CPS)-protein conjugate vaccines in healthy adults have demonstrated their safety and improved immunogenicity compared with uncoupled CPSs. Two recent trials sought to determine (i) whether adsorption of conjugate vaccine to aluminum hydroxide would improve immunogenicity and (ii) whether the CPS-specific immunoglobulin G (IgG) response could be boosted by administration of a second dose. Adsorption of GBS type III CPS-tetanus toxoid (III-TT) conjugate vaccine to alum did not improve the immune response to a 12.5-microg dose in healthy adult recipients. Four weeks after vaccination, the geometric mean antibody concentrations (GMCs) for the 15 recipients of III-TT with or without alum were 3.3 and 3.6 microg/ml, respectively. In the second trial, 36 healthy adults vaccinated previously with GBS III-TT conjugate were given a second 12.5-microg dose 21 months later. At 4 weeks after the second dose, the GMCs of type III CPS-specific IgG were similar to those measured 4 weeks after the primary vaccination, suggesting a lack of a booster response. However, 8 (22%) of the 36 participants who had undetectable III CPS-specific IgG (<0.05 microg/ml) before the first dose of III-TT conjugate exhibited a booster response to the second dose, with a fourfold-greater GMC of type III CPS-specific IgG than after the initial immunization. These results suggest that prior natural exposure to type III GBS or a related antigen may be responsible for the brisk IgG response to CPS noted in most adults after vaccination. However, a second dose of GBS III-TT conjugate vaccine may be required for adults whose initial CPS-specific IgG concentrations are very low and would also restore the initial peak-specific III CPS-IgG in responders to previous vaccination.

Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis.

Bacteroides fragilis, though only a minor component of the human intestinal commensal flora, is the anaerobe most frequently isolated from intra-abdominal abscesses. B. fragilis 9343 expresses at least three capsular polysaccharides-polysaccharide A (PS A), PS B, and PS C. Purified PS A and PS B have been tested in animal models and are both able to induce the formation of intra-abdominal abscesses. Mutants unable to synthesize PS B or PS C still facilitate abscess formation at levels comparable to those of wild-type 9343. To determine the contribution of PS A to abscess formation in the context of the intact organism, the PS A biosynthesis region was cloned, sequenced, and deleted from 9343 to produce a PS A-negative mutant. Animal experiments demonstrate that the abscess-inducing capability of 9343 is severely attenuated when the organism cannot synthesize PS A, despite continued synthesis of the other capsular polysaccharides. The PS A of 9343 contains an unusual free amino sugar that is essential for abscess formation by this polymer. PCR analysis of the PS A biosynthesis loci of 50 B. fragilis isolates indicates that regions flanking each side of this locus are conserved in all strains. The downstream conserved region includes two terminal PS A biosynthesis genes that homology-based analyses predict are involved in the synthesis and transfer of the free amino sugar of PS A. Conservation of these genes suggests that this sugar is present in the PS A of all serotypes and may explain the abscessogenic nature of B. fragilis.

Immunochemical and biological characterization of three capsular polysaccharides from a single Bacteroides fragilis strain.

Although Bacteroides fragilis accounts for only 0.5% of the normal human colonic flora, it is the anaerobic species most frequently isolated from intra-abdominal and other infections with an intestinal source. The capsular polysaccharides of B. fragilis are part of a complex of surface polysaccharides and are the organism's most important virulence factors in the formation of intra-abdominal abscesses. Two capsular polysaccharides from strain NCTC 9343, PS A1 and PS B1, have been characterized structurally. Their most striking feature is a zwitterionic charge motif consisting of both positively and negatively charged substituent groups on each repeating unit. This zwitterionic motif is essential for abscess formation. In this study, we sought to elucidate structural features of the capsular polysaccharide complex of a commonly studied B. fragilis strain, 638R, that is distinct from strain 9343. We sought a more general picture of the species to establish basic structure-activity and structure-biosynthesis relationships among abscess-inducing polysaccharides. Strain 638R was found to have a capsular polysaccharide complex from which three distinct carbohydrates could be isolated by a complex purification procedure. Compositional and immunochemical studies demonstrated a zwitterionic charge motif common to all of the capsular polysaccharides that correlated with their ability to induce experimental intra-abdominal abscesses. Of interest is the range of net charges of the isolated polysaccharides-from positive (PS C2) to balanced (PS A2) to negative (PS 3). Relationships among structural components of the zwitterionic polysaccharides and their molecular biosynthesis loci were identified.

Functional analysis in type Ia group B Streptococcus of a cluster of genes involved in extracellular polysaccharide production by diverse species of streptococci.

Several species of streptococci produce extracellular polysaccharides in the form of secreted exopolysaccharides or cell-associated capsules. Although the biological properties and repeating unit structures of these polysaccharides are diverse, sequence analysis of the genes required for their production has revealed a surprising degree of conservation among five genes found in the capsule gene cluster of each of several polysaccharide-producing streptococci. To determine the function of these conserved genes, we characterized a series of isogenic mutants derived from a wild-type strain of type Ia group B Streptococcus by selectively inactivating each gene. Inactivation of cpsIaE resulted in an acapsular phenotype, consistent with previous work that identified the cpsIaE product as the glycosyltransferase that initiates synthesis of the polysaccharide repeating unit. Mutants in cpsIaA, cpsIaB, cpsIaC, or cpsIaD produced type Ia capsular polysaccharide, but in reduced amounts compared with the wild type. Analysis of the mutant polysaccharides and of capsule gene transcription in the mutant strains provided evidence that cpsIaA encodes a transcriptional activator that regulates expression of the capsule gene operon. Mutants in cpsIaC or cpsIaD produced polysaccharide of reduced molecular size but with an identical repeating unit structure as the wild-type strain. We conclude that CpsA to -D are not required for polysaccharide repeating unit biosynthesis but rather that they direct the coordinated polymerization and export of high molecular weight polysaccharide.

Structural basis of the abscess-modulating polysaccharide A2 from Bacteroides fragilis.

Zwitterionic capsular polysaccharides from pathogenic bacteria have peculiar immunological properties. They are capable of eliciting T-cell proliferation and modulating the course of abscess formation. To understand the molecular basis of this characteristic immune response, we are conducting detailed structure-function studies on these polysaccharides. We have identified, purified, and characterized an abscess-modulating polysaccharide, PS A2, from the clinical strain Bacteroides fragilis 638R. Here, we report the elucidation of both the chemical and three-dimensional structures of PS A2 by NMR spectroscopy, chemical methods, gas chromatography-mass spectrometry, and restrained molecular dynamics calculations. PS A2 consists of a pentasaccharide repeating unit containing mannoheptose, N-acetylmannosamine, 3-acetamido-3,6-dideoxyglucose, 2-amino-4-acetamido-2,4,6-trideoxygalactose, fucose, and 3-hydroxybutanoic acid. PS A2 is zwitterionic and carries one cationic free amine and one anionic carboxylate in each repeating unit. It forms an extended right-handed helix with two repeating units per turn and a pitch of 20 A. Positive and negative charges are exposed on the outer surface of the polymer in a regularly spaced pattern, which renders them easily accessible to other molecules. The helix is characterized by repeated large grooves whose lateral boundaries are occupied by the charges. The three-dimensional structure of PS A2 explicitly suggests mechanisms of interaction between zwitterionic polysaccharides and proteins.

Bacteroides fragilis NCTC9343 produces at least three distinct capsular polysaccharides: cloning, characterization, and reassignment of polysaccharide B and C biosynthesis loci.

Bacteroides fragilis produces a capsular polysaccharide complex (CPC) that is directly involved in its ability to induce abscesses. Two distinct capsular polysaccharides, polysaccharide A (PS A) and PS B, have been shown to be synthesized by the prototype strain for the study of abscesses, NCTC9343. Both of these polysaccharides in purified form induce abscesses in animal models. In this study, we demonstrate that the CPC of NCTC9343 is composed of at least three distinct capsular polysaccharides: PS A, PS B, and PS C. A previously described locus contains genes whose products are involved in the biosynthesis of PS C rather than PS B as was originally suggested. The actual PS B biosynthesis locus was cloned, sequenced, and found to contain 22 genes in an operon-type structure. A mutant with a large chromosomal deletion of the PS B biosynthesis locus was created so that the contribution of PS B to the formation of abscesses could be assessed in a rodent model. Although purified PS B can induce abscesses, removal of this polysaccharide does not attenuate the organism's ability to induce abscesses.

Genetic diversity of the capsular polysaccharide C biosynthesis region of Bacteroides fragilis.

A genetic approach was used to assess the heterogeneity of the capsular polysaccharide C (PS C) biosynthesis locus of Bacteroides fragilis and to determine whether distinct loci contain genes whose products are likely to be involved in conferring charged groups that enable the B. fragilis capsular polysaccharides to induce abscesses. A collection of 50 B. fragilis strains was examined. PCR analysis demonstrated that the genes flanking the PS C biosynthesis region are conserved, whereas the genes within the loci are heterogeneous. Only cfiA(+) B. fragilis strains, which represent 3% of the clinical isolates of B. fragilis, displayed heterogeneity in the regions flanking the polysaccharide biosynthesis genes. Primers were designed in the conserved regions upstream and downstream of the PS C locus and were used to amplify the region from 45 of the 50 B. fragilis strains studied. Fourteen PS C genetic loci could be differentiated by a combination of PCR and extended PCR. These loci ranged in size from 14 to 26 kb. Hybridization analysis with genes from the PS C loci of strains 9343 and 638R revealed that the majority of strains contain homologs of wcgC (N-acetylmannosamine dehydrogenase), wcfF (putative dehydrogenase), and wcgP (putative aminotransferase). The data suggest that the synthesis of polysaccharides that have zwitterionic characteristics rendering them able to induce abscesses is common in B. fragilis.

Use of capsular polysaccharide-tetanus toxoid conjugate vaccine for type II group B Streptococcus in healthy women.

An estimated 15% of invasive group B streptococcal (GBS) disease is caused by type II capsular polysaccharide (II CPS). In developing a pentavalent vaccine for the prevention of GBS infections, individual GBS CPSs have been coupled to tetanus toxoid (TT) to prepare vaccines with enhanced immunogenicity. Type II GBS (GBS II) vaccine was created by direct, covalent coupling of II CPS to TT by reductive amination. In 2 clinical trials, 75 healthy nonpregnant women 18-45 years old were randomized to receive II CPS-TT (II-TT) conjugate (dose range, 3.6-57 microg of CPS component) or uncoupled II CPS vaccine. Both vaccines were well tolerated. II CPS-specific IgG serum concentrations (as well as IgM and IgA) peaked 2 weeks after immunization, being significantly higher in recipients of conjugated vaccine than in recipients of uncoupled CPS. Immunological responses to conjugate were dose dependent and correlated with opsonophagocytosis in vitro. These results support inclusion of II-TT conjugate when preparing a multivalent GBS vaccine.

T cells activated by zwitterionic molecules prevent abscesses induced by pathogenic bacteria.

Immunologic paradigms classify bacterial polysaccharides as T cell-independent antigens. However, these models fail to explain how zwitterionic polysaccharides (Zps) confer protection against intraabdominal abscess formation in a T cell-dependent manner. Here, we demonstrate that Zps elicit a potent CD4+ T cell response in vitro that requires available major histocompatibility complex class II molecules on antigen-presenting cells. Specific chemical modifications to Zps show that: 1) the activity is specific for carbohydrate structure, and 2) the proliferative response depends upon free amino and carboxyl groups on the repeating units of these polysaccharides. Peptides synthesized to mimic the zwitterionic charge motif associated with Zps also exhibited these biologic properties. Lysine-aspartic acid (KD) peptides with more than 15 repeating units stimulated CD4+ T cells in vitro and conferred protection against abscesses induced by bacteria such as Bacteroides fragilis and Staphylococcus aureus. Evidence for the biologic importance of T cell activation by these zwitterionic polymers was provided when human CD4+ T cells stimulated with these molecules in vitro and adoptively transferred to rats in vivo conferred protection against intraabdominal abscesses induced by viable bacterial challenge. These studies demonstrate that bacterial polysaccharides with a distinct charge motif activate T cells and that this activity confers immunity to a distinct pathologic response to bacterial infection.

Characterization of the linkage between the type III capsular polysaccharide and the bacterial cell wall of group B Streptococcus.

The capsular polysaccharide of group B Streptococcus 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 beta-N-acetylglucosaminidase, which catalyzes hydrolysis of the beta-D-GlcNAc(1-->4)beta-D-MurNAc subunit produced by mutanolysin digestion of peptidoglycan. Evidence from gas chromatography/mass spectrometry and (31)P NMR analysis of the separated polysaccharides supports a model of the group B Streptococcus 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.

Maternal antibody transfer in baboons and mice vaccinated with a group B streptococcal polysaccharide conjugate.

Two animal models were used to study maternal transfer of antibody to a group B Streptococcus (GBS) type III polysaccharide-tetanus toxoid (III-TT) conjugate. The III-TT vaccine protected all 27 mouse pups born to vaccinated dams against a GBS challenge. In a separate study of vaccinated mouse dams and pups, maternal sera contained all 4 subclasses of polysaccharide-specific IgG, with IgG1 accounting for 83% of total IgG. Specific IgG subclass distribution (IgG1>>IgG2a=IgG2b=IgG3) in newborn pups closely resembled that in their mothers. Seven of 9 female baboons given the III-TT vaccine had 5- to 36-fold increases in specific antibody from baseline levels; they transferred 26%-185% of specific antibody to their offspring. Matched maternal and neonatal sera obtained at delivery were functionally equivalent in an in vitro opsonophagocytosis assay. These preclinical studies provide further evidence for effective immunogenicity of GBS conjugate vaccine and efficient transport of functionally active maternal antibody.

Effect of molecular size on the ability of zwitterionic polysaccharides to stimulate cellular immunity.

The large-molecular-sized zwitterionic capsular polysaccharide of the anaerobe Bacteroides fragilis NCTC 9343, designated polysaccharide (PS) A, stimulates T cell proliferation in vitro and induces T cell-dependent protection against abscess formation in vivo. In the present study, we utilized a modification of a recently developed ozonolytic method for depolymerizing polysaccharides to examine the influence of the molecular size of PS A on cell-mediated immunity. Ozonolysis successfully depolymerized PS A into structurally intact fragments. PS A with average molecular sizes of 129.0 (native), 77.8, 46.9, and 17.1 kDa stimulated CD4+-cell proliferation in vitro to the same degree, whereas the 5.0-kDa fragment was much less stimulatory than the control 129.0-kDa PS A. Rats treated with 129.0-kDa, 46.9-kDa, and 17.1-kDa PS A molecules, but not those treated with the 5.0-kDa molecule, were protected against intraabdominal abscesses induced by challenge with viable B. fragilis. These results demonstrate that a zwitterionic polysaccharide as small as 22 repeating units (88 monosaccharides) elicits a T cell-dependent immune response. These findings clearly distinguish zwitterionic T cell-dependent polysaccharides from T cell-independent polysaccharides and give evidence of the existence of a novel mechanism for a polysaccharide-induced immune response.

Cognate stimulatory B-cell-T-cell interactions are critical for T-cell help recruited by glycoconjugate vaccines.

Covalent linkage of a bacterial polysaccharide to an immunogenic protein greatly enhances the carbohydrate's immunogenicity and induces polysaccharide-specific B-cell memory in vivo. These findings have spurred the development of glycoconjugate vaccines for serious bacterial infections. The specific B-cell-T-cell interactions responsible for recruitment of T-cell help by glycoconjugate vaccines are not well defined. We used mice deficient in molecules critical for stimulatory, cognate B-cell-T-cell interactions to study how T cells improve the immunogenicity of a glycoconjugate vaccine against group B streptococcal disease. Isotype switching to immunoglobulin G (IgG) was abrogated in mice deficient in major histocompatibility complex (MHC) class II antigen (Ag)-T-cell receptor (TCR), B7-CD28, or CD40-CD40L interactions. However, expression of either the B7-1 or the B7-2 molecule on antigen-presenting cells was sufficient for optimal T-cell costimulation. T cells activated by the vaccine also played a pivotal role in determining the magnitude of the IgM response to the polysaccharide. Comparable results were obtained with pathway antagonists. These data suggest that MHC class II Ag-TCR, B7-CD28, and CD40-CD40L interactions are critical for immune responses to glycoconjugate vaccines in vivo.

Ozonolytic depolymerization of polysaccharides in aqueous solution.

The selective oxidation of beta-D-glycosidic linkages of polysaccharides by ozone has great utility as a general method for depolymerization of polysaccharides. Here we describe a 'one-step' method whereby polysaccharides dissolved in water or basic solutions are depolymerized by ozonolysis. The oxidation of glycosidic linkages of unprotected carbohydrates by ozone is complicated by several side reactions. We describe here optimized conditions for carrying out ozonolysis degradation. We also characterize the major pathways for unwanted degradation by various side reactions. In the preferred oxidation pathway, the aldosidic linkage is oxidized to an aldonic ester function that hydrolyzes under the basic conditions employed to give a free aldonate, with cleavage of the polysaccharide chain. Nonselective degradation pathways include oxidative degradation by radical species that oxidize glycosyl residues to formic, acetic, and oxalic acids. The nonselective degradation caused by acids is minimized by basic buffers. The products of polysaccharide depolymerization form a size distribution around a nominal molecular weight, and the average molecular weight of the products can be controlled by the rate or amount of ozone passed through the reaction mixture. The ozonolysis method described herein provides a convenient, inexpensive, and controllable means for generating small polysaccharides or large oligosaccharide fragments.

Interstrain variation of the polysaccharide B biosynthesis locus of Bacteroides fragilis: characterization of the region from strain 638R.

The sequence and analysis of the capsular polysaccharide biosynthesis locus, PS B2, of Bacteroides fragilis 638R are described, and the sequence is compared with that of the PS B1 biosynthesis locus of B. fragilis NCTC 9343. Two genes of the region, wcgD and wcgC, are shown by complementation to encode a UDP-N-acetylglucosamine 2-epimerase and a UDP-N-acetylmannosamine dehydrogenase, respectively.

Synthesis and preclinical evaluation of glycoconjugate vaccines against group B Streptococcus types VI and VIII.

Group B Streptococcus (GBS) types VI and VIII are prevalent among serotypes isolated from pregnant women in Japan. Maternal vaccination with a safe and effective GBS vaccine has been proposed as a rational approach to prevent neonatal GBS disease. Because antibody specific for the capsular polysaccharide (CPS) antigens of GBS is protective, vaccines were developed with purified type VI and VIII CPS coupled to tetanus toxoid. In rabbits the newly synthesized conjugate vaccines elicited high-titered, type-specific antibody that was opsonically active in vitro. Moreover, litters born to mice actively vaccinated with the conjugate vaccines, in contrast to uncoupled CPS or saline, were protected against an ordinarily lethal challenge of GBS of homologous serotype. GBS types VI and VIII conjugate vaccines of the design presented may be important components of a multivalent GBS vaccine for use in regions where these serotypes predominate.