Molecular characterization of an IgA receptor from group B streptococci: sequence of the gene, identification of a proline-rich region with unique structure and isolation of N-terminal fragments with IgA-binding capacity.

Certain strains of group B streptococci express a cell surface protein that binds IgA and acts as a virulence factor. This IgA receptor is referred to here as protein Bac. The gene for protein Bac was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. The deduced amino acid sequence of 1134 residues includes a signal sequence of 37 amino acids and a putative membrane anchor region at the C-terminal end. The processed form of the receptor, 1097 residues, has a calculated molecular weight of 123,786. There are no cysteines in protein Bac, suggesting a fibrillar structure. The C-terminal half of the protein includes a 90 residues long region with a novel type of periodic structure, the "XPZ motif", in which every third amino acid is proline. Unlike other bacterial immunoglobulin-binding proteins, there are no long repeats in protein Bac. Clones which express only part of the protein Bac gene were used to show that IgA-binding takes place in the N-terminal part of the molecule. Protein Bac was originally described as an antigen called beta, but N-terminal fragments that bind IgA do not react with a reference serum against the beta antigen. These and other data indicate that protein Bac can be divided into two regions with different functions: an N-terminal IgA-binding region and a C-terminal region corresponding to the beta antigen. The IgA-binding region of protein Bac does not show any homology to protein Arp, the IgA receptor from group A streptococci, although these receptors have similar binding properties. This indicates that convergent evolution has favored the appearance of these two structurally different streptococcal IgA receptors.

Purification of the protein X of Streptococcus agalactiae with a monoclonal antibody.

The protein X of Steptococcus agalactiae is a surface antigen included in the typing scheme of group B streptococci (GBS). We have developed a monoclonal antibody to the protein X and used it to purify this antigen by affinity chromatography. Electrophoresis in polyacrylamide, and immunoblotting using the monoclonal antibody or a rabbit antiserum raised with the affinity purified protein X, revealed a major band in the region of 200 kDa and a smaller one at 100 kDa. The isolated protein X will make possible investigations of its potential role in virulence and protection.

Molecular analysis of lipoteichoic acid from Streptococcus agalactiae.

A method for the analysis of lipoteichoic acid (LTA) by polyacrylamide gel electrophoresis (PAGE) is described. Purified LTA from Streptococcus agalactiae tended to smear in the upper two-thirds of a 30 to 40% linear polyacrylamide gel, while the chemically deacylated form (cdLTA) migrated as a ladder of discrete bands, reminiscent of lipopolysaccharides. The deacylated polymer appeared to separate in this system on the basis of size, as evident from results obtained from PAGE analysis of cdLTA subjected to limited acid hydrolysis and LTA that had been fractionated by gel filtration. A survey of cdLTA from other streptococci revealed similarities in molecular weight ranges. The polymer from Enterococcus hirae was of a higher molecular weight. This procedure was used to examine the effect of penicillin and chloramphenicol on the synthesis, turnover, and heterogeneity of LTA in S. agalactiae. Penicillin appeared to enhance LTA synthesis while causing the release of this polymer into the supernatant fluid. In contrast, chloramphenicol inhibited the synthesis of this molecule and resulted in its depletion from the cell surface. Penicillin did not alter the heterogeneity of this polymer, but chloramphenicol caused an apparent shift to a lower-molecular-weight from of the LTA, as determined by PAGE. This shift in the heterogeneity of LTA did not appear to be due to increased carbohydrate substitution, since chloramphenicol did not alter the electrophoretic migration profile of LTA from E. hirae. From a pulse-chase study, it was determined that LTA was released as a consequence of deacylation.

Characterization of an IgA receptor from group B streptococci: specificity for serum IgA.

Some strains of group B streptococci express a cell surface protein which binds IgA. This report describes some properties of such an IgA receptor and compares it with a previously described IgA receptor from group A streptococci. The group B receptor was released in an almost pure form from bacteria incubated at elevated pH, and could be isolated by IgA-Sepharose affinity chromatography. The sequence of the N-terminal 19 amino acid residues was unique. The receptor preferentially binds IgA of human origin, as shown in immunoblotting experiments with purified IgA from nine different species. The affinity constant of the purified receptor for serum IgA was determined to be 3.5 x 10(8) M-1, but for secretory IgA it was too low to allow determination. This result indicates that secretory component and/or J chain interferes with the binding of IgA to this type of bacterial receptor, which may be one of the physiological functions of these polypeptides. A reduction in affinity was also observed for another complexed form of IgA, alpha 1-microglobulin-IgA. The group B receptor is antigenically unrelated to the IgA receptor from group A streptococci (protein Arp), but competitive inhibition experiments indicate that they bind to the same region in IgA. The implications of these findings, and the biological role of bacterial IgA receptors, are discussed.

Synthesis of a tri- and a tetra-saccharide fragment of the capsular polysaccharide of type III group B Streptococcus.

Syntheses of the propyl glycosides (1-3) of beta-D-Galp-(1----4)-beta-D-GlcpNAc, beta-D-Glcp-(1----6)-[beta-D-Galp-(1----4)]-beta-D-GlcpNAc, and beta-D-Galp-(1----4)-beta-D-Glcp-(1----6)-[beta-D-Galp-(1----)]-beta-D- GlcpNAc, respectively, are reported. Reaction of allyl 2-acetamido-3-O-benzyl-2-deoxy-6-O-(4-methoxybenzyl)-beta-D-glucopyranos ide with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide under Hg(CN)2 catalysis, followed by oxidative removal of the 4-methoxybenzyl group, gave allyl 2-acetamido-3-O-benzyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-beta-D-glucopyranoside (10) O-deacetylation of which, followed by hydrogenolysis/hydrogenation, gave 1. Reaction of 10 with beta-D-glucopyranose penta-acetate and beta-lactose octa-acetate, under catalysis by trimethylsilyl trifluoromethanesulfonate, and treatment of the products as for 10 gave 2 and 3, respectively. Attempted glycosylation of 10 with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl bromide under catalysis by Hg(CN)2 or silver trifluoromethanesulfonate gave an orthoester. Complete assignments of the 1H- and 13C-n.m.r. spectra of 1-3 are reported together with their carbon spin-lattice relaxation times which indicate that 3 assumes a compact instead of an extended shape.

Whole-cell protein patterns of nonhemolytic group B, type Ib, streptococci isolated from humans, mice, cattle, frogs, and fish.

Whole-cell protein and physiological patterns of nonhemolytic group B, type Ib, streptococci isolated from humans, cattle, frogs, fish, and mice were compared. Isolates from humans, fish, and mice were identical. Only minor differences were seen in the isolates from human, bovine, and frog sources. Nonhemolytic group B streptococci from humans, fish, and mice and, to a lesser extent, from cattle and frogs share several characteristics, including a high similarity in proteins (on the basis of molecular weight); this suggests that they may have a common ancestry.

Isolation and characterization of type IV group B Streptococcus capsular polysaccharide.

An antigenically distinct serotype, type IV, has recently been added to the recognized serotypes of group B streptococci (GBS). We isolated and purified the capsular polysaccharide antigen from a prototype type IV GBS strain. The type IV capsular polysaccharide formed a precipitin line with rabbit antiserum to type IV GBS organisms but not with antiserum to organisms of GBS serotype Ia, Ib, II, or III. Enzyme-linked immunosorbent assay inhibition experiments showed no cross-reaction between type IV antiserum and other GBS serotypes. Capsular polysaccharide released from the bacterial cells with mutanolysin and that isolated from the culture supernatant had similar elution profiles on Sepharose CL-6B, with a Kav of 0.30 and an estimated Mr of 200,000. The purified type IV polysaccharide was found to contain galactose, glucose, N-acetylglucosamine, and N-acetylneuraminic acid (sialic acid) as exclusive sugars. The polysaccharide contained 23% (by weight) sialic acid and galactose, glucose, and N-acetylglucosamine in a relative ratio of (1):1.10:0.55. These results are compatible with a repeating structure of six monosaccharide residues containing galactose, glucose, N-acetylglucosamine, and sialic acid in a molar ratio of 2:2:1:1. Unlike type Ia, II, and III GBS polysaccharides, desialylation of the type IV polysaccharide produced an antigen which formed a line of identity with the native type IV antigen in double diffusion in agar against homologous antiserum. This result suggests that sialic acid is not as critical to the immunodeterminant structure of the type IV antigen as it is for other GBS capsular types.

Cell electrophoresis of group B streptococci: separation of types Ia, Ib/c, II, III and IV before and after neuraminidase treatment.

Group B streptococcal strains of types Ia, Ib/c, II, III and IV could be characterized by distinct electrophoretic mobilities before and after neuraminidase treatment. By this method of cell electrophoresis in the type I a strain, two subpopulations could be detected; whereas in all strains the electrophoretic mobility is markedly reduced after enzymatic removal of neuraminic acid, type II remained unaffected. The method of "bacteriopheresis" offers a new approach to classification of bacteria with respect to the primary and secondary surface structures.

Preservation of capsular material of streptococcal cells by specific lectins determined by immunoelectron microscopy.

We describe the use of lectins as specific stabilizing agents for the polysaccharide capsular components of two Gram-positive bacteria, Streptococcus agalactiae and Streptococcus bovis. Treatment of bacterial suspensions with wheatgerm agglutinin and concanavalin A allowed better morphological preservation as well as immunoelectron microscopic localization of a capsular component (lipoteichoic acid) by employing specific antibodies and the protein A-gold technique. Data obtained indicate that lectins are useful agents in preserving highly water-soluble capsular components during the electron microscopy procedures for both unembedded and embedded samples.

Sensitivity and specificity of latex agglutination tests used to identify Streptococcus agalactiae and Staphylococcus aureus isolated from bulk tank milk.

Comparisons were made among rapid latex agglutination tests and conventional biochemical tests used to identify Streptococcus agalactiae and Staphylococcus aureus. Ninety-eight streptococci and 149 staphylococci isolated from bulk tank milk were tested. Sensitivity and specificity for the latex agglutination test used for identification of Str agalactiae were 97.6 and 98.2%, respectively. Sensitivity and specificity for the latex agglutination test used for identification of S aureus were 90.2 and 67.5%, respectively. Of 25 staphylococci considered false-positive by the latex agglutination test, 14 (56%) were considered tube coagulase-positive. Fifteen staphylococci considered false-positive by latex agglutination test had biotypes representative of S hyicus of S xylosus.

Complete amino acid sequence of protein B.

The complete amino acid sequence of protein B (= CAMP factor) of Streptococcus agalactiae has been determined. The sequence data were obtained mainly by manual sequencing of peptides derived from digestion with lysyl-peptidase, clostripain and Staphylococcus aureus protease and by solid phase sequencing of cyanogen bromide fragments. The protein contains 226 amino acids and has an Mr of 25,263. The sequence was compared with sequences of other Fc-binding proteins and partial sequence homology was found between protein B and the Fc-binding region of protein A.

Multiantennary group-specific polysaccharide of group B Streptococcus.

The group-specific antigen of group B Streptococcus is composed of four different oligosaccharide units of Mw 766 (III), 1277 (II), 1462 (IV), and 1788 (I). The major constituent sugars of the oligosaccharides are alpha-L-rhamnopyranose, alpha-D-galactopyranose, 2-acetamido-2-deoxy-beta-D-glucopyranosyl, and D-glucitol except that III does not contain alpha-D-galactopyranosyl or 2-acetamido-2-deoxy-beta-D-glucopyranosyl residues and IV contains no D-glucitol but has one additional beta-L-rhamnopyranosyl residue. The structures of II and III have been previously elucidated [Michon, F., Katzenellenbogen, E., Kasper, D. L., & Jennings, H. J. (1987) Biochemistry 26, 476-486]. In the group B antigen all the oligosaccharides are linked by one type of phosphodiester bond from O6 of the D-glucitol residue of one oligosaccharide to O6 of the alpha-D-galactopyranosyl residue of the next to form a complex and highly branched multiantennary structure. However, despite the heterogeneous nature of its component oligosaccharides, some order has been identified in the biosynthesis of the group B antigen from chemical and enzymatic sequence studies. Because III lacks an alpha-D-galactopyranosyl residue but has a D-glucitol residue, it is situated at the reducing terminus of all the branches of the group B antigen where it is always adjacent to a II moiety. Conversely, IV has an alpha-D-galactopyranosyl residue but has no D-glucitol and is therefore located at the reducing terminus of the group B antigen where it probably functions as a linker molecule between the group B polysaccharide and the cell wall peptidoglycan of the group B streptococcal organisms. Oligosaccharide I contains two alpha-D-galactopyranosyl residues and one D-glucitol residue and thus constitutes the branch point in the group B antigen, whereas II contains one of each of the above residues and therefore is situated in linear interchain positions. The group B antigen is highly branched and probably has a unique multiantennary structure.

Immuno-electronmicroscopic demonstration of capsules on group-B streptococci of new serotypes and type candidates.

The location of type polysaccharides on the cells of reference strains of group-B streptococci of serotypes IV and V and new type candidates NT6 and 7271 was investigated by electronmicroscopy of the bacteria after incubation with homologous type-specific antiserum. A distinct capsular layer was found on the surface of the cells of all these strains. Sialic acid, an integral part of all the conventional type polysaccharides of group-B streptococci, was also detected in all the strains examined.

[Isolation of group-specific polysaccharide complexes from group B streptococci by phenol-water extraction]. / Einsatz der Phenol-Wasser-Extraktion zur Isolierung des gruppenspezifischen Polysaccharidkomplexes aus B-Streptokokken.

The group specific polysaccharide of Group B streptococci was isolated either by means of phenol-water extraction or by Lancefield extraction procedures. Two groups of rabbits were immunized with these substances to induce polyclonal antibodies. The phenol-water extract induced antisera had a significantly higher titer against B streptococci than against A-, D- and G streptococci in the agglutination reaction. The B-specific titers in rabbits immunized with phenol-water extract were significantly higher than those obtained in the rabbits immunized with Lancefield extract. In counter immunoelectrophoresis, only the antisera prepared by immunization with phenol-water extract reacted with standard antigen from group B streptococci. The serological specificity of the phenol-water extract antigen isolated from B streptococci was confirmed by counterimmunoelectrophoresis. The antigen was a heterologous fraction with a molecular mass of 8 x 10(4)-2 x 10(6) Dalton.

Carbohydrate profiling of bacteria by gas chromatography-mass spectrometry: chemical derivatization and analytical pyrolysis.

Carbohydrate profiling by gas chromatography-mass spectrometry is a powerful tool for the identification and detection of bacteria. Its increasing applicability in the microbiology laboratory is illustrated by three examples. In the first, differentiation of legionellae by their sugar composition was determined with alditol acetate derivatization followed by selected- ion monitoring. In the second example, a carbohydrate pyrolysis product from Streptococcus agalactiae was used to differentiate group B streptococci from other Lancefield groups after direct sampling from culture plates. The third example employed the carbohydrates rhamnose and muramic acid as chemical markers for the direct detection of bacterial cell wall degradation products in mammalian tissues. The analysis of carbohydrate markers for bacteria by gas chromatography-mass spectrometry has great potential for use in clinical identification of isolated bacteria as well as in the rapid diagnosis of bacterial infections without prior culture.

Comparative analysis of the localization of lipoteichoic acid in Streptococcus agalactiae and Streptococcus pyogenes.

The cellular locations of deacylated lipoteichoic acid (dLTA) and lipoteichoic acid (LTA) were examined in late-exponential-phase cells of a serotype III strain of Streptococcus agalactiae (group B streptococci [GBS]) isolated from an infant with late-onset meningitis and compared with a fresh clinical isolate of Streptococcus pyogenes (group A streptococci [GAS]). LTA and dLTA were found to be associated with the protoplast membranes of both organisms, with only dLTA found in mutanolysin cell wall digests. Both organisms released dLTA during growth, but only the GAS released substantial levels of LTA into the culture medium. However, penicillin treatment (5 micrograms/ml for 60 min) of GBS resulted in the recovery of LTA in cell wall digests as well as in the culture medium. These results suggest that under normal growth conditions, the hydrophobic region (glycolipid) of LTA remains associated with the cytoplasmic membrane of GBS and unavailable for hydrophobic interactions at the cell surface with epithelial cells. In contrast, release of LTA into the environment by the GAS allows the fatty acid moieties to interact with hydrophobic domains on the surface of epithelial cells. These results may help explain the marked differences in the specificity of binding between these two major streptococcal pathogens for human fetal and adult epithelial cells.

Presumptive identification of group B streptococci by rapid detection of CAMP factor and pigment production.

Modified rapid versions of CAMP and pigment tests were developed for the identification of group B streptococci using the supernatant and cell deposit of organisms grown for 4 hr in a modified Todd-Hewitt broth. All but one of 203 group B streptococci were reliably differentiated from 148 nongroup B strains.

Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen.

We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus. Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----6 )]- beta-D-GlcpNAc-(1----3)-beta-D-Gal. The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit. High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58, 112-120). This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide. Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic. Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.