Immunochemistry of Capsular Type Polysaccharide and Virulence ...

INFECTION AND IMMUNITY, Apr. 1993, p. 1272-1280 0019-9567/93/041272-09$02.00/0 Copyright © 1993, American Society for Microbiology

Vol. 61, No. 4

Immunochemistry of Capsular Type Polysaccharide and Virulence Properties of Type VI Streptococcus agalactiae (Group B Streptococci) CHRISTINA VON HUNOLSTEIN,l SANDRO D'ASCENZI,1 BARBARA WAGNER,2 JARMILA JELINKOVA,3 GIOVANNA ALFARONE,1 SIMONA RECCHIA,l MANFRED WAGNER,2 AND GRAZIELLA OREFICIl* Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanitai, Viale Regina Elena 299, 00161 Rome, Italy'; Institute of Experimental Microbiology, Friedrich Schiller University, D-0-6900 Jena, Gennany2; and National Public Health Institute, Srobarova 48, 100 42 Prague 10, Czechoslovakia3 Received 2 September 1992/Accepted 6 January 1993

The immunochemistry of capsular type polysaccharide and virulence characteristics of group B streptococci (GBS), type VI, were studied. By high-pressure anion-exchange chromatography and pulsed amperometric detection, as well as by 13C nuclear magnetic resonance analysis, both extracellular and cell-bound polysaccharides were found to contain glucose, galactose, and N-acetylneuraminic acid in the molar ratio of 2:2:1, respectively. At variance with all other GBS serotypes described to date (Ia, Ib, II, Ill, IV, and V), no N-acetylglucosamine was present, whatever the source of the material (secreted or cell bound; reference or clinical isolate). Sialic acid was probably involved in the immunodeterminant structure of this new serotype since cleavage of this sugar from the polysaccharide gave rise to an antigen which reacted very weakly with type VI antiserum and to a precipitation line in immunodiffusion with no identity with the native type VI polysaccharide. By using type VI antiserum and the protein A-gold technique, a large capsule was observed in the type VI GBS reference strain by electron microscopy. All type VI strains examined were lethal for CD-1 mice, the 50% lethal dose after intraperitoneal challenge ranging from 1.0 (±0.9, standard deviation) x 105 to 2.5 (±1.5, standard deviation) x 10' CFU per mouse. A rabbit antiserum against capsular type polysaccharide exhibited both protective activity for mice injected intraperitoneally with type VI reference strain or with clinical isolates and opsonic activity in a phagocytosis assay.

While Streptococcus agalactiae (group B streptococci [GBS]) has long been recognized as a leading cause of neonatal sepsis and meningitis (1, 13), its importance as a pathogen in adults has recently been emphasized (32). GBS are classified into serotypes on the basis of specific capsular type polysaccharides (type polysaccharides). The strains usually isolated from clinical cases belong to one of the major capsular types Ia, Ib, II, and III (13), but recently two new serotypes (IV and V) have been described (20). A common feature of GBS type polysaccharides is their nature of high-molecular-weight polymers with a repeating unit composed of glucose, galactose, N-acetylglucosamine, and N-acetylneuraminic acid (sialic acid) (22, 48, 49). Despite structural relatedness, these type polysaccharides are immunologically distinct, and mechanisms of host response against the pathogens are based largely on recognition by antibodies of capsular antigen specificity (37, 53). Occasionally, nontypeable strains, i.e., strains bearing capsular type antigens serologically different from the afore-

(Some of these results were presented at the 92nd General Meeting of the American Society for Microbiology, New Orleans, La., 26-30 May 1992.) MATERIALS AND METHODS Bacterial strains. Type VI reference strain 118754 was obtained from the Czechoslovak National Type Culture Collection (Prague). Additional type VI GBS clinical isolates were used: 114852, 114862, 114866, and 118775 were from the National Public Health Institute (Prague, Czechoslovakia), and B 4589 and B 4645 were kindly supplied by E. Gunther (Institute of Experimental Microbiology, Jena, Germany). Other GBS strains included 090 (type Ia), NCTC 8187 (type Ib), NCTC 11079 (type II), and NCTC 11080 (type III) (kindly supplied by G. Colman [Colindale, London, United Kingdom]) and strains 1/82 (type IV) and 10/84 (type V) (Czechoslovak National Type Culture Collection). Extraction and purification of type polysaccharide. Bacteria were grown in 8 liters of ultrafiltered (DC 10 LA Amicon filtration system, with 10,000-Mr-cutoff hollow fiber cartridges [H5P10-43]; Amicon Corp., Danvers, Mass.) ToddHewitt broth containing 1.5% (wt/vol) Na2HPO4 and 2% (wt/vol) glucose with continuous titration to pH 7.2 with 5 N NaOH. Cultures were grown for 24 h at 37°C with continuous stirring (200 rpm) and aeration (8 liters min-'). Type polysaccharide was obtained from culture supernatants after the culture was heated at 60°C for 30 min and centrifuged at 8,000 x g for 20 min. The supernatant was concentrated to 10% of its original volume (with 30,000-Mrcutoff Amicon hollow fiber cartridges [H5P30-43]). The

mentioned classical serotypes, are isolated from either healthy subjects or patients (31, 35, 52). Here we report the pathogenicity and the immunochemical characterization of the type polysaccharide of a nontypeable GBS strain representing S. agalactiae type VI, a new GBS serotype. Type VI GBS are substantially pathogenic for mice, and their capsular type polysaccharide has the unique characteristic of lacking N-acetylglucosamine, a common sugar in all other GBS type strains (22, 48, 49). *

Corresponding author. 1272

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retentate was brought to 30% (vol/vol) with ethanol at 4°C, and the precipitate was discarded. The solution was then adjusted to 80% (vol/vol) ethanol and kept at 4°C for 18 h. The precipitate was dialyzed against water and lyophilized. The crude extract was dissolved in 10 mM Tris-HCl (pH 7.3) and loaded onto a Sepharose 4B column (2.6 by 90 cm) (Pharmacia Fine Chemicals, Uppsala, Sweden) preequilibrated with the same buffer. Fractions reacting in double diffusion with type-specific antiserum were pooled and further purified by DEAE-Sephacel chromatography, with a column of 2.5 by 60 cm (Pharmacia) equilibrated in 10 mM Tris-HCI (pH 8.3). The serotype polysaccharide was eluted with a linear gradient of 0 to 500 mM NaCl in the same buffer, dialyzed against water, and lyophilized. Type VI polysaccharide was also extracted from bacterial cells. To this end, cells were washed twice with saline solution and centrifuged (8,000 x g for 20 min), and the pellet was suspended in 50 mM potassium phosphate buffer (pH 7.5) containing 10 mM EDTA and 150 mM NaCl (1 g of bacteria per 2.5 ml of buffer). The polysaccharide was extracted by ultrasonication (Soniprep 150; MSE, Crawley, United Kingdom) for 20 min and then incubated overnight at room temperature under agitation in the presence of glass beads (diameter, 3 mm). Cells were subsequently removed by centrifugation, and the supernatant was fractionated with ethanol at a 30% (vol/vol) final concentration. The precipitate was discarded, and the resulting supernatant was brought to a concentration of 80% (vol/vol) ethanol and kept at 4°C overnight. The precipitate, recovered by centrifugation, was dissolved in water, dialyzed against water, and lyophilized. The sample was then solubilized in 100 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 10 mM CaC12, and 10 mM MgCl2 and treated with 0.1 mg of DNase ml-1 (Boehringer GmbH, Mannheim, Germany) and 0.5 mg of RNase ml-' (Sigma) at 37°C overnight. The digestion was repeated by incubating the mixture for 2 h. A subsequent overnight digestion at 37°C was performed with 1 mg of pronase ml-1 (Boehringer) and repeated for 2 h. The solution was then dialyzed against water and lyophilized. The polysaccharide was purified by DEAE-Sephacel chromatography as described above. Fractions reacting in immunodiffusion with type-specific antiserum were pooled and purified by Sepharose 4B gel filtration (Pharmacia) (see above). Molecular weight estimation. The molecular size of the type polysaccharide was estimated by Sepharose 4B (Pharmacia) gel filtration (2.6 by 90 cm) equilibrated in 10 mM Tris-HCl buffer (pH 7.3) by using dextrans of predetermined Mrs as the Mr standards. Analytical methods. Protein content was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Munich, Germany) with bovine serum albumin as the standard. All sugars and sialic acid were determined both by colorimetric assays and by chromatographic techniques (i.e., high-performance anion-exchange chromatography [HPAC]; see below). Hexoses were determined by the method of Dubois et al. (11). Sialic acid was quantitated by the thiobarbituric acid method of Warren (47), after hydrolysis of the sample in sulfuric acid at 80°C for 1 h, or by an enzymatic assay kit (Boehringer). HPAC. Analysis of the monosaccharide constituents was carried out by HPAC under alkaline conditions (17). Neutral and amino sugar residues of the type polysaccharide were identified after hydrolysis of 1 mg of carbohydrate with 1 ml of 2 N trifluoroacetic acid at 100°C for 5 h. The hydrolysate was dried by lyophilization and redissolved in 1 ml of water

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for HPAC analysis. Sialic acid was determined after hydrolysis of the sample with 100 mM HCl at 80°C for 1 h. The HPAC system used throughout was composed of a Dionex (Sunnyvale, Calif.) gradient pump module (GPM II) and a pulsed amperometric detector (PAD). Sample injections were via a Dionex autosampler equipped with a 25-,ul sample loop. Saccharides were separated on a CarboPac PAl column (250 by 4 mm) serially connected with a CarboPac PA guard column (25 by 3 mm) at a flow rate of 1 ml min-1. Isocratic analysis with 15 mM NaOH as the eluant was performed for the separation of neutral and amino monosaccharides, while the elution of acidic sugars was carried out with 100 mM NaOH plus 150 mM CH3COONa. Detection was by PAD with a gold working electrode (26). The following pulse potentials and durations were employed for detection: E1 = 0.1 V (t1 = 480 ms); E2 = 0.6 V (t2 = 120 ms); E3 = -0.8 V (t3 = 60 ms). For sialic acid determination, E1 was changed to 0.05 V. NMR. The 13C nuclear magnetic resonance (NMR) spectra were obtained with a Bruker AMX 400 spectrometer (Karlsruhe, Germany) operating at 100.3 MHz in the pulsed Fourier transform mode with complete proton decoupling. The sample was contained in 5-mm-diameter tubes at 27°C. Chemical shifts (b) were quoted relative to the CH3 signal of external 4,4-dimethy-4-silapentane-1-sulfonate. The polysaccharide was run as deuterium oxide solution (50 mg

ml-').

Neuraminidase treatment of type polysaccharide. One hundred seventy micrograms of type VI polysaccharide was treated with 77 mU of immobilized neuraminidase (from Clostndium perfringens; Sigma) under agitation at 37°C. After 3 h of incubation, the enzyme was removed by centrifugation. Sialic acid-binding lectin. For the cytochemical demonstration of the N-acetylneuraminic acid of the type-specific polysaccharide, a lectin from the snail Cepaea hortensis, previously characterized for its specificity for N-acetylneuraminic acid and other N-acetyl-bearing sialic acid derivatives (7), was used. The lectin was prepared from the albumen glands of snails and purified by affinity chromatography on fetuin-Sepharose as described previously (45). For the cytochemical reaction, the bacteria were incubated with a 0.1% solution of lectin in 100 mM Tris-HCl buffer, containing 100 mM NaCl and 10 mM CaCl2 (pH 7.2), for 1 h at room temperature, washed with the same buffer, and incubated with fetuin-gold as described previously (46). Antiserum preparation. Group- and type-specific antisera were prepared by immunizing New Zealand White rabbits with formalin-killed whole bacterial cells as described by Lancefield (27). The titers of the sera were determined by enzyme-linked immunosorbent assay (ELISA), using 1 ,ug of polysaccharide (type or group) per well for coating, as described previously (43). The titers achieved were 1:64,000 for type VI-specific antiserum and 1:32,000 for group B antiserum. Gold-labeled antibodies and other gold reagents. A monodisperse suspension of colloidal gold with a mean particle size of 16 nm was prepared by the method of Frens (14). Swine anti-rabbit immunoglobulin G (IgG) (Serovac, Prague, Czechoslovakia) was adsorbed to the colloidal gold at pH 9.0, while protein A (prepared by D. Gerlach, Jena, Germany) was adsorbed at pH 7.0, and fetuin (Sigma) was adsorbed at pH 5.4 (46). Electron microscopic demonstration of the capsule and the type-specific polysaccharide. For the demonstration of the capsule, the bacteria were incubated with type VI-specific

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antiserum diluted 1:100 and prepared for transmission electron microscopy as described previously (39). The typespecific polysaccharide was demonstrated by two techniques. (i) Preembedding labeling was done by incubation of the bacteria with homologous type-specific antiserum and then, after washing with phosphate-buffered saline (PBS; pH 7.2), by incubation with protein A-gold. Fixation, dehydration, and embedding in Epon were performed as described earlier (44). (ii) For postembedding labeling, the capsule was first stabilized with type-specific antiserum as described above. After being washed with PBS, the bacteria were fixed with a mixture of 0.05% glutaraldehyde and 0.5% formaldehyde in 100 mM cacodylate buffer (pH 7.2), dehydrated at progressively lower temperatures, and embedded in Lowicryl K4M at -35°C as described previously (44). Ultrathin sections were pretreated as described previously (44), incubated at 4°C overnight with diluted (1:100) typespecific rabbit antiserum, rinsed with buffer, and incubated with anti-rabbit IgG-gold (diluted 1:80) for 30 min at room temperature. After being washed, the samples were postfixed with 0.5% glutaraldehyde in double-distilled water and stained with uranyl acetate and lead citrate. Virulence determination. To evaluate the virulence of different strains of type VI GBS, groups of eight CD-1 outbred mice (Charles River Breeding Laboratories, Calco, Milan, Italy), 4 weeks of age (approximately 12 g), were injected intraperitoneally (i.p.) with 102 to 106 CFU per mouse, and mortality was recorded at 24-h intervals for 15 days. The 50% lethal dose (LD50), calculated by the method of Reed and Muench (38), represents the mean of three experiments. Mouse protection test. Groups of eight CD-1 mice were injected i.p. with a lethal dose (30 LD50) of GBS VI reference strain (0.25 ml) with the addition of 0.25 ml of type VIspecific antiserum diluted 1:10. Survivors were counted daily for 15 days. A control with absorbed serum was performed. Preparation of human polymorphonuclear leukocytes (PMN). Leukocyte buffy coats obtained from normal volunteers were diluted 1:2 in PBS and layered on 10 ml of Histopaque-1077 as described in the Sigma catalog. After centrifugation for 30 min at 400 x g at room temperature, the leukocyte layer lying on the surface of the erythrocyte pellet was collected, and the erythrocytes were lysed by hypotonic shock with sterile distilled water (30 s). The cells were washed twice in PBS before being adjusted to the desired cell density in RPMI 1640 (GIBCO) free of fetal calf serum. PMN preparations stained with May-Grunwald Giemsa showed more than 99% PMN by morphology. Phagocytosis assay. The phagocytosis assay was performed as previously described by Baltimore et al. (2). Briefly, PMN were mixed with log-phase bacteria in a ratio of 3:1 in the presence or absence of a final 10% guinea pig serum as the source of active complement and different dilutions of rabbit type VI-specific serum. Bacteria were enumerated on Columbia blood agar plates at the beginning and after 1 h of incubation at 37°C. Experiments were performed in triplicate, and results were reported as the percent decrease of the initial number of viable bacteria. Immunologic assay. The ELISA was performed by using 1 ,ug of type VI polysaccharide per well as the coating antigen as previously described (43). The immune serum was diluted in PBS plus Tween 20 (0.05%, vol/vol) and gelatin (0.01%, wt/vol). After 2 h of incubation at room temperature and three washings, 100 Rl of goat anti-rabbit IgG alkaline phosphatase-conjugated antiserum (Sigma) was added.

INFECT. IMMUN.

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36

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52

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Fraction number

FIG. 1. Separation of type VI and group B polysaccharides from culture supernatant of the reference strain on a Sepharose 4B column. For purification purposes, polysaccharides were monitored in the column fractions (5 ml) by immunodiffusion with specific type VI and group B antisera. VO, void volume estimated by blue dextran; V., total volume estimated by tryptophan.

ELISA inhibition experiments were performed by using type VI antiserum absorbed with whole bacterial cells of the different GBS serotypes. Absorption was carried out overnight at 4°C by incubating 3 parts of serum (diluted 1:500 in PBS) with 1 part of packed cells, previously washed twice with PBS. A double-diffusion test was performed in agar as described by Ouchterlony (34). RESULTS Purification of type polysaccharide. The isolation and purification of type polysaccharide were performed with 24-h culture supernatants of the type VI GBS reference strain. The precipitate obtained by fractionating supernatants with ethanol was chromatographed on a Sepharose 4B column, giving the typical separation shown in Fig. 1. The fractions 36 to 64, positive in immunodiffusion for the presence of the type antigen but negative for group B antigen and with no absorption at 280 nm, were pooled and further purified by DEAE-Sephacel. The type VI antigen eluted as a single peak at about 200 mM NaCl (Fig. 2). This procedure resulted in a high yield of pure polysaccharide: about 31 mg of polysaccharide was obtained from 8 liters of culture, corresponding to 0.69 ,ug/g (dry weight) of cells. The Kd of the native purified polysaccharide was 0.39 as calculated by the elution volume on a Sepharose 4B column. The void and bed volumes were determined by the elution volumes of blue dextran 2000 and tryptophan, respectively. By comparison with dextran molecular weight standards, the average Mr of the type polysaccharide was estimated to be approximately 260,000 (Fig. 3). Chemical composition and NMR spectra of the purified type VI antigen. The chemical composition of the native type VI polysaccharide purified from the supernatant broth is shown in Table 1: component sugars were determined in a typical batch of antigen and expressed as a percentage of dry weight. Negligible amounts of protein (less than 0.5% by the Bio-Rad protein assay) were detected. Nucleic acid, detected as UV absorption at 260 nm, gave an absorbance of less than 1% of that at 206 nm. The carbohydrate composition of the native polysaccharide was studied by HPAC.

TYPE VI GBS

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80

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FIG. 2. Elution profile of type VI polysaccharide on DEAESephacel column. The polysaccharide was eluted with a linear gradient of NaCl in 10 mM Tris-HCl (pH 8.3) and revealed in the fractions (5 ml) by immunodiffusion with type-specific antiserum.

The polysaccharide contained glucose (31.55%), galactose (31.55%), and N-acetylneuraminic acid (27.00%), which together accounted for 90% of the material weight (Table 1) and which were in an apparent molar ratio of 2:2:1, respectively. No N-acetylglucosamine and no contamination by the group B antigen (absence of rhamnose) and lipoteichoic acid (absence of glycerolphosphate) were found (Table 1). The results described above, in particular the molar ratio of 2:2:1 of the sugar components, were totally comparable in three separate batches of polysaccharide (data not shown). Since N-acetylglucosamine is present in all GBS type polysaccharides reported to date (22, 48, 49), the sugar composition of cell-bound type polysaccharide of the type VI reference strain was also analyzed. To this end, the polysaccharide was extracted by a mild procedure and chromatographically purified as described in Materials and Methods. As shown in Fig. 4, the extracted type antigen contained the same sugars as the extracellular type polysaccharide did and no N-acetylglucosamine (see above). The ratios between the single monosaccharides were also the

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Glucose Galactose N-Acetylglucosamine N-Acetylneuraminic acid

0.010

Rhamnosed

10

20

12

TABLE 1. Composition of native type VI GBS polysaccharidea

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30 40 50 60 70 80 90 100 Fraction number FIG. 3. Sepharose 4B elution profile of type VI GBS polysaccharide isolated from culture supernatant. Arrows indicate void volume (VO), bed volume (V,), and elution volumes of dextran T70,000 (T 70) and dextran T40,000 (T 40). 0

8

same (see above) as that in the secreted purified polysaccharide. To confirm the absence of N-acetylglucosamine in type VI antigen, the polysaccharide was isolated and purified from both cells and culture supernatant of the clinical isolate GBS 114852. The chemical composition of the antigen extracted from this isolate confirmed the monosaccharide composition of the reference type antigen strain with the same ratio among individual monosaccharides (data not shown). The 13C NMR spectrum (Fig. 5) of the native polysaccharide showed five anomeric signals, at 104.4, 103.9, 103.8, 103.5, and 100.7 ppm, which suggests that the type VI

c

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Retention time (min) FIG. 4. HPAC-PAD chromatograms of GBS type VI native polysaccharide extracted from the reference strain 118754. (A) Trifluoroacetic-acid-hydrolyzed polysaccharide, prepared as described in Materials and Methods, was dissolved in water, and a sample (25 ,ul) containing 1 ,ug was injected and eluted with 15 mM NaOH at 1 ml min-'. (B) Dried HCl-hydrolyzed polysaccharide was dissolved in water, and a sample (25 ,ul) containing 1.6 p.g was injected and eluted with 100 mM NaOH plus 150 mM CH3COONa at 1 ml min-'. Detection was by PAD at 300 nA full scale. Peaks: 1, solvent front; 2, by-products of hydrolysis.

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a Obtained from the culture supernatant. b Percentage of dry material. d, absent. d Representative of group B polysaccharide. e Representative of lipoteichoic acid.

Composition' p.mol/mg 31.55 31.55

1.7 1.7

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27.00