INFECTION
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IMMUNITY, June 1991, p. 2075-2082
Vol. 59, No. 6
0019-9567/91/062075-08$02.00/0 Copyright C) 1991, American Society for Microbiology
Immunochemical Characterization of Two Surface Polysaccharides of Bacteroides fragilis ANNALISA PANTOSTI,1t ARTHUR 0. TZIANABOS,1 ANDREW B. ONDERDONK,1 AND DENNIS L. KASPER' 2* Channing Laboratory, Brigham and Women's Hospital,' and Division of Infectious Diseases, Beth Israel Hospital,2 Harvard Medical School, 180 Longwood Avenue, Boston, Massachusetts 02115 Received 14 February 1991/Accepted 2 April 1991
Immunochemical analysis of the capsular polysaccharide from Bacteroides fragilis NCTC 9343 revealed a novel structure composed of two distinct polysaccharides. Immunoelectrophoresis of an extract of purified surface polysaccharide from fermenter-grown organisms showed a complex precipitin profile with varying anodal mobility. DEAE-Sephacel anion-exchange chromatography of the polysaccharide extract failed to separate the majority of this aggregate. Disaggregation of this complex was accomplished by very mild acid treatment; purification was achieved by DEAE-Sephacel anion-exchange chromatography. Polysaccharide A had a neutral charge at pH 7.3, a net negative charge at pH 8.6, and an average Mr = 110,000; chemical analysis showed it to contain galactose, galactosamine, and an unidentified amino sugar. Polysaccharide B eluted from the anion-exchange column with increased salt concentration; it had a net negative charge and an average Mr = 200,000, and contained fucose, galactose, quinovosamine, galacturonic acid, and glucosamine. Neither of these polysaccharides contained detectable 3-deoxy-D-manno-octolusonic acid, and both were recognized as distinct antigens on the basis of their reactivity with monoclonal antibodies CE3 and F10, which reacted with the complex before acid treatment. These data indicate that the capsule of B. fragilis NCTC 9343 comprises two discrete, surface-exposed polysaccharides with differing physicochemical properties that are distinct from the lipopolysaccharide of this organism. The finding of two surface polysaccharides has not been described for other bacteria pathogenic to humans. to be components found in what was previously termed
Bacteroides fragilis is the most common obligately anaerobic bacterial species isolated from serious human infections (36), yet it is not among the numerically dominant species of Bacteroides found as part of normal human colonic microflora (27). This dichotomy suggests that B. fragilis must have unique virulence properties compared with those of more numerically dominant Bacteroides species. Studies from several groups indicate that most B. fragilis strains are encapsulated (2, 4, 14, 23, 33). In an animal model of intraabdominal abscess formation, B. fragilis was shown to have the unique ability to promote the formation of abscesses when injected intraperitoneally along with sterile cecal contents (29). This ability has been attributed, in part, to the capsular polysaccharide (CP) of this species, which when used for subcutaneous immunization of rats and mice has also been found to induce a T-cell-dependent form of immunity that protects animals against the formation of intraabdominal abscesses by B. fragilis (30, 41). Chemical analysis of the B. fragilis capsule indicates that a complex structure may exist. Strain ATCC 23745 CP was found to contain nine sugar residues, and strain NCTC 9343 CP was found to contain six sugars: D-galactose, D-glucosamine, L-fucose, D- and L-quinovosamine, and galacturonic acid (18). The interesting biologic activities of these polysaccharides led us to further characterize their chemical nature. We were surprised to find that strain NCTC 9343 synthesizes two distinct polysaccharides in addition to its rough lipopolysaccharide (LPS). Each polysaccharide has epitopes exposed on the surface of the cell. These two polysaccharides appear
capsular polysaccharide (CP). For the purpose of this presentation, the term CP will be used to describe a polysaccharide complex extracted from B. fragilis and containing both of these surface polysaccharide antigens, which appear to exist primarily in a complex, perhaps bound to lipid moieties, embedded in the outer membrane of B. fragilis. We report the isolation of the two surface polysaccharides and define the chemical compositions of each. MATERIALS AND METHODS
Bacterial strains. B. fragilis NCTC 9343 was obtained from the National Collection of Type Cultures, London, England, and aliquots were kept frozen at -70°C in peptone-yeast broth until used. Aliquots were inoculated onto tryptic soy agar (Difco Laboratories, Detroit, Mich.) plates supplemented with 5% sheep blood and were incubated in an anaerobic jar (GasPak; BBL Microbiology Systems, Cockeysville, Md.) at 37°C for 24 h. Media and growth conditions. Organisms were grown in broth culture for extraction of bacterial antigens. The basal medium was a modification of the medium previously described (18) and contained 5 g of yeast extract, 20 g of proteose peptone, 5 g of NaCl, 60 g of glucose, 5 mg of hemin, and 0.5 mg of vitamin Kl per liter. B. fragilis NCTC 9343 was grown by batch culture in a 20-liter fermenter (Biolafitte, Poissy, France) containing 16 liters of basal medium at 37°C for 18 h. Anaerobiosis was maintained by flushing with N2, and a pH of 7.2 was maintained by titration with 10 N NaOH. The culture was checked for purity at the end of the growth cycle by aerobic and anaerobic techniques.
* Corresponding author. t Present address: Istituto Superiore Di Sanita, Via Regina Elena, 299, 00161 Roma, Italy.
Extraction and purification of the CP. The bacterial cells recovered by centrifugation (8,000 x g at 4°C for 20
were
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PANTOSTI ET AL.
min) and washed once with 0.15 M NaCl. The pellet was resuspended in 750 ml of water, and an equal volume of 75% phenol was added (18, 46). The mixture was kept at 68°C for 30 min with continuous stirring and was then centrifuged at 5,000 x g for 20 min to separate the phenol from the aqueous phase, which was extracted with an equal volume of ether, concentrated on a rotary evaporator, extensively dialyzed against distilled water, and lyophilized. The phenol-water extraction was repeated, and the aqueous phases from both extractions were pooled. Aliquots of the aqueous phase were suspended in 0.1 M sodium acetate buffer (pH 4.5) containing 10 mM CaCl2 and 10 mM MgCl2 and were subsequently treated with 2 mg of DNase and 10 mg of RNase (Worthington Biochemical Corp., Freehold, N.J.) at 37°C for 2 h. This treatment was repeated at 37°C overnight. After the pH was adjusted to 7.0 with NaOH, 20 mg of pronase (Calbiochem, La Jolla, Calif.) was added twice, once for 2 h at 37°C and again at 37°C overnight. The material was brought to a concentration of 80% (vol/vol) ethanol and allowed to stand at 4°C overnight. The alcohol-insoluble precipitate was recovered by centrifugation and dissolved in a buffer containing 0.5% sodium deoxycholate, 50 mM glycine, and 10 mM EDTA (pH 9.8) (14). Three milliliters of this material was loaded onto a Sephacryl S-300 column (1.5 by 80 cm) (Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated in the 0.5% sodium deoxycholate buffer described above. The fractions collected (1.6 ml) were examined for the presence of the CP by double diffusion in agar and for the presence of LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described below. The fractions that contained CP but did not contain LPS were pooled, precipitated in 80% ethanol, dialyzed against distilled water, and lyophilized. If the UV absorbance was above 0.100 at 280 nm, the extract was treated again with DNase, RNase, and pronase. To achieve separation of the different components of the CP, 100 mg of CP or 5% acetic acid-treated CP was solubilized in 5 ml of 50 mM Tris-HCl (pH 7.3) and loaded into a column (2.5 by 30 cm) of DEAE-Sephacel (Pharmacia Fine Chemicals) equilibrated in the same buffer. After washing with 250 ml of the same buffer, the material bound to the column was eluted with a linear gradient of 0 to 0.5 M NaCl or with 2 M NaCl in the starting buffer (600 ml); 5-ml fractions were collected, and each was tested by double diffusion in agarose against antiserum prepared to whole bacteria. Fractions that were eluted with the starting buffer and gave a precipitin line were pooled, concentrated, dialyzed, and lyophilized. Fractions eluted at the beginning of the salt gradient that gave a single precipitin line and later fractions showing double precipitins were separately pooled and concentrated. Preparation of CP from a dialysate broth. A 2-liter dialysate (10,000-Mr membrane, Pellicon Cassette System; Millipore Corp., Bedford, Mass.) of basal medium was inoculated with B. fragilis NCTC 9343 and cultured anaerobically at 37°C for 48 h. The supematant was recovered (following centrifugation of bacteria at 8,000 x g for 20 min), concentrated by lyophilization, and dialyzed against distilled water to remove medium constituents. Bacterial products from the dialyzed supernatant were precipitated with 80% ethanol. The precipitated material was solubilized in 2 ml of distilled water and tested for CP content by immunoelectrophoresis (IEP) without further processing. Chemical modification of the CP. Acid treatment of B. fragilis CP was performed with 2% acetic acid at 60°C for 20,
INFECT. IMMUN.
40, and 60 min or with 5% acetic acid at 100°C for 1 h. A separate sample was treated with 0.1 M HCl at 100°C for 1 h. Chemical analysis of polysaccharides. Uronic acid was quantitated by the method of Dische (9). Total phosphorus was measured by the method of Chen et al. (7). The fatty acids were extracted as methyl esters following acidification with 4 N methanolic HCI and detected by gas-liquid chromatography (47). The thiobarbiturate assay for the quantitation of 3-deoxyD-manno-octulosonic acid (KDO) was performed according to the method of Chaby and Szabo (6) following treatment of the sample with 2 M HCl at 100°C for 2 h (3) or with 48% HF at 4°C for 48 h (5) to remove the putative phosphate substituents of KDO reported for B. fragilis, which could account for a negative thiobarbituric acid assay (24). Analyses of the monosaccharide constituents were carried out by gas-liquid chromatography of alditol acetate derivatives (40), with use of xylose as an internal standard. The alditol acetates were analyzed on an SP 2330 capillary column (0.25 mm [internal diameter] by 30 m [length]) (Supelco, Inc., Bellefonte, Pa.) with an instrument (model 5880A; Hewlett-Packard, Andover, Mass.) equipped with a flame ionization detector at 225°C oven temperature. For identification of amino sugars, a Silar 10C capillary column (0.25 mm [internal diameter] by 25 m [length]) (Alltech Associates, Deerfield, Ill.) was used with an oven temperature program from 205°C to 250°C. The method of Taylor and Conrad (43) was used to reduce the carboxyl group of the uronic acid constituents in the polysaccharides before identification by gas-liquid chromatography of the alditol acetates. Molecular sizing of polysaccharides. The molecular sizes of polysaccharides A and B were estimated by Sephacryl S-300 (Pharmacia) gel filtration chromatography (1.6 by 89 cm) equilibrated in 50 mM Tris buffer, pH 7.3. The elution volumes were compared with those of dextran standards. Preparation of antisera. Antiserum to B. fragilis NCTC 9343 was prepared by immunization of New Zealand White rabbits with formalin-killed whole bacterial cells (109 for each injection), as described earlier (14). Antiserum to the CP was raised in New Zealand White rabbits immunized with purified CP according to procedures previously described (18). Monoclonal antibodies. Mouse monoclonal antibodies (MAbs) were generated by immunization of BALB/c mice with formalin-killed whole B. fragilis cells and a fusion of the splenocytes of immune animals with cells of the P3X63AG8.653 nonsecreting mouse myeloma cell line (10). The hybridoma obtained was screened for the production of antibodies directed to the CP by an enzyme-linked immunosorbent assay (ELISA) (unpublished data). Two MAbs were selected: CE3, an immunoglobulin G3 lambda chain, and F10, an immunoglobulin G2A kappa chain. The antigenic specificity of each MAb was demonstrated by ELISA inhibition. The supernatants of the hybridoma cell lines were used unprocessed. Immunologic assays. Double diffusion in agarose was performed by the method of Ouchterlony (32) with 1% agarose in 0.15 M saline. IEP was performed in 1% agarose with either 0.2 M barbital buffer (pH 8.6) or 50 mM Tris-HCl (pH 7.3) (13). For both assays, the polysaccharide antigens were used as 1-mg/ml solutions. SDS-PAGE and immunoblotting. SDS-PAGE was performed according to the method of Laemmli (21) with an 8% resolving gel and a 3% stacking gel and a mini-gel apparatus (Hoefer, San Francisco, Calif.). Gels were stained with a
VOL. 59, 1991
B. FRAGILIS SYNTHESIZES TWO SURFACE POLYSACCHARIDES
silver stain (Bio-Rad, Richmond, Calif.). Immunoblotting was performed by the method of Sturm et al. (42). In brief, SDS-PAGE samples were electrophoretically transferred onto nitrocellulose sheets at 60 mA overnight. The nitrocellulose was baked at 80°C for 2 h prior to blocking for 2 h at room temperature in a 50 mM Tris-HCl buffer (pH 7.4) containing 200 mM NaCl, 5% skim milk, and 0.3% sodium azide (blocking buffer). After being washed in Tris buffer, the nitrocellulose was allowed to react with a rabbit antiserum to the CP diluted 1:500 at room temperature for 2 h. After more washes, goat anti-rabbit immunoglobulin G alkaline phosphatase conjugate (Tago, Inc., Burlingame, Calif.) diluted 1:400 in blocking buffer was applied for 2 h at room temperature. In some cases, supernatants of mouse hybridoma cell lines and a goat anti-mouse immunoglobulin G alkaline phosphatase conjugate (Tago, Inc.) were used. The substrate used for detection was 5-bromo-4-chloro-3-indolyl phosphate (Sigma Chemical Co., St. Louis, Mo.). Dot blot experiments were performed with 2.5 ,ul of sample applied to the nitrocellulose paper and according to the same procedure used for immunoblotting. Colony blotting was performed by applying 2.5 ,ul of an overnight culture of B. fragilis NCTC 9343 to nitrocellulose paper, and then immunoblotting was performed as described above. Organisms were not heated or treated in any way that would disrupt surface structure. Immunofluorescence staining of B. fragilis. B. fragilis was grown in broth culture or on blood agar plates overnight, harvested with sterile saline, and washed thoroughly with phosphate-buffered saline (three times). Organisms were treated with either polyclonal antibodies or MAbs specific for the capsule of B. fragilis NCTC 9343 or an irrelevant antibody and incubated at 37°C for 1 h. Cells were then treated with goat anti-rabbit or anti-mouse serum conjugated to fluorescein isothiocyanate (Sigma Chemical Co.) and viewed with a Nikon DIAPHOT-TMD epifluorescent microscope (44). RESULTS Separation of polysaccharides. B. fragilis NCTC 9343 was grown in a fermenter; the cells were harvested by centrifugation and suspended in water. An equal volume of phenol (75%) was added, and the mixture was heated to 60°C for 30 min. The resultant aqueous phase was extracted with ether, concentrated, and treated twice with DNase, RNase, and pronase. This concentrate was chromatographed on a column of Sephacryl S-300 in a buffer containing 0.5% sodium deoxycholate; the fractions were tested for CP by double diffusion in agar against antiserum to whole bacteria. Fractions containing CP were pooled, dialyzed against distilled water, and lyophilized. CP was tested by double diffusion in agarose. A doubleprecipitin line was formed (Fig. la) with rabbit antiserum prepared by immunization with whole bacteria. CP was also examined by IEP (in barbital buffer at pH 8.6), which demonstrated the presence of distinct components migrating towards the anode with different degrees of mobility (Fig.
lb). To determine whether the extraction procedure modified the native polysaccharides, we studied the IEP profile of CP released from the bacteria into the growth medium. To accomplish this, B. fragilis was grown in a medium that was prepared as a dialysate (10,000-Mr membrane) of the basal medium. The bacteria were removed from the dialysate broth, and the supernatant was exhaustively dialyzed against
2077
a-
b._. FIG. 1. Immunoprecipitation of B. fragilis CP. (a) Double diffusion in agarose of B. fragilis CP with polyclonal rabbit antiserum raised against whole bacteria. (b) IEP of capsular polysaccharide in bacterial buffer, pH 8.6. Trough contains polyclonal rabbit antiserum raised against whole bacteria. Black bar indicates cathode.
water. IEP of this concentrate showed a profile identical to that seen with the phenol-water-extracted CP (as in Fig. lb). The CP was loaded onto a column containing DEAESephacel (50 mM Tris, pH 7.3). Antigens were eluted with an NaCl gradient. Fractions were tested for the presence of CP components by agarose double diffusion. The fractions that eluted with the starting buffer and contained material forming precipitins with the antiserum were pooled, concentrated, dialyzed against distilled water, and lyophilized. This material was called polysaccharide A. The fractions that eluted with a low NaCl concentration (approximately 0.1 M NaCl) and were positive for precipitins were pooled. This latter pool contained a polysaccharide, termed polysaccharide B, which formed a precipitin line distinct from that formed by polysaccharide A (not shown). Fractions eluted with a higher salt concentration contained materials which gave two precipitin lines. These fractions were pooled and called polysaccharide pool C. Approximately 79% of the materials recovered from the DEAE-Sephacel column were eluted as polysaccharide pool C. IEP (Fig. 2, top panel) in barbital buffer at pH 8.6 showed that polysaccharides A and B each formed a single immunoprecipitation arc migrating towards the anode, while polysaccharide pool C had a profile identical to that of the unfractionated CP. The faint second parallel precipitin lines likely represent the Liesegang phenomenon (8). Since polysaccharide A was not retained in the DEAE-Sephacel column, it appeared to lack a net negative charge at pH 7.3. Polysaccharide A migrated toward the anode, however, in IEP at pH 8.6, an indication of a net negative charge at a more basic pH. This conclusion was further supported by IEP performed with 50 mM Tris at pH 7.3. Under these latter conditions, polysaccharide A appeared to be neutral and remained at the origin, while polysaccharide B remained negatively charged (Fig. 2, bottom panel). There was a broad precipitin line (precipitin c) extending between polysaccharides A and B in the CP and in polysaccharide pool C (Fig. 2, top and bottom panels). For chemical analysis, polysaccharide A was rechromatographed on DEAE-Sephacel in Tris buffer at pH 8.6. Under these conditions, polysaccharide A was retained on the column and was eluted as a single peak with an NaCl gradient. An additional precipitin line was seen in the IEP profile of some preparations of CP and polysaccharide A that had a more cathodal migration and was clearly not identical with
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PANTOSTI ET AL.
+
CP
A kD 180-
A
a
B C
C
11 68458493727-
C
pH 7.3
polysaccharide A (Fig. 2, bottom panel). This material was separated from polysaccharide A by DEAE-Sephacel chromatography at pH 8.6. This cathodally migrating antigen was not retained by the DEAE column. Further chemical analysis revealed this antigen to be residual rough LPS (45). The CP and polysaccharides A, B, and pool C were analyzed by SDS-PAGE after silver staining or studied by Western blot (immunoblot), probing with a rabbit polyclonal antiserum to the CP. By both methods, the CP and pool C appeared as broad bands of high molecular weight (Fig. 3). Close examination revealed a tightly spaced ladder-like pattern with Mr between 80,000 and 120,000. Polysaccharides A and B could not be detected by silver staining but were faintly visualized by Western blot analysis. Characterization of polysaccharide pool C. Although polysaccharide pool C represented the majority of the material eluted from the DEAE-Sephacel column, it appeared by IEP to be identical to the CP. We wondered, therefore, whether it could be chemically disaggregated. CP was treated with 2% acetic acid at 60°C for increasing time periods (20, 40, and 60 min) and with 5% acetic acid at 1000C for 1 h. With increasing time or concentration of acetic acid, there was a progressive disappearance of the broad precipitin c line in the IEP profile of CP. The IEP profile of the 5% acetic acid-treated CP had only two distinct precipitin arcs, corresponding to polysaccharides A and B (Fig. 4). The precipitin representing polysaccharide B appeared more prominent after 5% acetic acid hydrolysis. Harsher hydrolysis with 0.1 M HCl at 1000C for 1 h caused the CP to lose reactivity in IEP. DEAE-Sephacel chromatography at pH 7.3 of 5% acetic
_.
CP
A
B
C
CP
ii s
FIG. 3. SDS-PAGE analysis of B. fragilis polysaccharides. SDSPAGE of polysaccharides A, B, pool C and CP was performed with an 8% uniform gel. Left panel, silver stain. Right panel, Western blot with polyclonal rabbit antiserum raised against whole bacteria.
B
FIG. 2. Immunoelectrophoretic mobility of B. fragilis polysaccharide. Top panel, IEP of CP and polysaccharides A, B, and pool C in barbital buffer, pH 8.6. Troughs contain polyclonal rabbit antiserum raised against whole bacteria. Note the anodal migration of polysaccharide A. (A) Polysaccharide A precipitin arc (a). (B) Polysaccharide B precipitin arc (b). (C) Precipitin c (c). Bottom panel, IEP of CP and polysaccharides A and B in Tris buffer pH 7.3. Trough contains polyclonal rabbit antiserum raised against whole bacteria. (A) Polysaccharide A precipitin arc (a). (B) Polysaccharide B precipitin arc (b). c, Precipitin c. Note the neutral charge of polysaccharide A.
C
O'.
pH 8.6
Ips
B
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acid-treated CP resulted in resolution of polysaccharides A and B as before, with almost no polysaccharide pool C eluted. The total recovery of polysaccharides A and B from 5% acetic acid-hydrolyzed CP run on the DEAE-Sephacel column was equivalent to the quantity of polysaccharides A, B, and pool C recovered before hydrolysis when eluted from the same column. In double-diffusion experiments, native and 5% acetic acid-treated polysaccharide As were observed to be identical, as were native and acetic acid-treated polysaccharide Bs. Furthermore, polysaccharides A and B were not identical with each other (Fig. 5). Antigens were probed with MAb CE3 (specific for polysaccharide A) or MAb F10 (specific for polysaccharide B). In Western blots of CP, polysaccharide A, and polysaccharide B, both MAbs strongly stained the high-molecularweight broad band in the CP. In addition, MAb CE3 stained some material at the top of the gel in the lane containing acetic acid-treated polysaccharide A but not in the lane containing acetic acid-treated polysaccharide B. MAb F10 showed some staining at the top of the gel in the lane containing acetic acid-treated polysaccharide B but not in the lane containing acetic acid-treated polysaccharide A (Fig. 6). Both A and B are contained in the broad band seen in SDS-PAGE gels of CP. Dot blots of polysaccharides A and B by the MAbs confirmed their specificity and demonstrated that both MAbs reacted with the CP. Surface location of polysaccharides A and B. In immunofluorescence experiments, B. fragilis were probed with polyclonal antibody specific for the CP and MAb CE3 or F10. In
2
4
;u
.._
FIG. 4. Immunoelectrophoretic analysis of acid-treated B. fragilis polysaccharide. IEP of B. fragilis CP was done at pH 7.3 in Tris buffer following acid treatment. Trough contains polyclonal rabbit antiserum raised against whole bacteria. (1) Untreated CP. (2) Acetic acid treatment (5%) for 1 h at 100°C.
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B. FRAGILIS SYNTHESIZES TWO SURFACE POLYSACCHARIDES
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TABLE 1. Chemical analysis of polysaccharides of B. fragilis Componenta
Polysaccharide A
Fucose Galactose Quinovosamine Galacturonic acid Glucosamine Galactosaminec d Unidentified amino sugarcd Protein Nucleic acids Phosphorus (,umol/mg) KDO (,ug/mg) Fatty acidc (no. of C)
_b 57 -
+b +
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IMMUNITY, June 1991, p. 2075-2082
Vol. 59, No. 6
0019-9567/91/062075-08$02.00/0 Copyright C) 1991, American Society for Microbiology
Immunochemical Characterization of Two Surface Polysaccharides of Bacteroides fragilis ANNALISA PANTOSTI,1t ARTHUR 0. TZIANABOS,1 ANDREW B. ONDERDONK,1 AND DENNIS L. KASPER' 2* Channing Laboratory, Brigham and Women's Hospital,' and Division of Infectious Diseases, Beth Israel Hospital,2 Harvard Medical School, 180 Longwood Avenue, Boston, Massachusetts 02115 Received 14 February 1991/Accepted 2 April 1991
Immunochemical analysis of the capsular polysaccharide from Bacteroides fragilis NCTC 9343 revealed a novel structure composed of two distinct polysaccharides. Immunoelectrophoresis of an extract of purified surface polysaccharide from fermenter-grown organisms showed a complex precipitin profile with varying anodal mobility. DEAE-Sephacel anion-exchange chromatography of the polysaccharide extract failed to separate the majority of this aggregate. Disaggregation of this complex was accomplished by very mild acid treatment; purification was achieved by DEAE-Sephacel anion-exchange chromatography. Polysaccharide A had a neutral charge at pH 7.3, a net negative charge at pH 8.6, and an average Mr = 110,000; chemical analysis showed it to contain galactose, galactosamine, and an unidentified amino sugar. Polysaccharide B eluted from the anion-exchange column with increased salt concentration; it had a net negative charge and an average Mr = 200,000, and contained fucose, galactose, quinovosamine, galacturonic acid, and glucosamine. Neither of these polysaccharides contained detectable 3-deoxy-D-manno-octolusonic acid, and both were recognized as distinct antigens on the basis of their reactivity with monoclonal antibodies CE3 and F10, which reacted with the complex before acid treatment. These data indicate that the capsule of B. fragilis NCTC 9343 comprises two discrete, surface-exposed polysaccharides with differing physicochemical properties that are distinct from the lipopolysaccharide of this organism. The finding of two surface polysaccharides has not been described for other bacteria pathogenic to humans. to be components found in what was previously termed
Bacteroides fragilis is the most common obligately anaerobic bacterial species isolated from serious human infections (36), yet it is not among the numerically dominant species of Bacteroides found as part of normal human colonic microflora (27). This dichotomy suggests that B. fragilis must have unique virulence properties compared with those of more numerically dominant Bacteroides species. Studies from several groups indicate that most B. fragilis strains are encapsulated (2, 4, 14, 23, 33). In an animal model of intraabdominal abscess formation, B. fragilis was shown to have the unique ability to promote the formation of abscesses when injected intraperitoneally along with sterile cecal contents (29). This ability has been attributed, in part, to the capsular polysaccharide (CP) of this species, which when used for subcutaneous immunization of rats and mice has also been found to induce a T-cell-dependent form of immunity that protects animals against the formation of intraabdominal abscesses by B. fragilis (30, 41). Chemical analysis of the B. fragilis capsule indicates that a complex structure may exist. Strain ATCC 23745 CP was found to contain nine sugar residues, and strain NCTC 9343 CP was found to contain six sugars: D-galactose, D-glucosamine, L-fucose, D- and L-quinovosamine, and galacturonic acid (18). The interesting biologic activities of these polysaccharides led us to further characterize their chemical nature. We were surprised to find that strain NCTC 9343 synthesizes two distinct polysaccharides in addition to its rough lipopolysaccharide (LPS). Each polysaccharide has epitopes exposed on the surface of the cell. These two polysaccharides appear
capsular polysaccharide (CP). For the purpose of this presentation, the term CP will be used to describe a polysaccharide complex extracted from B. fragilis and containing both of these surface polysaccharide antigens, which appear to exist primarily in a complex, perhaps bound to lipid moieties, embedded in the outer membrane of B. fragilis. We report the isolation of the two surface polysaccharides and define the chemical compositions of each. MATERIALS AND METHODS
Bacterial strains. B. fragilis NCTC 9343 was obtained from the National Collection of Type Cultures, London, England, and aliquots were kept frozen at -70°C in peptone-yeast broth until used. Aliquots were inoculated onto tryptic soy agar (Difco Laboratories, Detroit, Mich.) plates supplemented with 5% sheep blood and were incubated in an anaerobic jar (GasPak; BBL Microbiology Systems, Cockeysville, Md.) at 37°C for 24 h. Media and growth conditions. Organisms were grown in broth culture for extraction of bacterial antigens. The basal medium was a modification of the medium previously described (18) and contained 5 g of yeast extract, 20 g of proteose peptone, 5 g of NaCl, 60 g of glucose, 5 mg of hemin, and 0.5 mg of vitamin Kl per liter. B. fragilis NCTC 9343 was grown by batch culture in a 20-liter fermenter (Biolafitte, Poissy, France) containing 16 liters of basal medium at 37°C for 18 h. Anaerobiosis was maintained by flushing with N2, and a pH of 7.2 was maintained by titration with 10 N NaOH. The culture was checked for purity at the end of the growth cycle by aerobic and anaerobic techniques.
* Corresponding author. t Present address: Istituto Superiore Di Sanita, Via Regina Elena, 299, 00161 Roma, Italy.
Extraction and purification of the CP. The bacterial cells recovered by centrifugation (8,000 x g at 4°C for 20
were
2075
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PANTOSTI ET AL.
min) and washed once with 0.15 M NaCl. The pellet was resuspended in 750 ml of water, and an equal volume of 75% phenol was added (18, 46). The mixture was kept at 68°C for 30 min with continuous stirring and was then centrifuged at 5,000 x g for 20 min to separate the phenol from the aqueous phase, which was extracted with an equal volume of ether, concentrated on a rotary evaporator, extensively dialyzed against distilled water, and lyophilized. The phenol-water extraction was repeated, and the aqueous phases from both extractions were pooled. Aliquots of the aqueous phase were suspended in 0.1 M sodium acetate buffer (pH 4.5) containing 10 mM CaCl2 and 10 mM MgCl2 and were subsequently treated with 2 mg of DNase and 10 mg of RNase (Worthington Biochemical Corp., Freehold, N.J.) at 37°C for 2 h. This treatment was repeated at 37°C overnight. After the pH was adjusted to 7.0 with NaOH, 20 mg of pronase (Calbiochem, La Jolla, Calif.) was added twice, once for 2 h at 37°C and again at 37°C overnight. The material was brought to a concentration of 80% (vol/vol) ethanol and allowed to stand at 4°C overnight. The alcohol-insoluble precipitate was recovered by centrifugation and dissolved in a buffer containing 0.5% sodium deoxycholate, 50 mM glycine, and 10 mM EDTA (pH 9.8) (14). Three milliliters of this material was loaded onto a Sephacryl S-300 column (1.5 by 80 cm) (Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated in the 0.5% sodium deoxycholate buffer described above. The fractions collected (1.6 ml) were examined for the presence of the CP by double diffusion in agar and for the presence of LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described below. The fractions that contained CP but did not contain LPS were pooled, precipitated in 80% ethanol, dialyzed against distilled water, and lyophilized. If the UV absorbance was above 0.100 at 280 nm, the extract was treated again with DNase, RNase, and pronase. To achieve separation of the different components of the CP, 100 mg of CP or 5% acetic acid-treated CP was solubilized in 5 ml of 50 mM Tris-HCl (pH 7.3) and loaded into a column (2.5 by 30 cm) of DEAE-Sephacel (Pharmacia Fine Chemicals) equilibrated in the same buffer. After washing with 250 ml of the same buffer, the material bound to the column was eluted with a linear gradient of 0 to 0.5 M NaCl or with 2 M NaCl in the starting buffer (600 ml); 5-ml fractions were collected, and each was tested by double diffusion in agarose against antiserum prepared to whole bacteria. Fractions that were eluted with the starting buffer and gave a precipitin line were pooled, concentrated, dialyzed, and lyophilized. Fractions eluted at the beginning of the salt gradient that gave a single precipitin line and later fractions showing double precipitins were separately pooled and concentrated. Preparation of CP from a dialysate broth. A 2-liter dialysate (10,000-Mr membrane, Pellicon Cassette System; Millipore Corp., Bedford, Mass.) of basal medium was inoculated with B. fragilis NCTC 9343 and cultured anaerobically at 37°C for 48 h. The supematant was recovered (following centrifugation of bacteria at 8,000 x g for 20 min), concentrated by lyophilization, and dialyzed against distilled water to remove medium constituents. Bacterial products from the dialyzed supernatant were precipitated with 80% ethanol. The precipitated material was solubilized in 2 ml of distilled water and tested for CP content by immunoelectrophoresis (IEP) without further processing. Chemical modification of the CP. Acid treatment of B. fragilis CP was performed with 2% acetic acid at 60°C for 20,
INFECT. IMMUN.
40, and 60 min or with 5% acetic acid at 100°C for 1 h. A separate sample was treated with 0.1 M HCl at 100°C for 1 h. Chemical analysis of polysaccharides. Uronic acid was quantitated by the method of Dische (9). Total phosphorus was measured by the method of Chen et al. (7). The fatty acids were extracted as methyl esters following acidification with 4 N methanolic HCI and detected by gas-liquid chromatography (47). The thiobarbiturate assay for the quantitation of 3-deoxyD-manno-octulosonic acid (KDO) was performed according to the method of Chaby and Szabo (6) following treatment of the sample with 2 M HCl at 100°C for 2 h (3) or with 48% HF at 4°C for 48 h (5) to remove the putative phosphate substituents of KDO reported for B. fragilis, which could account for a negative thiobarbituric acid assay (24). Analyses of the monosaccharide constituents were carried out by gas-liquid chromatography of alditol acetate derivatives (40), with use of xylose as an internal standard. The alditol acetates were analyzed on an SP 2330 capillary column (0.25 mm [internal diameter] by 30 m [length]) (Supelco, Inc., Bellefonte, Pa.) with an instrument (model 5880A; Hewlett-Packard, Andover, Mass.) equipped with a flame ionization detector at 225°C oven temperature. For identification of amino sugars, a Silar 10C capillary column (0.25 mm [internal diameter] by 25 m [length]) (Alltech Associates, Deerfield, Ill.) was used with an oven temperature program from 205°C to 250°C. The method of Taylor and Conrad (43) was used to reduce the carboxyl group of the uronic acid constituents in the polysaccharides before identification by gas-liquid chromatography of the alditol acetates. Molecular sizing of polysaccharides. The molecular sizes of polysaccharides A and B were estimated by Sephacryl S-300 (Pharmacia) gel filtration chromatography (1.6 by 89 cm) equilibrated in 50 mM Tris buffer, pH 7.3. The elution volumes were compared with those of dextran standards. Preparation of antisera. Antiserum to B. fragilis NCTC 9343 was prepared by immunization of New Zealand White rabbits with formalin-killed whole bacterial cells (109 for each injection), as described earlier (14). Antiserum to the CP was raised in New Zealand White rabbits immunized with purified CP according to procedures previously described (18). Monoclonal antibodies. Mouse monoclonal antibodies (MAbs) were generated by immunization of BALB/c mice with formalin-killed whole B. fragilis cells and a fusion of the splenocytes of immune animals with cells of the P3X63AG8.653 nonsecreting mouse myeloma cell line (10). The hybridoma obtained was screened for the production of antibodies directed to the CP by an enzyme-linked immunosorbent assay (ELISA) (unpublished data). Two MAbs were selected: CE3, an immunoglobulin G3 lambda chain, and F10, an immunoglobulin G2A kappa chain. The antigenic specificity of each MAb was demonstrated by ELISA inhibition. The supernatants of the hybridoma cell lines were used unprocessed. Immunologic assays. Double diffusion in agarose was performed by the method of Ouchterlony (32) with 1% agarose in 0.15 M saline. IEP was performed in 1% agarose with either 0.2 M barbital buffer (pH 8.6) or 50 mM Tris-HCl (pH 7.3) (13). For both assays, the polysaccharide antigens were used as 1-mg/ml solutions. SDS-PAGE and immunoblotting. SDS-PAGE was performed according to the method of Laemmli (21) with an 8% resolving gel and a 3% stacking gel and a mini-gel apparatus (Hoefer, San Francisco, Calif.). Gels were stained with a
VOL. 59, 1991
B. FRAGILIS SYNTHESIZES TWO SURFACE POLYSACCHARIDES
silver stain (Bio-Rad, Richmond, Calif.). Immunoblotting was performed by the method of Sturm et al. (42). In brief, SDS-PAGE samples were electrophoretically transferred onto nitrocellulose sheets at 60 mA overnight. The nitrocellulose was baked at 80°C for 2 h prior to blocking for 2 h at room temperature in a 50 mM Tris-HCl buffer (pH 7.4) containing 200 mM NaCl, 5% skim milk, and 0.3% sodium azide (blocking buffer). After being washed in Tris buffer, the nitrocellulose was allowed to react with a rabbit antiserum to the CP diluted 1:500 at room temperature for 2 h. After more washes, goat anti-rabbit immunoglobulin G alkaline phosphatase conjugate (Tago, Inc., Burlingame, Calif.) diluted 1:400 in blocking buffer was applied for 2 h at room temperature. In some cases, supernatants of mouse hybridoma cell lines and a goat anti-mouse immunoglobulin G alkaline phosphatase conjugate (Tago, Inc.) were used. The substrate used for detection was 5-bromo-4-chloro-3-indolyl phosphate (Sigma Chemical Co., St. Louis, Mo.). Dot blot experiments were performed with 2.5 ,ul of sample applied to the nitrocellulose paper and according to the same procedure used for immunoblotting. Colony blotting was performed by applying 2.5 ,ul of an overnight culture of B. fragilis NCTC 9343 to nitrocellulose paper, and then immunoblotting was performed as described above. Organisms were not heated or treated in any way that would disrupt surface structure. Immunofluorescence staining of B. fragilis. B. fragilis was grown in broth culture or on blood agar plates overnight, harvested with sterile saline, and washed thoroughly with phosphate-buffered saline (three times). Organisms were treated with either polyclonal antibodies or MAbs specific for the capsule of B. fragilis NCTC 9343 or an irrelevant antibody and incubated at 37°C for 1 h. Cells were then treated with goat anti-rabbit or anti-mouse serum conjugated to fluorescein isothiocyanate (Sigma Chemical Co.) and viewed with a Nikon DIAPHOT-TMD epifluorescent microscope (44). RESULTS Separation of polysaccharides. B. fragilis NCTC 9343 was grown in a fermenter; the cells were harvested by centrifugation and suspended in water. An equal volume of phenol (75%) was added, and the mixture was heated to 60°C for 30 min. The resultant aqueous phase was extracted with ether, concentrated, and treated twice with DNase, RNase, and pronase. This concentrate was chromatographed on a column of Sephacryl S-300 in a buffer containing 0.5% sodium deoxycholate; the fractions were tested for CP by double diffusion in agar against antiserum to whole bacteria. Fractions containing CP were pooled, dialyzed against distilled water, and lyophilized. CP was tested by double diffusion in agarose. A doubleprecipitin line was formed (Fig. la) with rabbit antiserum prepared by immunization with whole bacteria. CP was also examined by IEP (in barbital buffer at pH 8.6), which demonstrated the presence of distinct components migrating towards the anode with different degrees of mobility (Fig.
lb). To determine whether the extraction procedure modified the native polysaccharides, we studied the IEP profile of CP released from the bacteria into the growth medium. To accomplish this, B. fragilis was grown in a medium that was prepared as a dialysate (10,000-Mr membrane) of the basal medium. The bacteria were removed from the dialysate broth, and the supernatant was exhaustively dialyzed against
2077
a-
b._. FIG. 1. Immunoprecipitation of B. fragilis CP. (a) Double diffusion in agarose of B. fragilis CP with polyclonal rabbit antiserum raised against whole bacteria. (b) IEP of capsular polysaccharide in bacterial buffer, pH 8.6. Trough contains polyclonal rabbit antiserum raised against whole bacteria. Black bar indicates cathode.
water. IEP of this concentrate showed a profile identical to that seen with the phenol-water-extracted CP (as in Fig. lb). The CP was loaded onto a column containing DEAESephacel (50 mM Tris, pH 7.3). Antigens were eluted with an NaCl gradient. Fractions were tested for the presence of CP components by agarose double diffusion. The fractions that eluted with the starting buffer and contained material forming precipitins with the antiserum were pooled, concentrated, dialyzed against distilled water, and lyophilized. This material was called polysaccharide A. The fractions that eluted with a low NaCl concentration (approximately 0.1 M NaCl) and were positive for precipitins were pooled. This latter pool contained a polysaccharide, termed polysaccharide B, which formed a precipitin line distinct from that formed by polysaccharide A (not shown). Fractions eluted with a higher salt concentration contained materials which gave two precipitin lines. These fractions were pooled and called polysaccharide pool C. Approximately 79% of the materials recovered from the DEAE-Sephacel column were eluted as polysaccharide pool C. IEP (Fig. 2, top panel) in barbital buffer at pH 8.6 showed that polysaccharides A and B each formed a single immunoprecipitation arc migrating towards the anode, while polysaccharide pool C had a profile identical to that of the unfractionated CP. The faint second parallel precipitin lines likely represent the Liesegang phenomenon (8). Since polysaccharide A was not retained in the DEAE-Sephacel column, it appeared to lack a net negative charge at pH 7.3. Polysaccharide A migrated toward the anode, however, in IEP at pH 8.6, an indication of a net negative charge at a more basic pH. This conclusion was further supported by IEP performed with 50 mM Tris at pH 7.3. Under these latter conditions, polysaccharide A appeared to be neutral and remained at the origin, while polysaccharide B remained negatively charged (Fig. 2, bottom panel). There was a broad precipitin line (precipitin c) extending between polysaccharides A and B in the CP and in polysaccharide pool C (Fig. 2, top and bottom panels). For chemical analysis, polysaccharide A was rechromatographed on DEAE-Sephacel in Tris buffer at pH 8.6. Under these conditions, polysaccharide A was retained on the column and was eluted as a single peak with an NaCl gradient. An additional precipitin line was seen in the IEP profile of some preparations of CP and polysaccharide A that had a more cathodal migration and was clearly not identical with
2078
PANTOSTI ET AL.
+
CP
A kD 180-
A
a
B C
C
11 68458493727-
C
pH 7.3
polysaccharide A (Fig. 2, bottom panel). This material was separated from polysaccharide A by DEAE-Sephacel chromatography at pH 8.6. This cathodally migrating antigen was not retained by the DEAE column. Further chemical analysis revealed this antigen to be residual rough LPS (45). The CP and polysaccharides A, B, and pool C were analyzed by SDS-PAGE after silver staining or studied by Western blot (immunoblot), probing with a rabbit polyclonal antiserum to the CP. By both methods, the CP and pool C appeared as broad bands of high molecular weight (Fig. 3). Close examination revealed a tightly spaced ladder-like pattern with Mr between 80,000 and 120,000. Polysaccharides A and B could not be detected by silver staining but were faintly visualized by Western blot analysis. Characterization of polysaccharide pool C. Although polysaccharide pool C represented the majority of the material eluted from the DEAE-Sephacel column, it appeared by IEP to be identical to the CP. We wondered, therefore, whether it could be chemically disaggregated. CP was treated with 2% acetic acid at 60°C for increasing time periods (20, 40, and 60 min) and with 5% acetic acid at 1000C for 1 h. With increasing time or concentration of acetic acid, there was a progressive disappearance of the broad precipitin c line in the IEP profile of CP. The IEP profile of the 5% acetic acid-treated CP had only two distinct precipitin arcs, corresponding to polysaccharides A and B (Fig. 4). The precipitin representing polysaccharide B appeared more prominent after 5% acetic acid hydrolysis. Harsher hydrolysis with 0.1 M HCl at 1000C for 1 h caused the CP to lose reactivity in IEP. DEAE-Sephacel chromatography at pH 7.3 of 5% acetic
_.
CP
A
B
C
CP
ii s
FIG. 3. SDS-PAGE analysis of B. fragilis polysaccharides. SDSPAGE of polysaccharides A, B, pool C and CP was performed with an 8% uniform gel. Left panel, silver stain. Right panel, Western blot with polyclonal rabbit antiserum raised against whole bacteria.
B
FIG. 2. Immunoelectrophoretic mobility of B. fragilis polysaccharide. Top panel, IEP of CP and polysaccharides A, B, and pool C in barbital buffer, pH 8.6. Troughs contain polyclonal rabbit antiserum raised against whole bacteria. Note the anodal migration of polysaccharide A. (A) Polysaccharide A precipitin arc (a). (B) Polysaccharide B precipitin arc (b). (C) Precipitin c (c). Bottom panel, IEP of CP and polysaccharides A and B in Tris buffer pH 7.3. Trough contains polyclonal rabbit antiserum raised against whole bacteria. (A) Polysaccharide A precipitin arc (a). (B) Polysaccharide B precipitin arc (b). c, Precipitin c. Note the neutral charge of polysaccharide A.
C
O'.
pH 8.6
Ips
B
INFECT. IMMUN.
acid-treated CP resulted in resolution of polysaccharides A and B as before, with almost no polysaccharide pool C eluted. The total recovery of polysaccharides A and B from 5% acetic acid-hydrolyzed CP run on the DEAE-Sephacel column was equivalent to the quantity of polysaccharides A, B, and pool C recovered before hydrolysis when eluted from the same column. In double-diffusion experiments, native and 5% acetic acid-treated polysaccharide As were observed to be identical, as were native and acetic acid-treated polysaccharide Bs. Furthermore, polysaccharides A and B were not identical with each other (Fig. 5). Antigens were probed with MAb CE3 (specific for polysaccharide A) or MAb F10 (specific for polysaccharide B). In Western blots of CP, polysaccharide A, and polysaccharide B, both MAbs strongly stained the high-molecularweight broad band in the CP. In addition, MAb CE3 stained some material at the top of the gel in the lane containing acetic acid-treated polysaccharide A but not in the lane containing acetic acid-treated polysaccharide B. MAb F10 showed some staining at the top of the gel in the lane containing acetic acid-treated polysaccharide B but not in the lane containing acetic acid-treated polysaccharide A (Fig. 6). Both A and B are contained in the broad band seen in SDS-PAGE gels of CP. Dot blots of polysaccharides A and B by the MAbs confirmed their specificity and demonstrated that both MAbs reacted with the CP. Surface location of polysaccharides A and B. In immunofluorescence experiments, B. fragilis were probed with polyclonal antibody specific for the CP and MAb CE3 or F10. In
2
4
;u
.._
FIG. 4. Immunoelectrophoretic analysis of acid-treated B. fragilis polysaccharide. IEP of B. fragilis CP was done at pH 7.3 in Tris buffer following acid treatment. Trough contains polyclonal rabbit antiserum raised against whole bacteria. (1) Untreated CP. (2) Acetic acid treatment (5%) for 1 h at 100°C.
VOL. 59, 1991
B. FRAGILIS SYNTHESIZES TWO SURFACE POLYSACCHARIDES
2079
TABLE 1. Chemical analysis of polysaccharides of B. fragilis Componenta
Polysaccharide A
Fucose Galactose Quinovosamine Galacturonic acid Glucosamine Galactosaminec d Unidentified amino sugarcd Protein Nucleic acids Phosphorus (,umol/mg) KDO (,ug/mg) Fatty acidc (no. of C)
_b 57 -
+b +
