INFECTION AND IMMUNITY, Dec. 1978, p. 934-944
Vol. 22, No. 3
0019-9567/78/0022-0934$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Modification of Surface Composition of Actinomyces viscosus T14V and T14AV JAMES T. POWELL, WERNER FISCHLSCHWEIGER, AND DALE C. BIRDSELL* Department of Basic Dental Sciences and Department of Immunology and Medical Microbiology, University of Florida, Gainesville, Florida 32610 Received for publication 31 August 1978
The morphology and serology of Actinomyces viscosus T14V and T14AV were compared. When grown in supplemented tryptic soy broth, the virulent strain (T14V) possessed an extensive network of cell surface fibrils. In this medium, the avirulent strain (T14AV) possessed a microcapsule, absent on strain T14V, and a comparatively small number of surface fibrils. Mild acid extraction (Lancefield procedure) solubilized common antigenic components on both strains as well as components detectable only in the virulent strain T14V (virulence-associated antigens 1 and 2). When grown in Socransky chemically defined medium or Carlsson complex medium, the avirulent strain possessed increased amounts of surface fibrils and virulence-associated antigens. Whole cells and extracts of avirulent cells grown in Socransky medium absorbed antibodies to virulenceassociated antigens with approximately the same efficiency as did whole cells and extracts of strain T14V, suggesting antigenic similarity between the two cell types. The results strongly support the hypothesis that observable differences between A. viscosus strains T14V and T14AV represent quantitative, rather than qualitative, differences in particular cell surface components. In addition, the magnitude of these differences can be modified by changing growth conditions.
Actinomyces viscosus, a branching, gram-positive, nonsporeforming rod, has been strongly implicated in the etiology of the human oral diseases gingivitis (7, 9) and periodontitis (8, 9). Studies utilizing germfree animal model systems have shown that colonization of the oral cavity solely with A. viscosus results in excessive plaque formation, root surface caries, and progressive bone loss characteristic of periodontal disease (6, 8, 9). Differential virulence in gnotobiotic rats has been reported utilizing an A. viscosus strain of human origin, T14V, and its laboratory-derived avirulent variant, T14AV (5). It has been suggested that the differences observed between T14V and T14AV strains may represent quantitative, rather than qualitative, differences in the amounts of particular surface components (1). The avirulent strain, T14AV, has been shown to produce copious amounts of extracellular polysaccharide compared with T14V when grown with glucose as the primary carbon and energy source (5). Also, quantitative differences in the amounts of fibrillar material present on the surface of T14V compared with T14AV have been reported (1). To utilize the T14V-T14AV system in examining mechanisms of periodontal pathology, physiological and metabolic characterization of each strain is essential. For such studies, param934
eters of growth environment, physiological age of cells, and preparation of antigens need to be strictly controlled. In this report, methods are compared for analyzing the antigenic composition ofA. viscosus. Evidence is presented which indicates that differences between strains T14V and T14AV can be attributed to quantitative differences in cell surface components. Data are also presented which suggest that the surface composition of A. viscosus T14AV cells varies dramatically in response to alterations in the growth environment. Moreover, batch culture conditions are defined in which T14AV cells appear nearly indistinguishable morphologically and serologically from T14V cells. MATERIALS AND METHODS Bacterial strains. A. viscosus T14V and T14AV were obtained from B. F. Hammond, University of Pennsylvania, Philadelphia. Cultures were stored either as lyohilized stocks or as multiple frozen stocks at -30°C in tryptic soy broth (Difco Laboratories, Detroit, Mich.) containing 20% glycerol. Media and growth. Batch cultures were grown in tryptic soy broth without dextrose (Difco) supplemented with 0.1% yeast extract (Difco) and 1% glucose (TSBS), Carlsson complex medium (CCM) plus 1% glucose (4), or Socransky chemically defined medium (CDM) plus 1% glucose (S. S. Socransky, C. Smith, and A. D. Manganiello, J. Dent. Res. 52[special
VOL. 22, 1978
A. VISCOSUS CELL SURFACE VARIATIONS
issue]:88, Abstr. no. 120, 1973; Socransky, personal communication). Cultures were incubated under microaerophilic conditions (90% N2-10% CO2) at 370C in a Psycrotherm (New Brunswick Scientific Co., New Brunswick, N.J.) shaker incubator (150 rpm). Electron microscopy. Cells in the late exponential phase of growth were placed in 2% glutaraldehyde for 2 h at 40C either in the growth medium or after harvesting and washing with water or buffer (0.05 M KH2PO4, pH 7.5). Cells were collected by centrifugation, and secondary fixation was carried out overnight at room temperature in 1% osmium tetroxide in Kellenberger buffer, pH 6.1 (10). Cells were dehydrated through a graded ethanol series and embedded in Epon-Araldite (Ladd, Burlington, Vt.). Thin sections were cut with diamond knives on a Porter-Blume MT2B ultramicrotome (Norwalk, Conn.), stained with lead citrate (16), and studied in a Zeiss EM9S-2 or EM-10 transmission electron microscope. For examination of unfixed whole cells, exponentialphase cells were washed twice in 10 volumes of saline and placed on carbon-stabilized Parlodion films. Excess cells were removed, and a drop of 1% uranyl acetate in water was added. Excess stain was removed, and the grids were examined at 60 kV in the Zeiss EM9S-2 or at 80 kV in the EM1OA electron microscope. To best visualize the microcapsule in the Zeiss EM1OA electron microscope, the accelerating voltage was decreased to 40 kV. Chemical extraction of whole cells. A modification of the methods of Lancefield and Perlmann (12) was used for chemical extraction of whole cells. Washed whole cells of A. viscosus T14V and T14AV were suspended in saline (0.85% NaCl) with 0.04 N HC1 (5 ml/g [wet weight] of whole cells) and placed in a boiling water bath for 15 min. After cooling in an ice bath, the suspension was titrated to neutrality by dropwise addition of 2.0 N NaOH in saline. Insoluble material was removed by centrifugation at 25,000 x g for 15 min. The supernatant fluid was lyophilized. Alternatively, whole cells were suspended at 0.2 g/ml in saline and autoclaved for 15 min by the procedure of Rantz and Randall (15). After removal of the cells by centrifugation (25,000 x g for 15 min), the supernatant fluid was lyophilized. Hot formamide extraction of whole cells was carried out by the method of Wetherell and Bleiweis (18). Preparation of antisera. Hyperimmune sera were prepared by intraveneous injections of A. viscosus antigen preparations into New Zealand white rabbits. Cells were grown in TSBS or CDM to late exponential phase, harvested, and washed with saline. Cells were suspended at 5 mg (wet weight) per ml in saline and placed in a boiling water bath for 15 min. Four rabbits were initially injected with 0.1 ml of killed whole-cell suspension. Beginning with week 2 and continuing at weekly intervals, rabbits were injected intravenously with 1.0 ml of the whole-cell suspension. Rabbits were bled from the marginal ear vein or by cardiac puncture once per week beginning 7 weeks after the initial immunization. The sera from individual rabbits were pooled before use. No immune reactions were detected between antigen preparations and serum taken before immunization. Immunoelectrophoresis. Antigens present in the various extracts were detected by Laurell rocket im-
935
munoelectrophoresis (LRI) (13). Wells were cut approximately 1 cm from the edge of a glass slide (2 by 3 inches [ca. 5 by 7.6 cm]) containing 4 ml of 0.75% agarose in 0.043 M sodium barbital buffer, pH 8.3. Up to 10 pl of the desired antigen was added to the wells. The agarose 2 mm above the wells was removed and replaced with 3 ml of agarose containing 10 to 50 [1I of antiserum per ml of agarose. In some cases, the Osserman modification (11, 14) was used as an aid in detecting antigenic identity between preparations. For this modification, a trough containing 50 pl of agarose plus 50 pl of the desired reference antigen (10-mg/ml agarose solution) was positioned 1 to 2 mm below the wells. One well containing a Lancefield extract of known composition was included as an internal standard. After electrophoresis at 8 mA/slide, gels were placed in saline overnight and then dialyzed in water for 2 h. Gels were dried and subsequently stained with 0.5% Coomassie brilliant blue R (Sigma Chemical Co., St. Louis, Mo.) in 95% ethanol-glacial acetic acid-water (4.5:1.0:4.5, vol/vol). Destaining was carried out in the above solvent system. Antibody absorptions. A. viscosus T14V and T14AV whole cells (10 mg) or Lancefield extracts (10 mg) were added to microfuge tubes (Bel-Art Products, Paquannock, N.J.) containing 400 1d of antiserum prepared against T14V CDM cells. The tubes were incubated for 1 to 2 h at 37°C and then overnight at 4°C. Precipitates were pelleted with a Beckman Microfuge tabletop centrifuge (Palo Alto, Calif.), and the supernatant fluids, containing unabsorbed antibody, were analyzed by LRI for their reactivity with a standard antigen preparation. The standard antigen preparation was an A. viscosus T14V Lancefield extract (50 mg/ml) prepared from TSBS-grown cells. Fifty percent absorption levels of the antisera were defined as the peak heights obtained when 10 pl of the standard antigen preparation was run in LRI, using a 1:2 dilution of whole anti-T14V antiserum. A standard curve relating antiserum dilution to standard antigen peak height was done with each absorption experiment.
RESULTS Morphology of cells grown in TSBS. Distinct morphological differences were observed between TSBS-grown A. viscosus strains T14V and T14AV by both light and electron microscopy. Examination of unfixed suspensions by phase-contrast microscopy revealed that strain T14V cells were pleiomorphic and grew in large clumps (Fig. 1A), whereas strain T14AV grew in small clumps or singly (Fig. 1C). Examination of thin sections by transmission electron microscopy revealed a delicate fibrillar network on the surface of washed cells of strain T14V (Fig. 1B). The surfaces of similarly prepared strain T14AV cells displayed few or no fibrils (Fig. 1D). Positively stained, washed, and unfixed whole cells of strains T14V and T14AV are shown in Fig. 2A and B. The extensive network of cell-associated fibrils was again evident on the surface of strain T14V. In sharp
POWELL, FISCHLSCHWEIGER, AND BIRDSELL
936
'N
A
INFECT. IMMUN
i
't I
14
B .,;. K-6 lw
FIG. 1. Light and electron micrographs of TSBS-grown A. viscosus T14V and T14AV. (A) Phase-contrast micrograph of unfixed A. viscosus T14V; (B) thin section ofA. viscosus T14V; (C) phase-contrast micrograph of unfixed A. viscosus T14A V; (D) thin section of A. viscosus T14A V. The bar in all micrographs represents 0.5 pmn.
contrast, the avirulent strain possessed few fibrils. A distinct cell-associated microcapsule, as well as a more loosely associated extracellular
component (Fig. 2B), was observed with the avirulent cells. Morphology of cells grown in CDM.
VOL. 22, 1978
-. r *
A. VISCOSUS CELL SURFACE VARIATIONS
'1 11 t.
I.-...
il
F-':
.
_.....
937
.:
ikj t
J,
-
i
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a
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I...
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,,.
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.
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FIG. 2. Transmission electron micrographs of uranyl acetate-stained TSBS-grown A. viscosus T14V and T14A V. (A) A. viscosus T14V; (B) A. viscosus T14A V; f, fibrils; mc, microcapsule; ec, extracellular component.
Phase-contrast and electron microscopic observations of strains T14V and T14AV grown in CDM are shown in Fig. 3. In CDM, both strains exhibited extensive clumping. Electron microscopy of uranyl acetate-stained whole cells of
both strains revealed large quantities of cellassociated fibrils (Fig. 3A and C). High-magnification micrographs of these fibrils (Fig. 3E) revealed their average diameter to be 2.5 nm and showed their tendency to aggregate (arrow).
938
POWELL, FISCHLSCHWEIGER, AND BIRDSELL
INFECT. IMMUN.
FIG. 3. Light and electron micrographs of CDM-grown A. viscosus T14V and T14A V. (A) Uranyl acetatestained A. viscosus T14V; (B) phase-contrast micrograph of A. viscosus T14V; (C) uranyl acetate-stained A. viscosus T14A V; (D) phase-contrast micrograph of A. viscosus T14A V; (E) uranyl acetate-stained A. viscosus T14V; (F) thin section of A. viscosus T14AV.
In thin section (Fig. 3F), the fibrils of strain T14AV appeared morphologically similar to those of strain T14V (Fig. 1B). No microcapsule was evident on CDM-grown strain T14AV cells.
Comparison of several methods of antigen extraction. Immunoelectrophoresis of soluble extracts of TSBS-grown whole cells of A. viscosus T14V prepared by the Lancefield,
A. VISCOSUS CELL SURFACE VARIATIONS
VOL. 22, 1978
Rantz and Randall, and hot-formamide procedures are shown in Fig. 4. The largest number and quantity of antigenic components were extracted by the Lancefield procedure (well 2). At least six components were identified. Four components were extracted by the Rantz and Randall procedure (well 3), two of which had zones of identity with those released by the Lancefield procedure. The hot-formnamide procedure (well 1), commonly used for the extraction of carbohydrate antigens (18), did not release any detectable antigenic components. No antigens detectable with the sera utilized were observed with hot or cold 10% trichloroacetic acid or 0.1 N NaOH extraction procedures (data not
939
shown). Based on these findings, the Lancefield procedure was designated as being the most efficient and was utilized exclusively for all subsequent soluble antigen preparations. Extractable antigens of cells grown in TSBS. The application of quantitative serological techniques to extracts of strain T14V and T14AV whole cells grown in TSBS revealed distinct differences in antigenic composition between the strains. Four predominant antigenic components extracted from T14V solubilized by the Lancefield procedure formed precipitin lines with anti-T14V serum (Fig. 5, well 2). These have been designated (from top to bottom of gel) top common, virulence-associated antigens 1 and 2 (VA 1 and VA 2), and bottom common. The VA antigens were present in extremely low concentrations in Lancefield extracts of T14AV (well 4). Both the top and bottom common antigens were observed in Lancefield extracts
.z.
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FIG. 4. Comparison of antigens extracted from whole cells of A. viscosus T14 Vby various procedures. (1) 250 pg of hot-formamide extract; (2) 250 jig of Lancefield extract; (3) 250 pg of Rantz-Randall extract. Anti-TJ4V (TSBS) whole-cell serum was used at 25 pil/ml of agarose. The reference antigen in the Osserman trough was a Lancefield extract of A. viscosus T14V.
FIG. 5. LRI of extracts from A. viscosus T14V and T14AV. (1) Rantz-Randall extract of A. viscosus T14V; (2) Lancefield extract of A. viscosus T14V; (3) Rantz-Randall extract of T14AV; (4) Lancefield extract of T14AV. Extract and antiserum concentrations were the same as in Fig. 4. The Rantz-Randall extracts were provided by B. F. Hammond (University of Pennsylvania, Philadelphia). TC, Top common; VA 1 and VA 2, virulence-associated antigens 1 and 2; BC, bottom common.
940
POWELL, FISCHLSCHWEIGER, AND BIRDSELL
from both strains. The VA antigens had lines of identity with the Rantz and Randall extracts of strain T14V obtained from B. F. Hammond (well 1) and were not detectable in similar extracts for T14AV (well 3). Effect of growth medium on extractable antigen patterns. Antigens present in Lancefield extracts of T14V and T14AV cells grown in CDM and CCM are shown in Fig. 6. When grown in either medium, strain T14AV cells (wells 2 and 4) displayed extractable antigens that showed identity with the VA antigens of T14V cells grown in TSBS (well 1). In addition, antigens were observed in the CDM (well 2) and CCM (well 4) extracts of strain T14AV which did not show identity with Lancefield extracts of T14V cells grown in TSBS. Absorption of anti-T14V antibodies. The ability of whole-cell suspensions or cell extracts to absorb antibodies is a direct function of their antigenic composition. As Fig. 7 shows, the peak height, representing a quantitative relationship between the standard antigen and the precipitating antibody, increases in a linear fashion after preabsorption of the antisera with increasing concentrations of antigen (T14V TSBS Lancefield extract). For convenience, the increase in peak height after absorption with different concentrations of extracts or whole cells
INFECT. IMMUN.
equivalent to a 1:2 dilution of serum (i.e., the absorption 50%) was used to compare absorption efficiencies (Table 1). As presented in Table 1, TSBS-grown strain T14V whole cells and Lancefield extracts showed a greater ability to absorb anti-T14V antibodies than did TSBS-grown strain T14AV antigen preparations. However, 35
30
EE 25 2
--20 a) 5
(LC
Iv(Y-
1.0 2.0 3.0 4.0 Conc. of Antigen (mg)
5.0
FIG. 7. Effect of absorption of anti-T14V antisewith antigen (T14V TSBS Lancefield extract) on peak height in LRI. Symbols: VA 1; 0, bottom rum
common.
A
fV
i .:,c
4.
4.'.
I L
1
2
3
5
FIG. 6. Effect of culture medium on Lancefield extractable antigens from A. viscosus T14V and T14A V. (1) TSBS-grown A. viscosus T14V; (2) CDM-grown A. viscosus T14AV; (3) CDM-grown A. viscosus T14V; (4) CCM-grown A. viscosus T14AV; (5) CCM-grown A. viscosus T14V. Extract and antiserum concentrations were the same as in Fig. 4.
VOL. 22, 1978
A. VISCOSUS CELL SURFACE VARIATIONS
941
concentrated solutions of strain T14AV TSBS antigen preparations were capable of absorbing antibodies reactive with strain T14V TSBS antigens. Strain T14V CDM-grown whole cells and Lancefield extracts behaved similarly to strain T14V TSBS preparations. In addition, strain T14AV CDM-grown whole cells and Lancefield extracts displayed a two- to fourfold-greater ability to absorb anti-T14V antibody than did the
T14AV TSBS antigen preparations. The reproducibility of data from one absorption experiment to another with the same antigen preparation was ±10%. Anti-T14V precipitating antibody could be removed completely by absorption with appropriate amounts of A. viscosus T14V or T14AV whole cells or Lancefield extracts under all conditions examined (data not shown). Effects of a mixed TSBS-CDM growth enThe transition between TSBSvironment. TABLE 1. Absorption of antibodies with whole cells grown T14AV (absence of VA antigens) and and extracts of A. viscosus T14V and T14AV CDM-grown T14AV (presence of VA antigens) Antiserum absorbed with: Absorption 50% (mg)a was examined in an experiment in which the proportions of TSBS and CDM in the growth Bottom medium were varied. common VA antigen Strain Medium antigen Avirulent cells were grown to late exponential phase in CDM. Cell suspensions were then inWhole cells oculated (1%, vol/vol) to cultures containing T14V
Vol. 22, No. 3
0019-9567/78/0022-0934$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Modification of Surface Composition of Actinomyces viscosus T14V and T14AV JAMES T. POWELL, WERNER FISCHLSCHWEIGER, AND DALE C. BIRDSELL* Department of Basic Dental Sciences and Department of Immunology and Medical Microbiology, University of Florida, Gainesville, Florida 32610 Received for publication 31 August 1978
The morphology and serology of Actinomyces viscosus T14V and T14AV were compared. When grown in supplemented tryptic soy broth, the virulent strain (T14V) possessed an extensive network of cell surface fibrils. In this medium, the avirulent strain (T14AV) possessed a microcapsule, absent on strain T14V, and a comparatively small number of surface fibrils. Mild acid extraction (Lancefield procedure) solubilized common antigenic components on both strains as well as components detectable only in the virulent strain T14V (virulence-associated antigens 1 and 2). When grown in Socransky chemically defined medium or Carlsson complex medium, the avirulent strain possessed increased amounts of surface fibrils and virulence-associated antigens. Whole cells and extracts of avirulent cells grown in Socransky medium absorbed antibodies to virulenceassociated antigens with approximately the same efficiency as did whole cells and extracts of strain T14V, suggesting antigenic similarity between the two cell types. The results strongly support the hypothesis that observable differences between A. viscosus strains T14V and T14AV represent quantitative, rather than qualitative, differences in particular cell surface components. In addition, the magnitude of these differences can be modified by changing growth conditions.
Actinomyces viscosus, a branching, gram-positive, nonsporeforming rod, has been strongly implicated in the etiology of the human oral diseases gingivitis (7, 9) and periodontitis (8, 9). Studies utilizing germfree animal model systems have shown that colonization of the oral cavity solely with A. viscosus results in excessive plaque formation, root surface caries, and progressive bone loss characteristic of periodontal disease (6, 8, 9). Differential virulence in gnotobiotic rats has been reported utilizing an A. viscosus strain of human origin, T14V, and its laboratory-derived avirulent variant, T14AV (5). It has been suggested that the differences observed between T14V and T14AV strains may represent quantitative, rather than qualitative, differences in the amounts of particular surface components (1). The avirulent strain, T14AV, has been shown to produce copious amounts of extracellular polysaccharide compared with T14V when grown with glucose as the primary carbon and energy source (5). Also, quantitative differences in the amounts of fibrillar material present on the surface of T14V compared with T14AV have been reported (1). To utilize the T14V-T14AV system in examining mechanisms of periodontal pathology, physiological and metabolic characterization of each strain is essential. For such studies, param934
eters of growth environment, physiological age of cells, and preparation of antigens need to be strictly controlled. In this report, methods are compared for analyzing the antigenic composition ofA. viscosus. Evidence is presented which indicates that differences between strains T14V and T14AV can be attributed to quantitative differences in cell surface components. Data are also presented which suggest that the surface composition of A. viscosus T14AV cells varies dramatically in response to alterations in the growth environment. Moreover, batch culture conditions are defined in which T14AV cells appear nearly indistinguishable morphologically and serologically from T14V cells. MATERIALS AND METHODS Bacterial strains. A. viscosus T14V and T14AV were obtained from B. F. Hammond, University of Pennsylvania, Philadelphia. Cultures were stored either as lyohilized stocks or as multiple frozen stocks at -30°C in tryptic soy broth (Difco Laboratories, Detroit, Mich.) containing 20% glycerol. Media and growth. Batch cultures were grown in tryptic soy broth without dextrose (Difco) supplemented with 0.1% yeast extract (Difco) and 1% glucose (TSBS), Carlsson complex medium (CCM) plus 1% glucose (4), or Socransky chemically defined medium (CDM) plus 1% glucose (S. S. Socransky, C. Smith, and A. D. Manganiello, J. Dent. Res. 52[special
VOL. 22, 1978
A. VISCOSUS CELL SURFACE VARIATIONS
issue]:88, Abstr. no. 120, 1973; Socransky, personal communication). Cultures were incubated under microaerophilic conditions (90% N2-10% CO2) at 370C in a Psycrotherm (New Brunswick Scientific Co., New Brunswick, N.J.) shaker incubator (150 rpm). Electron microscopy. Cells in the late exponential phase of growth were placed in 2% glutaraldehyde for 2 h at 40C either in the growth medium or after harvesting and washing with water or buffer (0.05 M KH2PO4, pH 7.5). Cells were collected by centrifugation, and secondary fixation was carried out overnight at room temperature in 1% osmium tetroxide in Kellenberger buffer, pH 6.1 (10). Cells were dehydrated through a graded ethanol series and embedded in Epon-Araldite (Ladd, Burlington, Vt.). Thin sections were cut with diamond knives on a Porter-Blume MT2B ultramicrotome (Norwalk, Conn.), stained with lead citrate (16), and studied in a Zeiss EM9S-2 or EM-10 transmission electron microscope. For examination of unfixed whole cells, exponentialphase cells were washed twice in 10 volumes of saline and placed on carbon-stabilized Parlodion films. Excess cells were removed, and a drop of 1% uranyl acetate in water was added. Excess stain was removed, and the grids were examined at 60 kV in the Zeiss EM9S-2 or at 80 kV in the EM1OA electron microscope. To best visualize the microcapsule in the Zeiss EM1OA electron microscope, the accelerating voltage was decreased to 40 kV. Chemical extraction of whole cells. A modification of the methods of Lancefield and Perlmann (12) was used for chemical extraction of whole cells. Washed whole cells of A. viscosus T14V and T14AV were suspended in saline (0.85% NaCl) with 0.04 N HC1 (5 ml/g [wet weight] of whole cells) and placed in a boiling water bath for 15 min. After cooling in an ice bath, the suspension was titrated to neutrality by dropwise addition of 2.0 N NaOH in saline. Insoluble material was removed by centrifugation at 25,000 x g for 15 min. The supernatant fluid was lyophilized. Alternatively, whole cells were suspended at 0.2 g/ml in saline and autoclaved for 15 min by the procedure of Rantz and Randall (15). After removal of the cells by centrifugation (25,000 x g for 15 min), the supernatant fluid was lyophilized. Hot formamide extraction of whole cells was carried out by the method of Wetherell and Bleiweis (18). Preparation of antisera. Hyperimmune sera were prepared by intraveneous injections of A. viscosus antigen preparations into New Zealand white rabbits. Cells were grown in TSBS or CDM to late exponential phase, harvested, and washed with saline. Cells were suspended at 5 mg (wet weight) per ml in saline and placed in a boiling water bath for 15 min. Four rabbits were initially injected with 0.1 ml of killed whole-cell suspension. Beginning with week 2 and continuing at weekly intervals, rabbits were injected intravenously with 1.0 ml of the whole-cell suspension. Rabbits were bled from the marginal ear vein or by cardiac puncture once per week beginning 7 weeks after the initial immunization. The sera from individual rabbits were pooled before use. No immune reactions were detected between antigen preparations and serum taken before immunization. Immunoelectrophoresis. Antigens present in the various extracts were detected by Laurell rocket im-
935
munoelectrophoresis (LRI) (13). Wells were cut approximately 1 cm from the edge of a glass slide (2 by 3 inches [ca. 5 by 7.6 cm]) containing 4 ml of 0.75% agarose in 0.043 M sodium barbital buffer, pH 8.3. Up to 10 pl of the desired antigen was added to the wells. The agarose 2 mm above the wells was removed and replaced with 3 ml of agarose containing 10 to 50 [1I of antiserum per ml of agarose. In some cases, the Osserman modification (11, 14) was used as an aid in detecting antigenic identity between preparations. For this modification, a trough containing 50 pl of agarose plus 50 pl of the desired reference antigen (10-mg/ml agarose solution) was positioned 1 to 2 mm below the wells. One well containing a Lancefield extract of known composition was included as an internal standard. After electrophoresis at 8 mA/slide, gels were placed in saline overnight and then dialyzed in water for 2 h. Gels were dried and subsequently stained with 0.5% Coomassie brilliant blue R (Sigma Chemical Co., St. Louis, Mo.) in 95% ethanol-glacial acetic acid-water (4.5:1.0:4.5, vol/vol). Destaining was carried out in the above solvent system. Antibody absorptions. A. viscosus T14V and T14AV whole cells (10 mg) or Lancefield extracts (10 mg) were added to microfuge tubes (Bel-Art Products, Paquannock, N.J.) containing 400 1d of antiserum prepared against T14V CDM cells. The tubes were incubated for 1 to 2 h at 37°C and then overnight at 4°C. Precipitates were pelleted with a Beckman Microfuge tabletop centrifuge (Palo Alto, Calif.), and the supernatant fluids, containing unabsorbed antibody, were analyzed by LRI for their reactivity with a standard antigen preparation. The standard antigen preparation was an A. viscosus T14V Lancefield extract (50 mg/ml) prepared from TSBS-grown cells. Fifty percent absorption levels of the antisera were defined as the peak heights obtained when 10 pl of the standard antigen preparation was run in LRI, using a 1:2 dilution of whole anti-T14V antiserum. A standard curve relating antiserum dilution to standard antigen peak height was done with each absorption experiment.
RESULTS Morphology of cells grown in TSBS. Distinct morphological differences were observed between TSBS-grown A. viscosus strains T14V and T14AV by both light and electron microscopy. Examination of unfixed suspensions by phase-contrast microscopy revealed that strain T14V cells were pleiomorphic and grew in large clumps (Fig. 1A), whereas strain T14AV grew in small clumps or singly (Fig. 1C). Examination of thin sections by transmission electron microscopy revealed a delicate fibrillar network on the surface of washed cells of strain T14V (Fig. 1B). The surfaces of similarly prepared strain T14AV cells displayed few or no fibrils (Fig. 1D). Positively stained, washed, and unfixed whole cells of strains T14V and T14AV are shown in Fig. 2A and B. The extensive network of cell-associated fibrils was again evident on the surface of strain T14V. In sharp
POWELL, FISCHLSCHWEIGER, AND BIRDSELL
936
'N
A
INFECT. IMMUN
i
't I
14
B .,;. K-6 lw
FIG. 1. Light and electron micrographs of TSBS-grown A. viscosus T14V and T14AV. (A) Phase-contrast micrograph of unfixed A. viscosus T14V; (B) thin section ofA. viscosus T14V; (C) phase-contrast micrograph of unfixed A. viscosus T14A V; (D) thin section of A. viscosus T14A V. The bar in all micrographs represents 0.5 pmn.
contrast, the avirulent strain possessed few fibrils. A distinct cell-associated microcapsule, as well as a more loosely associated extracellular
component (Fig. 2B), was observed with the avirulent cells. Morphology of cells grown in CDM.
VOL. 22, 1978
-. r *
A. VISCOSUS CELL SURFACE VARIATIONS
'1 11 t.
I.-...
il
F-':
.
_.....
937
.:
ikj t
J,
-
i
_r
0
V h;1
a
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s > A. >
I...
"
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,,.
S
.
A
So; *'
FIG. 2. Transmission electron micrographs of uranyl acetate-stained TSBS-grown A. viscosus T14V and T14A V. (A) A. viscosus T14V; (B) A. viscosus T14A V; f, fibrils; mc, microcapsule; ec, extracellular component.
Phase-contrast and electron microscopic observations of strains T14V and T14AV grown in CDM are shown in Fig. 3. In CDM, both strains exhibited extensive clumping. Electron microscopy of uranyl acetate-stained whole cells of
both strains revealed large quantities of cellassociated fibrils (Fig. 3A and C). High-magnification micrographs of these fibrils (Fig. 3E) revealed their average diameter to be 2.5 nm and showed their tendency to aggregate (arrow).
938
POWELL, FISCHLSCHWEIGER, AND BIRDSELL
INFECT. IMMUN.
FIG. 3. Light and electron micrographs of CDM-grown A. viscosus T14V and T14A V. (A) Uranyl acetatestained A. viscosus T14V; (B) phase-contrast micrograph of A. viscosus T14V; (C) uranyl acetate-stained A. viscosus T14A V; (D) phase-contrast micrograph of A. viscosus T14A V; (E) uranyl acetate-stained A. viscosus T14V; (F) thin section of A. viscosus T14AV.
In thin section (Fig. 3F), the fibrils of strain T14AV appeared morphologically similar to those of strain T14V (Fig. 1B). No microcapsule was evident on CDM-grown strain T14AV cells.
Comparison of several methods of antigen extraction. Immunoelectrophoresis of soluble extracts of TSBS-grown whole cells of A. viscosus T14V prepared by the Lancefield,
A. VISCOSUS CELL SURFACE VARIATIONS
VOL. 22, 1978
Rantz and Randall, and hot-formamide procedures are shown in Fig. 4. The largest number and quantity of antigenic components were extracted by the Lancefield procedure (well 2). At least six components were identified. Four components were extracted by the Rantz and Randall procedure (well 3), two of which had zones of identity with those released by the Lancefield procedure. The hot-formnamide procedure (well 1), commonly used for the extraction of carbohydrate antigens (18), did not release any detectable antigenic components. No antigens detectable with the sera utilized were observed with hot or cold 10% trichloroacetic acid or 0.1 N NaOH extraction procedures (data not
939
shown). Based on these findings, the Lancefield procedure was designated as being the most efficient and was utilized exclusively for all subsequent soluble antigen preparations. Extractable antigens of cells grown in TSBS. The application of quantitative serological techniques to extracts of strain T14V and T14AV whole cells grown in TSBS revealed distinct differences in antigenic composition between the strains. Four predominant antigenic components extracted from T14V solubilized by the Lancefield procedure formed precipitin lines with anti-T14V serum (Fig. 5, well 2). These have been designated (from top to bottom of gel) top common, virulence-associated antigens 1 and 2 (VA 1 and VA 2), and bottom common. The VA antigens were present in extremely low concentrations in Lancefield extracts of T14AV (well 4). Both the top and bottom common antigens were observed in Lancefield extracts
.z.
t-.
-~~~~~~~~Ww
FIG. 4. Comparison of antigens extracted from whole cells of A. viscosus T14 Vby various procedures. (1) 250 pg of hot-formamide extract; (2) 250 jig of Lancefield extract; (3) 250 pg of Rantz-Randall extract. Anti-TJ4V (TSBS) whole-cell serum was used at 25 pil/ml of agarose. The reference antigen in the Osserman trough was a Lancefield extract of A. viscosus T14V.
FIG. 5. LRI of extracts from A. viscosus T14V and T14AV. (1) Rantz-Randall extract of A. viscosus T14V; (2) Lancefield extract of A. viscosus T14V; (3) Rantz-Randall extract of T14AV; (4) Lancefield extract of T14AV. Extract and antiserum concentrations were the same as in Fig. 4. The Rantz-Randall extracts were provided by B. F. Hammond (University of Pennsylvania, Philadelphia). TC, Top common; VA 1 and VA 2, virulence-associated antigens 1 and 2; BC, bottom common.
940
POWELL, FISCHLSCHWEIGER, AND BIRDSELL
from both strains. The VA antigens had lines of identity with the Rantz and Randall extracts of strain T14V obtained from B. F. Hammond (well 1) and were not detectable in similar extracts for T14AV (well 3). Effect of growth medium on extractable antigen patterns. Antigens present in Lancefield extracts of T14V and T14AV cells grown in CDM and CCM are shown in Fig. 6. When grown in either medium, strain T14AV cells (wells 2 and 4) displayed extractable antigens that showed identity with the VA antigens of T14V cells grown in TSBS (well 1). In addition, antigens were observed in the CDM (well 2) and CCM (well 4) extracts of strain T14AV which did not show identity with Lancefield extracts of T14V cells grown in TSBS. Absorption of anti-T14V antibodies. The ability of whole-cell suspensions or cell extracts to absorb antibodies is a direct function of their antigenic composition. As Fig. 7 shows, the peak height, representing a quantitative relationship between the standard antigen and the precipitating antibody, increases in a linear fashion after preabsorption of the antisera with increasing concentrations of antigen (T14V TSBS Lancefield extract). For convenience, the increase in peak height after absorption with different concentrations of extracts or whole cells
INFECT. IMMUN.
equivalent to a 1:2 dilution of serum (i.e., the absorption 50%) was used to compare absorption efficiencies (Table 1). As presented in Table 1, TSBS-grown strain T14V whole cells and Lancefield extracts showed a greater ability to absorb anti-T14V antibodies than did TSBS-grown strain T14AV antigen preparations. However, 35
30
EE 25 2
--20 a) 5
(LC
Iv(Y-
1.0 2.0 3.0 4.0 Conc. of Antigen (mg)
5.0
FIG. 7. Effect of absorption of anti-T14V antisewith antigen (T14V TSBS Lancefield extract) on peak height in LRI. Symbols: VA 1; 0, bottom rum
common.
A
fV
i .:,c
4.
4.'.
I L
1
2
3
5
FIG. 6. Effect of culture medium on Lancefield extractable antigens from A. viscosus T14V and T14A V. (1) TSBS-grown A. viscosus T14V; (2) CDM-grown A. viscosus T14AV; (3) CDM-grown A. viscosus T14V; (4) CCM-grown A. viscosus T14AV; (5) CCM-grown A. viscosus T14V. Extract and antiserum concentrations were the same as in Fig. 4.
VOL. 22, 1978
A. VISCOSUS CELL SURFACE VARIATIONS
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concentrated solutions of strain T14AV TSBS antigen preparations were capable of absorbing antibodies reactive with strain T14V TSBS antigens. Strain T14V CDM-grown whole cells and Lancefield extracts behaved similarly to strain T14V TSBS preparations. In addition, strain T14AV CDM-grown whole cells and Lancefield extracts displayed a two- to fourfold-greater ability to absorb anti-T14V antibody than did the
T14AV TSBS antigen preparations. The reproducibility of data from one absorption experiment to another with the same antigen preparation was ±10%. Anti-T14V precipitating antibody could be removed completely by absorption with appropriate amounts of A. viscosus T14V or T14AV whole cells or Lancefield extracts under all conditions examined (data not shown). Effects of a mixed TSBS-CDM growth enThe transition between TSBSvironment. TABLE 1. Absorption of antibodies with whole cells grown T14AV (absence of VA antigens) and and extracts of A. viscosus T14V and T14AV CDM-grown T14AV (presence of VA antigens) Antiserum absorbed with: Absorption 50% (mg)a was examined in an experiment in which the proportions of TSBS and CDM in the growth Bottom medium were varied. common VA antigen Strain Medium antigen Avirulent cells were grown to late exponential phase in CDM. Cell suspensions were then inWhole cells oculated (1%, vol/vol) to cultures containing T14V
