0019-9567/78/0019-0265$02.00/0 INFECTION AND IMMUNITY, Jan. 1978, p. 265-271 Copyright © 1978 American Society for Microbiology
Vol. 19, No. 1
Printed in U.S.A.
Immunoelectron Microscopic Localization of Lipopolysaccharides in the Cell Wall of Bacteroides oralis and Fusobacterium nucleatum G. DAHLEN,1 H. NYGREN,l* AND H.-A. HANSSON2
Department of Oral Microbiology, Institute of Medical Microbiology,' and Department of Histology,2 University of Goteborg, Goteborg, Sweden Received for publication 8 June 1977
Lipopolysaccharides (LPS) have been extracted and purified from two anaerobic gram-negative bacteria: Bacteroides oralis and Fusobacterium nucleatum. Chemical analysis of the preparations showed a great proportion of neutral sugars, mainly glucose, in LPS of B. oralis. In rabbits, LPS of B. oralis induced both immunoglobulin M and G antibodies in contrast to LPS of F. nucleatum, to which only immunoglobulin M antibodies were produced. An immunohistochemical method with horseradish peroxidase-labeled antibodies was used to localize LPS antigens at the ultrastructural level. An electron-dense reaction product, representing an immune complex consisting of bacterial surface antigens and specific rabbit immunoglobulin labeled with peroxidase, was surrounding the cell wall, whereas appropriate controls were negative. The findings of the present study show that LPS of Bacteroides are probably bound to a complex, including glucans, in the outer membrane of the cell wall. LPS of Fusobacterium resemble LPS of other gram-negative bacteria.
(i) the chemical composition, (ii) the immunogenicity, and (iii) the ultrastructural localization of LPS in the cell wall of Bacteroides and Fusobacterium were investigated.
The outer membrane of the cell wall in gram-
negative microorganisms consists of a macromolecular complex of lipopolysaccharides (LPS) with biological activities collectively known as endotoxic. The anaerobic gram-negative bacteria Bacteroides and Fusobacterium have gathered increasing interest as possible pathogenic organisms in many infections (2). These bacteria are frequently found in association with many oral infections, e.g., in periodontal disease (25) and periapical inflammation of teeth (G. Sundqvist, Ph.D. thesis, Umea University, Umea, Sweden, 1976), in which endotoxin may participate as a pathogenetic factor. Bacteroides strains differ from other gram-negative bacteria, including Fusobacterium, in their chemical composition of LPS. They do not contain heptose or 2-keto-3-deoxyoctulosonic acid (KDO) (13); they have no typical lipid A (T. Hofstad, personal communication); and the endotoxic activity is low (12; G. Dahlen and T. Hofstad, Scand. J. Dent. Res., in press). Another difference concerning LPS of Bacteroides strains in contrast to Fusobacterium has appeared to be the content of glucans in LPS preparations, when extraction with the phenol-water method is used (11). By immunoelectron microscopic technique, it should be possible to obtain an indication whether these glucans occur in the cytoplasm or as a part of the cell wall. In the present study
MATERIALS AND METHODS Bacterial strains. Bacteroides oralis (strain BactMC3) and Fusobacterium nucleatum (strain FusMC8), originally isolated from the root canal of a monkey tooth, were used. Cultivation. The culture medium was described by Dahlen and Hofstad (in press). The cultivation was carried out in a fermentor with stirring at a constant pH 7.2. The cells were harvested at the end of the logarithmic phase. Isolation of LPS. LPS were extracted with 45% phenol for 15 min at room temperature (11) from packed wet cells of strain Bact-MC3 and from crushed, defatted, and buffer-extracted cells of strain Fus-MC8 (18). After dialyzing against water, the LPS preparations were purified by centrifugation at 100,000 x g for 1 h, treated with deoxyribonuclease (DNase, 0.05 mg/ml, pH 7.0, Sigma Chemical Co., St. Louis, Mo.) and ribonuclease (RNase, 0.05 mg/ml, pH 7.0, Sigma), recentrifuged, and lyophilized. Chemical analysis. The procedures for paper chromatography and quantitative estimations for protein, carbohydrates, fatty acid esters, glucosamine, and KDO content were carried out according to Dahlen and Hofstad (in press). Serological method. For estimation of specific antibody activity, indirect hemagglutination (HA) and
265
266
DAHLRN, NYGREN, AND HANSSON
inhibition of hemagglutination were used, according to the modification described by Hofstad (14). Preparation and conjugation of antibodies. Rabbits were injected intravenously with.1 ml of an LPS solution from strain Bact-MC3 or Fus-MC8 in increasing concentrations from 0.1 to 1.5 mg/ml three times a week in 3 weeks, and were then bled 1 week after the last injection. The serum was collected and fractioned by gel chromatography (Sephadex G-200, Pharmacia, Uppsala, Sweden). Each fraction was analyzed for specific anti-LPS antibodies. Pooled fractions were concentrated 10 times against 30% polyethylene glycol. Immunoglobulin G and M (IgG; IgM) antibodies to Bact-MC3 LPS and IgM antibodies to Fus-MC8 LPS were conjugated with peroxidase. The antibody solution (200,ul) was mixed under continuous shaking with 0.25% glutaraldehyde (25 pl) and horseradish peroxidase (12.5 mg, type VI, Sigma) at room temperature for 45 to 90 min (1). The antibodies conjugated with peroxidase were separated from free peroxidase on a Sephadex G-100 column (Pharmacia). The fractions containing protein were identified by their adsorption at 280 and 405 nm. The specific antibody activity after conjugation was found in pilot studies to decrease to about 5% of the initial activity. Fixation of bacteria. The bacterial cells were separated from the culture medium by centrifugation and were fixed in a cacodylate-buffered solution (0.1 M, pH 7.2) containing either 4% freshly prepared formaldehyde with 0.1% picric acid (10) or 3% purified glutaraldehyde. After aldehyde fixation, the bacteria were rinsed in 0.15 M cacodylate buffer for 12 h. The bacteria used in the morphological experiments were postfixed in 2% osmium tetroxide in Veronal-acetate buffer directly after the glutaraldehyde fixation. Immunohistochemical analysis. The bacteria, either freshly isolated or fixed in aldehyde only, as described above, were incubated for 20 to 30 min in peroxidase-conjugated antibodies diluted 1:10 to 1:200 with 0.15 M cacodylate buffer, rinsed thoroughly, and incubated in cacodylate buffer containing 0.05% 3.3diaminobenzidine-0.01% hydrogen peroxidase for 45 min. After rinsing, the bacteria were postfixed for 1 h in 2% osmium tetroxide, dehydrated in ethanol, and embedded in Epon. Control incubations comprised (i) Bact-MC3 cells in anti-Fus-MC8 LPS antibodies conjugated with peroxidase and (ii) Fus-MC8 cells in antiBact-MC3 LPS antibodies conjugated with peroxidase. Furthermore, cells of both strains were incubated with (iii) goat anti-rabbit immunoglobulins conjugated with peroxidase, (iv) antiserum adsorbed with homologous antigen after conjugation, (v) peroxidase, and (vi) diaminobenzidine without preincubation with antibodies. Electron microscopy. Sections were cut on an LKB Ultrotome III ultramicrotome and examined in a Siemens Elmiskop IA electron microscope. Unstained sections and sections stained with uranyl acetate and lead citrate were used.
RESULTS Chemical analysis. LPS from Bact-MC3 contained 2.6% protein, 57.6% carbohydrates, 5.5% fatty acid esters, and 2.0% hexosamine.
INFECT. IMMUN.
KDO was lacking. The main sugar was glucose, but small amounts of glucosamine, galactose, rhamnose, and ribose were also found. LPS from Fus-MC8 contained 5.9% protein, 29.7% carbohydrates, 22.7% fatty acid esters, 6.3% glucosamine, and 2.0% KDO. The main sugars were heptose, glucose, glucosamine, and rhamnose. Immunogenicity. Gel chromatography of antiserum revealed two separate peaks for titers of Bact-MC3 LPS (Fig. 1), on the basis of the hemagglutination activity of each fraction. The peaks corresponded to the elution pattern of IgM and IgG antibodies, respectively. By contrast, LPS of Fus-MC8 gave only an IgM response, as indicated by a single peak in Fig. 2. The antibody titers of the pooled- and concentrated-antibody fractions showed no significant cross-reaction to LPS between Bact-MC3 and Fus-MC8 antibodies by the hemagglutination test or by hemagglutination inhibition. Ultrastructural localization of LPS. The morphology of the outer membrane of the cell wall of Bact-MC3 showed the triple-laminar structure typical of gram-negative bacteria (Fig. 3a). In Fus-MC8, the outer membrane of the A__ 2 !80
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FIG. 3. (a) Electron micrograph of cells of B. oralis. The trilaminated outer membrane of the cell wall (arrow) and the cytoplasmic membrane enclose the granular and filamentous structures of the cell. The inner cell waU is very thin and can only be seen in some areas (arrow head). Counterstained with uranyl acetate and lead citrate. x160,000. (b) Electron micrograph of cells of F. nucleatum counterstained with uranyl acetate and lead citrate. The cell waU consists of a trilamellar outer membrane (arrow) that is separated from the electron-dense inner ceU wall and the cytoplasmic membrane proper (arrow head). Numerous granules, vesicles, and filaments are seen within the cytoplasm. x200,000.
cell wall was distinctly stained by uranyl acetate and lead citrate (Fig. 3b). The outer membrane of the cell wall stripped off easily and piled up in double layers, or it was completely lost. No conclusive difference in- ultrastructure of the outer membrane was found between the two strains. The inner lamina of the cell wall of Bact-MC3, however, was thinner and less electron dense than that of Fus-MC8. Incubation of homologous cells in anti-Bact-MC3 LPS, conjugated with peroxidase, resulted in a granular irregular staining of the outer membrane of the cell wall (Fig. 4a). In some sites, the immune reaction produced larger dots, with a cross-sectional area 10 to 100 times that of the small granules (Fig. 4a, b, and c). The immunohistochemical reaction was restricted to the membrane of the cell wall. No other part of the cell reacted. After formaldehyde fixation, the immunohistochemical reaction was diminished (Fig. 4b) and was almost abolished by glutaraldehyde fix-
ation (Fig. 4c). The IgG antibodies to Bact-MC3 LPS (Fig. 4d) showed a distribution similar to the IgM antibodies (Fig. 4a). In Fig. 5a, the outer membrane of the cell wall of an unfixed cell is seen together with the immunohistochemical reaction product, which is irregularly distributed. The reaction is restricted to the outer membrane of the cell wall. Glutaraldehyde fixation (Fig. 5b) almost abolished the reaction. Control incubations (see i through vi in Materials and Methods) resulted in very slight activity (Fig. 4e). DISCUSSION The high glucose content in the LPS of Bacteroides has been considered to be a contamination of intracellular glucans (11, 16; Dahlen and Hofstad, in press). Attempts to prepare glucogen-free LPS from different Bacteroides strains with different extraction methods have failed in the sense that a high glucose content could not be avoided (T. Hofstad, K. Sveen, and
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VOL. 19, 1978
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5b FIG. 5. (a) Electron micrograph of F. nucleatum incubated in peroxidase-conjugated IgM antibodies to LPS and further processed for immunoelectron microscopy. The reactivity is limited to the outer membrane of the cell wall, and there are no reaction products to be observed in the interior of the bacteria cell. Note the granular distribution of the reaction products indicating that parts of the covering LPS were stripped off during the process of the bacteria. The three lamellar structures of the cell wall are seen through some of the granules (arrow). X70,000. (b) Electron micrograph of a glutaraldehyde-fixed cell of F. nucleatum incubated in peroxidase-conjugated antibodies to LPS of B. oralis. There is no significant reaction to be observed in the cell wall. x60,000.
G. Dahl6n, Acta Pathol. Microbiol. Scand. Sect. B, in press). This glucan content of LPS BactMC3 may explain the diverse immunological pattern resulting in both IgM and IgG antibody production observed in the present study. It is well known that antibodies against somatic antigen of Salmonella species and Escherichia coli in animals (20) and humans (8, 21, 24) are 19S macroglobulins. Somatic antigen refers to the polysaccharide-containing 0-antigen, which is supposed to be the main antigenic determinant of LPS endotoxin. It might thus be argued that the LPS preparation of Bact-MC3 contains two immunologically different substances and
does not correspond immunologically to the endotoxin of Enterobacteriaceae and Fusobacterium. Recently Hofstad (15) has succeeded in separating LPS from Bacteroides fragilis into two different types responsible for agglutinating and precipitating antibodies, respectively. It is not yet clear whether or not these two types have different chemical compositions with respect to their glucose content. Both IgM and IgG antibodies conjugated with peroxidase were found to be attached to the outer membrane of the cell wall of Bact-MC3 cells (Fig. 4c and d). This fmding indicates that, even if an antigenic contamination is responsible for the diverse im-
FIG. 4. (a) Cells of B. oralis incubated in peroxidase-conjugated IgM antibodies to LPS. There is no reaction to be observed in this except in the outer cell wall. (Unfixed.) Note the granular distribution of the reaction products and the larger dots (arrow). x 70,000. (b) A cell of B. oralis fixed in formaldehyde before incubation with IgM antibodies to LPS as described in (a). There is a decrease in the reactivity indicating partial loss of antigenic activity due to the Formalin fixation. There is no reactivity in the cytoplasmic membrane or in the cytoplasm. Note the larger dot of antibody reaction (arrow). x70,000. (c) Electron micrograph of a glutaraldehyde-fixed cell of B. oralis incubated in peroxidase conjugated with IgM antibodies to LPS. There is a faint reaction to be observed in the outer membrane of the cell wall. x 70,000. (d) Electron micrograph of B. oralis freshly incubated in peroxidase-conjugated IgG antibodies to LPS. The distribution of reaction products is similar although less intense than that in (a). x 75,000. (e) Electron micrograph of a oralis cell treated with peroxidase-conjugated antibodies to LPS of F. nucleatum. There is hardly any reactivity to be observed. x 75,000.
270
DAHLEN, NYGREN, AND HANSSON
munological response, it originates from the same outer membrane as the LPS. This supports the theory that glucans are a part of the LPS molecular complex. The cell wall of gram-negative bacteria consists of a number of distinct layers with differing chemical compositions. The outer membrane, which contains LPS, appears to be triple layered when examined with the electron microscope (6). Various Bacteroides species have been found to have this triple-layered outer membrane (4, 5, 27). The present study confirms that the cell walls of B. oralis and F. nucleatum also have these structures. The staining of the LPS complex of Fus-MC8 was not constant along the whole-cell periphery. In spite of the fact that the cells were harvested in the logarithmic phase of growth, the outer membrane of the cell wall was lost in some areas. The explanation for this difference in stability of the cell wall between strains Bact-MC3 and Fus-MC8 may be the chemical differences in LPS composition, but more probably it may be the result of an artifact produced by centrifugation; Fus-MC8 is a rather filamentous cell, which possibly makes it more sensitive to mechanical forces than the more coccoid BactMC3. Location of the 0 antigen-containing LPS in the cell wall of gram-negative bacteria has been studied by other methods (9, 22, 23, 26). Utilizing the peroxidase-conjugated method of other studies for localization of bacterial surface antigens (3, 19), the location of LPS in the outer cell membrane was confirmed for both Bacteroides and Fusobacterium. No immunohistochemical reaction was observed inside the cells or in the inner parts of the cell wall. This does not exclude the possibility of antigenic sites inside the cell that could not be demonstrated because the conjugated immunoglobulins could not penetrate through the cell wall. However, it should be noted that no reaction product was seen within cells undergoing lysis where penetration should have been facilitated. This indicates a low probability for the existence of intracellular antigens cross-reacting with LPS. The immunohistochemical reactions were highly reproducible. The controls showed almost no reactivity. There was no cross-reaction between the different antisera in either the indirect hemagglutination test or the immunohistochemical reaction. This demonstrates the high reliability of the method. Two different patterns were demonstrated with regard to the distribution of the reaction product on Bact-MC3 cells. One of these, the fine, granular type, also appeared on the cell wall of Fus-MC8 (Fig. 4a and
INFECT. IMMUN.
5a). No difference was seen in the appearance of the label when either peroxidase-conjugated IgG or IgM antibodies against Bact-MC3 LPS were used. The coarser, dotlike precipitates were formed irrespective of the method of fixation. The reaction products were localized exclusively on the outer part of the trilaminated membrane of the cell wall. The appearance of the larger dots was reminiscent of the "blebs" on the cell wall of B. fragilis described by Kasper (17). The granular pattern of the reactive sites indicates that these sites are randomly distributed on the cell wall. Aldehyde fixation diminished the intensity of the immunohistochemical reaction, but the distribution was the same in freshly isolated and aldehyde-fixed cells. This means that there was no redistribution due to lack of fixation of the LPS. Edebo and Holme (7) showed that LPS are extracted from the cell wall even in saline and buffer solutions. In the fresh preparations used in the present study, LPS may have been partially extracted, but, as the distribution of the reaction products was the same in freshly prepared and fixed cells, this was not considered to be a significant source of error. ACKNOWLEDGMENTS This work was supported by Goteborgs Tandliikare-SalIskap and by grants from the Faculty of Odontology, Goteborg, and Swedish Medical Research Council Project B 77-254309.
LiMRATURE CMD 1. Avrameas, S. 1969. Coupling of enzymes to proteins with glutaraldehyde. Use of conjugates for the detection of antigens and antibodies. Immunochemistry 6:43-48. 2. Balows, A., R. M. Dehaan, V. R. Dowell, and L. B. Guze. 1974. Anaerobic bacteria. Role in disease.
Charles C Thomas, Publisher, Springfield, Ill. 3. Berthold, P., D. Bratthall, and C.-H. Berthold. 1974. Immunoperoxidase staining of Streptococcus mutans. Arch. Oral Biol. 19:1227-1230. 4. Bladen, H. A., and J. F. Waters. 1963. Electron microscopic study of some strains of Bacteroides. J. Bacteriol. 86:1339-1344. 5. Costerton, J. W., H. N. Damgaard, and K.-J. Cheng. 1974. Cell envelope morphology of rumen bacteria. J. Bacteriol. 118:1132-1143. 6. Costerton, J. W., J. M. Ingram, and K.-J. Cheng. 1974. Structure and function of the cell envelope of gram-negative bacteria. Bacteriol. Rev. 38:87-110. 7. Edebo, L., and T. Holme. 1965. Preparation of biologically active fractions of Salmonella typhimurium. Acta Pathol. Microbiol. Scand. 63:228-234. 8. Evans, R. T., S. Spaeth, and S. E. Morgenhagen. 1966. Bacterial antibody in mammalian serum to obligatorily anaerobic gram-negative bacteria. J. Immunol. 97:112-119. 9. Fukushi, K., F. Ariji, J. Yamaguchi, and S. Oka. 1966. Electron microscopic localization of endotoxin of Escherichia coli by the use of ferritin-conjugated antibody. J. Electron Microsc. 15:137-142. 10. Haglid, K., A. Hamberger, H.-A. Hansson, H. Hyden, L. Persson, and L. Ronnback. 1976. Cellular and
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LPS IN BACTEROIDES AND FUSOBACTERIUM
subcellular distribution of the S-100 protein in rabbit and rat central nervous system. J. Neurosci. Res. 2:175-191. 11. Hofstad, T. 1968. Chemical characteristics of Bacteroides melaninogenicus endotoxin. Arch. Oral Biol. 13:1149-1155. 12. Hofstad, T. 1970. Biological activities of endotoxin from Bacteroides melaninogenicus. Arch. Oral Biol. 15:343-348. 13. Hofstad, T. 1974. The distribution of heptose and 2-keto3-deoxyoctonate in Bacteroidaceae. J. Gen. Microbiol. 85:314-320. 14. Hofstad, T. 1974. Antibodies reacting with lipopolysaccharides from Bacteroides melaninogenicus, Bacteroides fragilis, and Fusobacterium nucleatum in serum from normal human subjects. J. Infect. Dis. 129:349-351. 15. Hofstad, T. 1976. Purification of the 0 antigen of Bacteroides fragilis ss. fragilis NCTC 9343 from phenolwater extracts by gel filtration and chromatography on DEAE-cellulose and hydroxylapatite. Acta Pathol. Microbiol. Scand. Sect. B. 84:229-234. 16. Hofstad, T., and T. Kristoffersen. 1970. Chemical characteristics of endotoxin from Bacteroides fragilis NCTC 9343. J. Gen. Microbiol. 61:15-19. 17. Kasper, D. L. 1976. The polysaccharide capsule of Bacteroides fragilis subspecies fragilis: immunochemical and morphologic definition. J. Infect. Dis. 133:79-87. 18. Kristoffersen, T. 1969. Immunochemical studies of oral fusobacteria. 3. Purification of a group reactive precipitinogens. Acta Pathol. Microbiol. Scand. 77:447-456. 19. Lai, C.-H., M. A. Listgarten, and B. Rosan. 1975. Immunoelectron microscopic identification and locali-
20.
21. 22.
23. 24.
25. 26.
27.
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zation of Streptococcus sanguis with peroxidase-labeled antibody: localization of surface antigens in pure cultures. Infect. Immun. 11:193-199. Landy, M., and W. P. WVeidanz. 1964. Natural antibodies against gram-negative bacteria, p. 275-290. In M. Landy and W. Braun (ed.), Bacterial endotoxins. Rutgers University Press, New Brunswick, N.J. Michael, J. G., and F. S. Rosen. 1963. Association of "natural antibodies" to gram-negative bacteria with the y-macroglobulins. J. Exp. Med. 118:619-626. Milner, K. C., R. L. Anacker, K. Fukushi, W. T. Haskins, M. Landy, B. Malmgren, and E. Ribi. 1963. Symposium on relationship of structure of microorganisms to their immunological properties. III. Structure and biological properties of surface antigens from gram-negative bacteria. Bacteriol. Rev. 27:352-368. Shands, J. W. 1966. Localization of somatic antigen on gram-negative bacteria using ferritin antibody conjugates. Ann. N.Y. Acad. Sci. 133:292-298. Smith, R. T. 1960. Immunity in infancy. Pediatr. Clin. North Am. 7:269-293. Socransky, S. S. 1970. Relationship of bacteria to the etiology of periodontal disease. J. Dent. Res. 49:203-222. Ushijima, T. 1970. Morphology and chemistry of the bacterial cell wall. 1. The location of mucopeptide in the cell wall of Bacteroides convexus and its chemical composition. Jpn. J. Microbiol. 14:15-25. Ushijima, T., K. Ueno, S. Suzuki, and U. Kurimoto. 1971. Morphology and chemistry of the cell wall. II. Sugar composition and location of 0-antigen in the cell wall of Bacteroides convexus. J. Electron Microsc. 20:32-39.
Vol. 19, No. 1
Printed in U.S.A.
Immunoelectron Microscopic Localization of Lipopolysaccharides in the Cell Wall of Bacteroides oralis and Fusobacterium nucleatum G. DAHLEN,1 H. NYGREN,l* AND H.-A. HANSSON2
Department of Oral Microbiology, Institute of Medical Microbiology,' and Department of Histology,2 University of Goteborg, Goteborg, Sweden Received for publication 8 June 1977
Lipopolysaccharides (LPS) have been extracted and purified from two anaerobic gram-negative bacteria: Bacteroides oralis and Fusobacterium nucleatum. Chemical analysis of the preparations showed a great proportion of neutral sugars, mainly glucose, in LPS of B. oralis. In rabbits, LPS of B. oralis induced both immunoglobulin M and G antibodies in contrast to LPS of F. nucleatum, to which only immunoglobulin M antibodies were produced. An immunohistochemical method with horseradish peroxidase-labeled antibodies was used to localize LPS antigens at the ultrastructural level. An electron-dense reaction product, representing an immune complex consisting of bacterial surface antigens and specific rabbit immunoglobulin labeled with peroxidase, was surrounding the cell wall, whereas appropriate controls were negative. The findings of the present study show that LPS of Bacteroides are probably bound to a complex, including glucans, in the outer membrane of the cell wall. LPS of Fusobacterium resemble LPS of other gram-negative bacteria.
(i) the chemical composition, (ii) the immunogenicity, and (iii) the ultrastructural localization of LPS in the cell wall of Bacteroides and Fusobacterium were investigated.
The outer membrane of the cell wall in gram-
negative microorganisms consists of a macromolecular complex of lipopolysaccharides (LPS) with biological activities collectively known as endotoxic. The anaerobic gram-negative bacteria Bacteroides and Fusobacterium have gathered increasing interest as possible pathogenic organisms in many infections (2). These bacteria are frequently found in association with many oral infections, e.g., in periodontal disease (25) and periapical inflammation of teeth (G. Sundqvist, Ph.D. thesis, Umea University, Umea, Sweden, 1976), in which endotoxin may participate as a pathogenetic factor. Bacteroides strains differ from other gram-negative bacteria, including Fusobacterium, in their chemical composition of LPS. They do not contain heptose or 2-keto-3-deoxyoctulosonic acid (KDO) (13); they have no typical lipid A (T. Hofstad, personal communication); and the endotoxic activity is low (12; G. Dahlen and T. Hofstad, Scand. J. Dent. Res., in press). Another difference concerning LPS of Bacteroides strains in contrast to Fusobacterium has appeared to be the content of glucans in LPS preparations, when extraction with the phenol-water method is used (11). By immunoelectron microscopic technique, it should be possible to obtain an indication whether these glucans occur in the cytoplasm or as a part of the cell wall. In the present study
MATERIALS AND METHODS Bacterial strains. Bacteroides oralis (strain BactMC3) and Fusobacterium nucleatum (strain FusMC8), originally isolated from the root canal of a monkey tooth, were used. Cultivation. The culture medium was described by Dahlen and Hofstad (in press). The cultivation was carried out in a fermentor with stirring at a constant pH 7.2. The cells were harvested at the end of the logarithmic phase. Isolation of LPS. LPS were extracted with 45% phenol for 15 min at room temperature (11) from packed wet cells of strain Bact-MC3 and from crushed, defatted, and buffer-extracted cells of strain Fus-MC8 (18). After dialyzing against water, the LPS preparations were purified by centrifugation at 100,000 x g for 1 h, treated with deoxyribonuclease (DNase, 0.05 mg/ml, pH 7.0, Sigma Chemical Co., St. Louis, Mo.) and ribonuclease (RNase, 0.05 mg/ml, pH 7.0, Sigma), recentrifuged, and lyophilized. Chemical analysis. The procedures for paper chromatography and quantitative estimations for protein, carbohydrates, fatty acid esters, glucosamine, and KDO content were carried out according to Dahlen and Hofstad (in press). Serological method. For estimation of specific antibody activity, indirect hemagglutination (HA) and
265
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DAHLRN, NYGREN, AND HANSSON
inhibition of hemagglutination were used, according to the modification described by Hofstad (14). Preparation and conjugation of antibodies. Rabbits were injected intravenously with.1 ml of an LPS solution from strain Bact-MC3 or Fus-MC8 in increasing concentrations from 0.1 to 1.5 mg/ml three times a week in 3 weeks, and were then bled 1 week after the last injection. The serum was collected and fractioned by gel chromatography (Sephadex G-200, Pharmacia, Uppsala, Sweden). Each fraction was analyzed for specific anti-LPS antibodies. Pooled fractions were concentrated 10 times against 30% polyethylene glycol. Immunoglobulin G and M (IgG; IgM) antibodies to Bact-MC3 LPS and IgM antibodies to Fus-MC8 LPS were conjugated with peroxidase. The antibody solution (200,ul) was mixed under continuous shaking with 0.25% glutaraldehyde (25 pl) and horseradish peroxidase (12.5 mg, type VI, Sigma) at room temperature for 45 to 90 min (1). The antibodies conjugated with peroxidase were separated from free peroxidase on a Sephadex G-100 column (Pharmacia). The fractions containing protein were identified by their adsorption at 280 and 405 nm. The specific antibody activity after conjugation was found in pilot studies to decrease to about 5% of the initial activity. Fixation of bacteria. The bacterial cells were separated from the culture medium by centrifugation and were fixed in a cacodylate-buffered solution (0.1 M, pH 7.2) containing either 4% freshly prepared formaldehyde with 0.1% picric acid (10) or 3% purified glutaraldehyde. After aldehyde fixation, the bacteria were rinsed in 0.15 M cacodylate buffer for 12 h. The bacteria used in the morphological experiments were postfixed in 2% osmium tetroxide in Veronal-acetate buffer directly after the glutaraldehyde fixation. Immunohistochemical analysis. The bacteria, either freshly isolated or fixed in aldehyde only, as described above, were incubated for 20 to 30 min in peroxidase-conjugated antibodies diluted 1:10 to 1:200 with 0.15 M cacodylate buffer, rinsed thoroughly, and incubated in cacodylate buffer containing 0.05% 3.3diaminobenzidine-0.01% hydrogen peroxidase for 45 min. After rinsing, the bacteria were postfixed for 1 h in 2% osmium tetroxide, dehydrated in ethanol, and embedded in Epon. Control incubations comprised (i) Bact-MC3 cells in anti-Fus-MC8 LPS antibodies conjugated with peroxidase and (ii) Fus-MC8 cells in antiBact-MC3 LPS antibodies conjugated with peroxidase. Furthermore, cells of both strains were incubated with (iii) goat anti-rabbit immunoglobulins conjugated with peroxidase, (iv) antiserum adsorbed with homologous antigen after conjugation, (v) peroxidase, and (vi) diaminobenzidine without preincubation with antibodies. Electron microscopy. Sections were cut on an LKB Ultrotome III ultramicrotome and examined in a Siemens Elmiskop IA electron microscope. Unstained sections and sections stained with uranyl acetate and lead citrate were used.
RESULTS Chemical analysis. LPS from Bact-MC3 contained 2.6% protein, 57.6% carbohydrates, 5.5% fatty acid esters, and 2.0% hexosamine.
INFECT. IMMUN.
KDO was lacking. The main sugar was glucose, but small amounts of glucosamine, galactose, rhamnose, and ribose were also found. LPS from Fus-MC8 contained 5.9% protein, 29.7% carbohydrates, 22.7% fatty acid esters, 6.3% glucosamine, and 2.0% KDO. The main sugars were heptose, glucose, glucosamine, and rhamnose. Immunogenicity. Gel chromatography of antiserum revealed two separate peaks for titers of Bact-MC3 LPS (Fig. 1), on the basis of the hemagglutination activity of each fraction. The peaks corresponded to the elution pattern of IgM and IgG antibodies, respectively. By contrast, LPS of Fus-MC8 gave only an IgM response, as indicated by a single peak in Fig. 2. The antibody titers of the pooled- and concentrated-antibody fractions showed no significant cross-reaction to LPS between Bact-MC3 and Fus-MC8 antibodies by the hemagglutination test or by hemagglutination inhibition. Ultrastructural localization of LPS. The morphology of the outer membrane of the cell wall of Bact-MC3 showed the triple-laminar structure typical of gram-negative bacteria (Fig. 3a). In Fus-MC8, the outer membrane of the A__ 2 !80
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FIG. 3. (a) Electron micrograph of cells of B. oralis. The trilaminated outer membrane of the cell wall (arrow) and the cytoplasmic membrane enclose the granular and filamentous structures of the cell. The inner cell waU is very thin and can only be seen in some areas (arrow head). Counterstained with uranyl acetate and lead citrate. x160,000. (b) Electron micrograph of cells of F. nucleatum counterstained with uranyl acetate and lead citrate. The cell waU consists of a trilamellar outer membrane (arrow) that is separated from the electron-dense inner ceU wall and the cytoplasmic membrane proper (arrow head). Numerous granules, vesicles, and filaments are seen within the cytoplasm. x200,000.
cell wall was distinctly stained by uranyl acetate and lead citrate (Fig. 3b). The outer membrane of the cell wall stripped off easily and piled up in double layers, or it was completely lost. No conclusive difference in- ultrastructure of the outer membrane was found between the two strains. The inner lamina of the cell wall of Bact-MC3, however, was thinner and less electron dense than that of Fus-MC8. Incubation of homologous cells in anti-Bact-MC3 LPS, conjugated with peroxidase, resulted in a granular irregular staining of the outer membrane of the cell wall (Fig. 4a). In some sites, the immune reaction produced larger dots, with a cross-sectional area 10 to 100 times that of the small granules (Fig. 4a, b, and c). The immunohistochemical reaction was restricted to the membrane of the cell wall. No other part of the cell reacted. After formaldehyde fixation, the immunohistochemical reaction was diminished (Fig. 4b) and was almost abolished by glutaraldehyde fix-
ation (Fig. 4c). The IgG antibodies to Bact-MC3 LPS (Fig. 4d) showed a distribution similar to the IgM antibodies (Fig. 4a). In Fig. 5a, the outer membrane of the cell wall of an unfixed cell is seen together with the immunohistochemical reaction product, which is irregularly distributed. The reaction is restricted to the outer membrane of the cell wall. Glutaraldehyde fixation (Fig. 5b) almost abolished the reaction. Control incubations (see i through vi in Materials and Methods) resulted in very slight activity (Fig. 4e). DISCUSSION The high glucose content in the LPS of Bacteroides has been considered to be a contamination of intracellular glucans (11, 16; Dahlen and Hofstad, in press). Attempts to prepare glucogen-free LPS from different Bacteroides strains with different extraction methods have failed in the sense that a high glucose content could not be avoided (T. Hofstad, K. Sveen, and
268
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VOL. 19, 1978
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5b FIG. 5. (a) Electron micrograph of F. nucleatum incubated in peroxidase-conjugated IgM antibodies to LPS and further processed for immunoelectron microscopy. The reactivity is limited to the outer membrane of the cell wall, and there are no reaction products to be observed in the interior of the bacteria cell. Note the granular distribution of the reaction products indicating that parts of the covering LPS were stripped off during the process of the bacteria. The three lamellar structures of the cell wall are seen through some of the granules (arrow). X70,000. (b) Electron micrograph of a glutaraldehyde-fixed cell of F. nucleatum incubated in peroxidase-conjugated antibodies to LPS of B. oralis. There is no significant reaction to be observed in the cell wall. x60,000.
G. Dahl6n, Acta Pathol. Microbiol. Scand. Sect. B, in press). This glucan content of LPS BactMC3 may explain the diverse immunological pattern resulting in both IgM and IgG antibody production observed in the present study. It is well known that antibodies against somatic antigen of Salmonella species and Escherichia coli in animals (20) and humans (8, 21, 24) are 19S macroglobulins. Somatic antigen refers to the polysaccharide-containing 0-antigen, which is supposed to be the main antigenic determinant of LPS endotoxin. It might thus be argued that the LPS preparation of Bact-MC3 contains two immunologically different substances and
does not correspond immunologically to the endotoxin of Enterobacteriaceae and Fusobacterium. Recently Hofstad (15) has succeeded in separating LPS from Bacteroides fragilis into two different types responsible for agglutinating and precipitating antibodies, respectively. It is not yet clear whether or not these two types have different chemical compositions with respect to their glucose content. Both IgM and IgG antibodies conjugated with peroxidase were found to be attached to the outer membrane of the cell wall of Bact-MC3 cells (Fig. 4c and d). This fmding indicates that, even if an antigenic contamination is responsible for the diverse im-
FIG. 4. (a) Cells of B. oralis incubated in peroxidase-conjugated IgM antibodies to LPS. There is no reaction to be observed in this except in the outer cell wall. (Unfixed.) Note the granular distribution of the reaction products and the larger dots (arrow). x 70,000. (b) A cell of B. oralis fixed in formaldehyde before incubation with IgM antibodies to LPS as described in (a). There is a decrease in the reactivity indicating partial loss of antigenic activity due to the Formalin fixation. There is no reactivity in the cytoplasmic membrane or in the cytoplasm. Note the larger dot of antibody reaction (arrow). x70,000. (c) Electron micrograph of a glutaraldehyde-fixed cell of B. oralis incubated in peroxidase conjugated with IgM antibodies to LPS. There is a faint reaction to be observed in the outer membrane of the cell wall. x 70,000. (d) Electron micrograph of B. oralis freshly incubated in peroxidase-conjugated IgG antibodies to LPS. The distribution of reaction products is similar although less intense than that in (a). x 75,000. (e) Electron micrograph of a oralis cell treated with peroxidase-conjugated antibodies to LPS of F. nucleatum. There is hardly any reactivity to be observed. x 75,000.
270
DAHLEN, NYGREN, AND HANSSON
munological response, it originates from the same outer membrane as the LPS. This supports the theory that glucans are a part of the LPS molecular complex. The cell wall of gram-negative bacteria consists of a number of distinct layers with differing chemical compositions. The outer membrane, which contains LPS, appears to be triple layered when examined with the electron microscope (6). Various Bacteroides species have been found to have this triple-layered outer membrane (4, 5, 27). The present study confirms that the cell walls of B. oralis and F. nucleatum also have these structures. The staining of the LPS complex of Fus-MC8 was not constant along the whole-cell periphery. In spite of the fact that the cells were harvested in the logarithmic phase of growth, the outer membrane of the cell wall was lost in some areas. The explanation for this difference in stability of the cell wall between strains Bact-MC3 and Fus-MC8 may be the chemical differences in LPS composition, but more probably it may be the result of an artifact produced by centrifugation; Fus-MC8 is a rather filamentous cell, which possibly makes it more sensitive to mechanical forces than the more coccoid BactMC3. Location of the 0 antigen-containing LPS in the cell wall of gram-negative bacteria has been studied by other methods (9, 22, 23, 26). Utilizing the peroxidase-conjugated method of other studies for localization of bacterial surface antigens (3, 19), the location of LPS in the outer cell membrane was confirmed for both Bacteroides and Fusobacterium. No immunohistochemical reaction was observed inside the cells or in the inner parts of the cell wall. This does not exclude the possibility of antigenic sites inside the cell that could not be demonstrated because the conjugated immunoglobulins could not penetrate through the cell wall. However, it should be noted that no reaction product was seen within cells undergoing lysis where penetration should have been facilitated. This indicates a low probability for the existence of intracellular antigens cross-reacting with LPS. The immunohistochemical reactions were highly reproducible. The controls showed almost no reactivity. There was no cross-reaction between the different antisera in either the indirect hemagglutination test or the immunohistochemical reaction. This demonstrates the high reliability of the method. Two different patterns were demonstrated with regard to the distribution of the reaction product on Bact-MC3 cells. One of these, the fine, granular type, also appeared on the cell wall of Fus-MC8 (Fig. 4a and
INFECT. IMMUN.
5a). No difference was seen in the appearance of the label when either peroxidase-conjugated IgG or IgM antibodies against Bact-MC3 LPS were used. The coarser, dotlike precipitates were formed irrespective of the method of fixation. The reaction products were localized exclusively on the outer part of the trilaminated membrane of the cell wall. The appearance of the larger dots was reminiscent of the "blebs" on the cell wall of B. fragilis described by Kasper (17). The granular pattern of the reactive sites indicates that these sites are randomly distributed on the cell wall. Aldehyde fixation diminished the intensity of the immunohistochemical reaction, but the distribution was the same in freshly isolated and aldehyde-fixed cells. This means that there was no redistribution due to lack of fixation of the LPS. Edebo and Holme (7) showed that LPS are extracted from the cell wall even in saline and buffer solutions. In the fresh preparations used in the present study, LPS may have been partially extracted, but, as the distribution of the reaction products was the same in freshly prepared and fixed cells, this was not considered to be a significant source of error. ACKNOWLEDGMENTS This work was supported by Goteborgs Tandliikare-SalIskap and by grants from the Faculty of Odontology, Goteborg, and Swedish Medical Research Council Project B 77-254309.
LiMRATURE CMD 1. Avrameas, S. 1969. Coupling of enzymes to proteins with glutaraldehyde. Use of conjugates for the detection of antigens and antibodies. Immunochemistry 6:43-48. 2. Balows, A., R. M. Dehaan, V. R. Dowell, and L. B. Guze. 1974. Anaerobic bacteria. Role in disease.
Charles C Thomas, Publisher, Springfield, Ill. 3. Berthold, P., D. Bratthall, and C.-H. Berthold. 1974. Immunoperoxidase staining of Streptococcus mutans. Arch. Oral Biol. 19:1227-1230. 4. Bladen, H. A., and J. F. Waters. 1963. Electron microscopic study of some strains of Bacteroides. J. Bacteriol. 86:1339-1344. 5. Costerton, J. W., H. N. Damgaard, and K.-J. Cheng. 1974. Cell envelope morphology of rumen bacteria. J. Bacteriol. 118:1132-1143. 6. Costerton, J. W., J. M. Ingram, and K.-J. Cheng. 1974. Structure and function of the cell envelope of gram-negative bacteria. Bacteriol. Rev. 38:87-110. 7. Edebo, L., and T. Holme. 1965. Preparation of biologically active fractions of Salmonella typhimurium. Acta Pathol. Microbiol. Scand. 63:228-234. 8. Evans, R. T., S. Spaeth, and S. E. Morgenhagen. 1966. Bacterial antibody in mammalian serum to obligatorily anaerobic gram-negative bacteria. J. Immunol. 97:112-119. 9. Fukushi, K., F. Ariji, J. Yamaguchi, and S. Oka. 1966. Electron microscopic localization of endotoxin of Escherichia coli by the use of ferritin-conjugated antibody. J. Electron Microsc. 15:137-142. 10. Haglid, K., A. Hamberger, H.-A. Hansson, H. Hyden, L. Persson, and L. Ronnback. 1976. Cellular and
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LPS IN BACTEROIDES AND FUSOBACTERIUM
subcellular distribution of the S-100 protein in rabbit and rat central nervous system. J. Neurosci. Res. 2:175-191. 11. Hofstad, T. 1968. Chemical characteristics of Bacteroides melaninogenicus endotoxin. Arch. Oral Biol. 13:1149-1155. 12. Hofstad, T. 1970. Biological activities of endotoxin from Bacteroides melaninogenicus. Arch. Oral Biol. 15:343-348. 13. Hofstad, T. 1974. The distribution of heptose and 2-keto3-deoxyoctonate in Bacteroidaceae. J. Gen. Microbiol. 85:314-320. 14. Hofstad, T. 1974. Antibodies reacting with lipopolysaccharides from Bacteroides melaninogenicus, Bacteroides fragilis, and Fusobacterium nucleatum in serum from normal human subjects. J. Infect. Dis. 129:349-351. 15. Hofstad, T. 1976. Purification of the 0 antigen of Bacteroides fragilis ss. fragilis NCTC 9343 from phenolwater extracts by gel filtration and chromatography on DEAE-cellulose and hydroxylapatite. Acta Pathol. Microbiol. Scand. Sect. B. 84:229-234. 16. Hofstad, T., and T. Kristoffersen. 1970. Chemical characteristics of endotoxin from Bacteroides fragilis NCTC 9343. J. Gen. Microbiol. 61:15-19. 17. Kasper, D. L. 1976. The polysaccharide capsule of Bacteroides fragilis subspecies fragilis: immunochemical and morphologic definition. J. Infect. Dis. 133:79-87. 18. Kristoffersen, T. 1969. Immunochemical studies of oral fusobacteria. 3. Purification of a group reactive precipitinogens. Acta Pathol. Microbiol. Scand. 77:447-456. 19. Lai, C.-H., M. A. Listgarten, and B. Rosan. 1975. Immunoelectron microscopic identification and locali-
20.
21. 22.
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25. 26.
27.
271
zation of Streptococcus sanguis with peroxidase-labeled antibody: localization of surface antigens in pure cultures. Infect. Immun. 11:193-199. Landy, M., and W. P. WVeidanz. 1964. Natural antibodies against gram-negative bacteria, p. 275-290. In M. Landy and W. Braun (ed.), Bacterial endotoxins. Rutgers University Press, New Brunswick, N.J. Michael, J. G., and F. S. Rosen. 1963. Association of "natural antibodies" to gram-negative bacteria with the y-macroglobulins. J. Exp. Med. 118:619-626. Milner, K. C., R. L. Anacker, K. Fukushi, W. T. Haskins, M. Landy, B. Malmgren, and E. Ribi. 1963. Symposium on relationship of structure of microorganisms to their immunological properties. III. Structure and biological properties of surface antigens from gram-negative bacteria. Bacteriol. Rev. 27:352-368. Shands, J. W. 1966. Localization of somatic antigen on gram-negative bacteria using ferritin antibody conjugates. Ann. N.Y. Acad. Sci. 133:292-298. Smith, R. T. 1960. Immunity in infancy. Pediatr. Clin. North Am. 7:269-293. Socransky, S. S. 1970. Relationship of bacteria to the etiology of periodontal disease. J. Dent. Res. 49:203-222. Ushijima, T. 1970. Morphology and chemistry of the bacterial cell wall. 1. The location of mucopeptide in the cell wall of Bacteroides convexus and its chemical composition. Jpn. J. Microbiol. 14:15-25. Ushijima, T., K. Ueno, S. Suzuki, and U. Kurimoto. 1971. Morphology and chemistry of the cell wall. II. Sugar composition and location of 0-antigen in the cell wall of Bacteroides convexus. J. Electron Microsc. 20:32-39.
