Vol. 11, No. 1

AND IMMUNITY, Jan. 1975, p. 193-199 Copyright 0 1975 American Society for Microbiology

INFECTION

Printed in U.S.A.

Immunoelectron Microscopic Identification and Localization of Streptococcus sanguis with Peroxidase-Labeled Antibody: Localization of Surface Antigens in Pure Cultures CHERN-HSIUNG LAI,* MAX A. LISTGARTEN, AND BURTON ROSAN Center for Oral Health Research and School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174

Received for publication 12 August 1974

An indirect method of localizing antigens with horseradish peroxidase-labeled antibody was used to identify and localize surface antigens of Streptococcus sanguis at the ultrastructural level. An electron dense layer surrounding the cell wall could be distinguished without any additional electron microscope staining. This labeled layer represents an immune complex consisting of bacterial surface antigens, specific rabbit antisera, and peroxidase-labeled goat anti-rabbit globulins. Although with undiluted antisera slight cross-reactions occurred with S. salivarius and S. miteor (mitis), these could be readily distinguished from the more intense homologous reaction by their patchiness and the difference in distribution of the label. These cross-reactions were eliminated by appropriate dilutions of the antiserum. No cross-reactions occurred with S. mutans, S. faecalis, Actinomyces species, or Bacterionema, microorganisms which are likely to be present in dental plaque along with S. sanguis. Control experiments indicated that horseradish peroxidase can become non-specifically adsorbed to the membrane of certain bacterial cells. Appropriate controls must, therefore, be included for the localization of membrane associated antigens with horseradish peroxidase. Marion Gilmour, Eastman Dental Center, Rochester, N.Y.); Actinomyces viscosus, strain T,4; A. naeslundii, strain I; and A. israelii, strain ATCC 10048. The cultures of S. sanguis, S. salivarius, and B. matruchotii were grown aerobically; S. miteor was grown anaerobically in brain heart infusion broth (Difco) and Trypticase soy agar supplemented with 5% sheep blood (BBL) at 37 C for 18 h. Cultures of A. viscosus, A. naeslundii, and A. israelii were grown aerobically; S. miteor was grown anaerobically in brain heart infusion broth (Difco) and Trypticase soy agar supplemented with 5% sheep blood (BBL) at 37 C for 18 h. Cultures of A. viscosus, A. naeslundii, and A. israelii were grown anaerobically in Trypticase soy broth (BBL) using gas generating packs (BBL). Serological techniques. Antisera against S. sanguis, strain M-5, were produced from rabbits by immunization with formalinized cells as described previously (4, 10); preimmune sera were used for MATERIALS AND METHODS controls. Commercial goat anti-rabbit immunoglobuBacterial strains and media. The bacteria used lin G (IgG) (Research Division, Miles Laboratories, for this study were S. sanguis, strain M-5, isolated Kankakee, Ill.) was conjugated with horseradish perfrom dental plaque, a reference strain used in previous oxidase (HP) (Type II, Sigma Chemical Co.) accordstudies on the serology of S. sanguis (4, 10). The ing to the method of Nakane and Pierce (7). following heterologous strains were included as conProcedures for localization of bacterial antigen. trols: S. mutans, strain LM-7; S. faecalis, strain S161; Localization of bacterial antigens in cells obtained S. salivarius, strains 9652 and CM-6; S. miteor from pure cultures was carried out by means of a (mitis), strain RE-7 (the latter three strains were modification of the indirect method initially used for obtained from Sigmund Socransky, Forsyth Dental tissue localization (7). The procedure is summarized Center, Boston, Mass.); Bacterionema matruchotii, in Fig. 1 (experimental). strains 47 (ATCC 14266) and 208 (obtained from The cells of strain M-5 were harvested from 18-h

In a previous study, we reported the development of a labeling technique for the localization of bacterial surface antigens at the ultrastructural level, without the use of electron dense labels (4). In attempting to apply this immunocoating reaction to samples of dental plaque, it was sometimes difficult to distinguish the immuno-coating from normal coatings found on the surfaces of some plaque bacteria. Therefore, it was deemed desirable to utilize some type of electron dense label which, coupled with specific antibody, could be used to identify specific bacteria in plaque. The present study describes a modification of the peroxidase-labeled antibody technique for the specific tagging of Streptococcus sanguis.

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VOL. ll, 1975

S. SANGUIS IDENTIFICATION AND LOCALIZATION

cultures by centrifugation at 1,500 x g for 10 min; the cells were resuspended in 0.05 M phosphate-buffered saline (PBS), pH 7.2. The cells were again centrifuged and either used directly or prefixed at 4 C in a solution of 4% paraformaldehyde-picric acid (11). Prefixed cells were washed overnight with 0.01 M PBS at pH 7.1. Pellets (about 1 mm3 in size) were then incubated with M-5 antisera at various dilutions (up to 1:20) for 30 min at room temperature; samples were washed in PBS to remove excess antibody and incubated with peroxidase-labeled goat anti-rabbit IgG (HP-anti-rabbit IgG) for 30 min. The excess labeled antibody was removed by washing in PBS and the cells were fixed in phosphate-buffered 5% glutaraldehyde, pH 7.2, for 1 h at 4 C. The samples were washed overnight 4 C in 0.05 M phosphate buffer, pH 7.2, containing 4.5% sucrose. The peroxidase label was developed by incubating cells for 1 h in a 3,3'-diamino-benzidine substrate solution (80 mg/100 ml of 0.05 M tris(hydroxymethyl)aminomethane buffer, pH 76); this was followed by incubation for 30 min in the same substrate solution containing 0.005% hydrogen peroxide (3). The labeled cells were washed in distilled water and exposed to 2% osmium tetroxide in veronal-acetate buffer (8) for 1 h at room temperature. The cells were dehydrated in graded aqueous ethanol solutions and embedded in Epon for electron microscopy (6). The sections were cut at 0.1 um and examined in a Philips EM-300 electron microscope either without additional stain or after staining with lead citrate (9) only or uranyl acetate and lead citrate. The controls for serological specificity of the reaction were: (i) cells treated with preimmune rabbit sera (Fig. 1, control a); (ii) heterologous cells listed above treated in the same manner as M-5 cells (Fig. 1, experimental and control a). The controls for the histochemical specificity of the reaction were: (i) M-5 cells washed in PBS and fixed in phosphate-buffered 5% glutaraldehyde for 1 h at 4 C. After washing overnight in 0.05 M phosphate buffer containing 4.5% sucrose, the cells were treated with 3,3'-diaminobenzidine and hydrogen peroxide and processed for electron microscopy (Fig. 1, control b); (ii) cells treated as in (i) but then incubated with either 3,3'-diaminobenzidine (Fig. 1, control c) or hydrogen peroxide (Fig. 1, control d). The cells were again washed and processed for electron microscopy; (iii) washed cells treated with a solution containing just the HP (10 mg/ml in 0.05 M PBS) for 1 h at room temperature. The cells were washed in PBS and fixed in 5% glutaraldehyde fixative for 1 h at 4 C. After washing overnight they were incubated with 3,3'diaminobenzidine and hydrogen peroxide and prepared for electron microscopy (Fig. 1, control e); (iv) the cells were treated as in (iii) but incubations with 3,3'-diaminobenzidine and hydrogen peroxide were omitted (Fig. 1, control f).

195

also observed in the region of the plasma membrane. The outermost layer of HP-labeled material was made more prominent by staining with lead citrate (Fig. 2b) or with uranyl acetate followed by lead citrate (Fig. 2c). The control sample of cells treated with preimmune sera followed by HP-labeled antirabbit IgG did not contain an electron dense layer around the cell wall (Fig. 2d). However, the electron dense layer associated with the cell membrane persisted in this control sample. Similar results were obtained with control e in which M-5 cells were treated with the HP solution followed by treatment with 3,3'diaminobenzidine and hydrogen peroxide (Fig. 2e). The electron dense coating on the cell wall or the membrane associated electron density were not observed in control samples b, c, d, and f (Fig. 2f). Controls for specificity of M-5 antisera with heterologous organisms are shown in Table 1. The only cells which cross-reacted with the M-5 antisera were S. salivarius and S. miteor. As shown in Fig. 3a and b, these cells were surrounded normally by a well-defined fringe of hair-like appendages (0.1 um) which radiated outward, perpendicularly from the cell wall. These hair-like structures were much longer than those occasionally visible in cells of S. sanguis (4). A comparison of HP-labeled cells of S. salivarius, S. miteor, and S. sanguis is shown in Fig. 4a, b, and c. The S. salivarius and S. miteor cells exhibited some nonspecific patchy labeling at the tip of the hair-like structures when they were incubated with undiluted M-5 antisera followed by HP-anti-rabbit IgG (Fig. 4a and b). It is clear that the relative intensity of the reaction is much greater with S. sanguis which exhibits a more homogeneous coating than S. miteor (Fig. 4c). The nonspecific labeling was much reduced and eventually eliminated when the cells were incubated in increasingly more diluted solutions of M-5 antisera (up to 1:20) followed by HP-anti-rabbit IgG.

DISCUSSION The nonspecific labeling at the top of the hair-like appendages of S. salivarius and S. miteor could only be seen when these cells were treated with M-5 antisera. It was not present on cells treated with normal rabbit sera. Therefore, it is likely that the labeling resulted from a between certain surface antigens cross-reaction RESULTS of S. sanguis and antigens located near the tip A distinct electron dense coating was noted of the hair-like appendages of S. salivarius and on cells of S. sanguis treated with specific rabbit S. miteor. This conclusion is also supported by antisera followed by HP-anti-rabbit IgG (Fig. results from slide agglutination tests in which S. 2a). In addition, an electron dense layer was sanguis antisera caused agglutination of S.

196

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FIG. 2. (a) Electron micrograph of unstained thin section of S. sanguis, strain M-5, treated with diluted (1:20) rabbit anti-M-5 serum, HP-labeled goat anti-rabbit IgG followed by histochemical treatment. Note electron dense coating on the outer surface of the cell wall (black arrows). A dense layer is also associated with the cell membrane (white arrows). x32,800. (b) Similar section stained with lead citrate. x32,800. (c) Similar section stained with uranyl acetate and lead citrate. x32,800. (d) Unstained thin section of S. sanguis, strain M-5, treated with control normal rabbit serum, HP-labeled anti-rabbit IgG followed by histochemical treatment. Outermost electron dense coating is not seen. Only the electron dense layer associated with the cell membrane is visible (arrows). x32,800. (e) Unstained thin section of S. sanguis, strain M-5, treated with peroxidase followed by histochemical treatment. Note resemblance to Fig. 2d. x32,800. (f) Unstained thin section of S. sanguis, strain M-5, treated with peroxidase without histochemical treatment (control f). A similar appearance was observed with controls b, c and d (Fig. 1). x32,800.

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FIG. 3. (a) Electron micrograph of typical morphology of S. salivarius, strain 9652. Note the distinctive fringe surrounding the cell. x68,000. (b) Electron micrograph of typical morphology of S. miteor, strain RE-7. Hair-like processes on the periphery of the cell are not as well developed as in S. salivarius. x54,000. 197

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TABLE 1. Results of cross-reactions among undiluted M-5 antisera and strains of heterologous species by means of the indirect HP-labeled antibody method

Species

S. sanguis S. mutansa S. faecalis S. salivarius S. miteor B. matruchotii A. viscosus A. naeslundii A. israelii

Strain

Result

M-5 FA-1, PK-1, LM-7, HS-6, GS-5 S 161 9652, CM-6 RE-7 47 (ATCC 14266), 208

Coating layer No reactionb

T14

No reaction No reaction No reaction

I 10048

No reactionb Patchy deposits Patchy deposits No reaction

a Strains tested previously by the immuno-coating reaction (4). bNeither coating layers not patchy deposits.

miteor and S. salivarius, as well as S. sanguis. It has been shown previously (4) that M-5 antisera absorbed with certain M-5 antigenic components will label M-5 cells in a pattern similar to that seen with S. salivarius and S. miteor, that is, only at the tip of these hair-like extensions from the cell wall. These observations suggest that certain common antigens may exist in association with these hair-like structures among strains of S. sanguis, S. salivarius, and S. miteor. Such hair-like structures are believed to play an important role in the adherence of S. salivarius to epithelial cells (2) and in the adherence of certain bacteria to one another (5). They also resemble the structures associated with the M protein of virulent strains of S. pyogenes (1, 12). The appearance of the electron dense precipitate associated with the cell membrane was first thought to be due to peroxidase activity associated with the cell membrane. However, the precipitate could not be demonstrated in cells treated with 3,3'-diaminobenzidine solution followed by hydrogen peroxide. From the results, it is most likely that this precipitate resulted from a nonspecific adsorption of HP to the cell membrane. This conclusion is supported by the observation that such electron dense precipimicrograph of lead citrate stained thin section of S. miteor, strain RE-7, treated with undiluted rabbit anti-M-5 serum and HP-labeled anti-rabbit IgG. FIG. 4. (a) Electron micrograph of lead citrate x86,000. (c) Electron micrograph of lead citrate stained thin section of S. salivarius, strain 9652, stained thin section of S. sanguis, treated with untreated with undiluted rabbit anti-M-5 serum and diluted rabbit anti-M-5 serum and HP labeled antiHP-labeled anti-rabbit IgG. x86,000. (b) Electron rabbit IgG. x86,000.

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S. SANGUIS IDENTIFICATION AND LOCALIZATION

tates appeared when cells were incubated first with HP, followed by 3,3'-diaminobenzidine and hydrogen peroxide, but did not form in other controls. The immuno-coating reaction results from the binding of rabbit antibody to antigens on the bacterial cell wall surface (4). After staining with heavy metal salts, it appeared electron microscopically as a dense layer, up to 60 nm thick. The layer specifically labeled with HPlabeled antibody contained, in addition to antiM-5 antibody, HP-labeled goat anti-rabbit IgG. The total thickness of the layer labeled by the indirect method measured up to 130 nm. Although the peroxidase-labeled antibody technique is more time consuming, the label is more prominent and can be identified without the need of additional staining for electron microscopy. Furthermore, the more thickly labeled antigen-antibody complex around the individual cells is less likely to be mistaken for extracellular substances in dental plaque. This error would be more likely if the immuno-coating reaction alone were used for the identification of specific bacteria in dental plaque.

LITERATURE CITED 1. Ellen, R. P., and R. J. Gibbons. 1972. M proteinassociated adherence of Streptococcus pyogenes to epithelial surfaces: prerequisite for virulence. Infect. Immunity 5:826-830. 2. Gibbons. R. J., J. VanHoute, and W. F. Liljemark. 1972.

3.

4. 5.

6.

7. 8. 9.

10. ACKNOWLEDGMENTS The excellent technical assistance of R. Tremblav and J. Smith is gratefully acknowledged. This work was supported by Public Health Service grants no. DE 02623 and DE 03180, both from the National Institute of Dental Research.

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Parameters that effect the adherence of Streptococcus saliLariu.s to oral epithelial surfaces. J. Dent. Res. 51:424-435. Graham, R. C.. and M. J. Karnovsky. 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14:291-302. Lai, C., M. Listgarten, and B. Rosan. 1973. Serology of Streptococcus sanguis: localization of antigen with unlabeled antisera. Infect. Immunity 8:475-481. Listgarten, M., H. Mayo, and M. Amsterdam. 1973. Ultrastructure of the attachment device between coccal and filamentous microorganisms in "corn cob" formations of dental plaque. Arch. Oral Biol. 18:651-656. Luft, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409-414. Nakane, P. K., and G. B. Pierce. 1967. Enzyme-labeled antibodies for light and electron microscopic localization of tissue antigens. J. Cell Biol. 33:307-318. Palade, G. E. 1952. A study of fixation for electron microscopy. J. Exp. Med. 95:285-298. Reynolds. E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17:208-213. Rosan, B. 1973. Antigens of Streptococcus sanguis. Infect. Immunity 7:205-211. Stefanini, M., C. DeMatino, and L. Zamboni. 1967. Fixation of ejaculated spermatozoa for electron microscopy. Nature (London) 216:173-174. Swanson, J., K. C. Hsu, and E. C. Gotschlich. 1969. Electron microscopic studies on Streptococcus. I. M antigen. J. Exp. Med. 130:1063-1069.