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Entrance of Actinobacillus actinomycetemcomitans into HEp-2 Cells In Vitro Inger J. Schytte Blix, * Renate Hars,+ Hans

R.

Preus,* and Kristen Helgeland*

strain of actinobacillus actinomycetemcomitans, freshly isolated from a juvenile Periodontitis patient, and the FDC Y4 laboratory strain of Aa were tested for their capacity to adhere to and enter the epithelial cell line HEp-2 cells in vitro. Immunofluorescence microscopy as well as scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that both strains adhered to the outer surface of the HEp-2 cells. In the TEM studies, the specimens were also treated with Aa specific antibodies and gold labeled protein A. These examinations showed that only the freshly isolated strain of Aa was found within the HEp-2 cells. The intracellular Aa were found to be viable, and in one case one of them was seen to undergo division. It is concluded that freshly isolated Aa has the ability to enter epithelial HEp-2 cells in vitro, and it is tentatively suggested that this may play a role in the pathogenesis of periodontal disease. / Periodontal 1992; 63:723-728.

A

Key Words: Periodontitis/pathogenesis; Actinobacillus actinomycetemcomitans; epithelial cells, HEp-2.

of oral bacteria such as Actinobacillus actinomycetemcomitans (Aa) to invade tissue and their role in the pathogenesis of periodontal diseases have been a matter of debate. Localization of bacteria within gingival tissues has been described in both advanced adult Periodontitis1,2 and localized juvenile Periodontitis (UP)3,4 by transmission (TEM) and scanning electron microscopy (SEM) on biopsies from diseased tissue. In some cases these microorganisms have been identified as Aa both by immunoperoxidase and immunofluorescence techniques.5,6 This postulated tissue invasion has been regarded as an important virulence factor. Bacteria have also been found in association with the pocket epithelium,13 gingival connective tissue,3,4 alveolar bone2 and oral epithelium.7 It has also been indicated that Aa are viable in the periodontal tissue,8 but the true nature of the interaction between bacteria and the epithelial cells has not been revealed. The mechanisms involved in the invasion of Aa into tissue are therefore still unknown. The invasive capacity of Enterobacteriaceae into epithelial cell lines in vitro, has been studied in great detail913 and good correlation between invasion in vitro and virulence in vivo has been demonstrated. The

ability

'Department of Microbiology, Dental Faculty, University of Oslo, Oslo, Norway. department of Anatomy. Department of Oral Biology and Periodontal Disease Clinical Research Center, State University of New York at Buffalo, Buffalo, NY.

In the present study we have investigated the potential of a freshly isolated strain of Aa to enter an epithelial cell line in vitro.

MATERIAL AND METHODS Aa were recovered from patients with

rapidly destructive Periodontitis by culturing dispersed (30 seconds) and diluted dental plaque samples on TSBV-medium.14 Aa colonies were recognized by colony morphology,15 Gram staining, and the positive catalase test. Diagnosis was confirmed by fermentation of selected carbohydrates16

and DNA restriction endonuclease mapping.17 The FDC Y4 laboratory strain of Aa was used in parallel experiments. A clinical isolate of Neisseria pharyngis obtained from The National Institute of Public Health, Oslo, Norway was used for test of specificity of the immunofluorescence assay. Cultivation of Cells Monolayers of HEp-2 cells were grown on glass coverslips 13 mm in diameter in 24 well tissue culture plates. To each 105 cells in 1 ml of RPMI 1640§ supplemented well 1 with 5 mM glutamine,11 5% fetal bovine serum (FBS)11, 100 µg streptomycin, 100 U penicillin and 2.5 µg fungizone §Gibco Ltd., Parsley, Scotland. "Gibco Laboratories, Grand Island, NY.

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Sorensen phosphate buffer, pH 7.4, at 4°C; washed in the buffer, critical point dried in carbon dioxide, sputtercoated

per ml were added. Cultures were incubated for 48 hours at 37°C in a humidified atmosphere of 5% C02 in air prior to inoculation with bacteria.

with 300 A gold palladium a Philips SEM 515.ft

of Bacterial Inoculum and Inoculation of Cell Cultures The cell cultures were inoculated with bacteria when they formed a nearly confluent monolayer. Prior to inoculation the cell layer was washed 3 times with 0.15 M phosphate buffered saline, pH 7.2, (PBS) (37°C), incubated for 1 hour in medium without antibiotics and then washed 3 times with PBS to minimize the concentration of antibiotics. Bacteria grown for 3 to 5 days on TSBV agar14 were harvested with a platinum loop and transferred to PBS, centrifuged at 500 g for 10 minutes, washed in PBS, and finally suspended in RPMI medium supplemented with 1% FBS, but without IO7 antibiotics. One ml of this suspension, containing 1 bacteria, was inoculated onto 1 105 HEp-2 cells.

Preparation

Microscopy

Staining of Specimens

Scanning Electron Microscopy Glass coverslips containing HEp-2

cells and bacteria were fixed for 12 hours in 2.5% glutaraldehyde buffered with

finally

-

electron

microscope.n

Viability of Intracellular Aa

freshly isolated strain of Aa was grown to mid-log phase in 1/2 Strength Brain Heart Infusion Broth (BHI)*** supplemented with 5 mg/1 Hemine and 0.5mg/ml vitamin Kx and 0.1% sodium bicarbonate, pelleted and resuspended The

in RPMI medium. The extent to which Aa were able to enter the epithelial cells was then determined on the basis of their ability to survive treatment with gentamicin.20 Such experiments indicated that less than 0.1% of the Aa organisms were capable of epithelial cell entry.

"Philips TEM 400, Philips Export, "Hüls A6, Marl, Germany.

. V.

Eindhoven, The Netherlands.

S5Ultrotome Nova, LKB Products, Bromma, Sweden. "Thermanox, Lux Scientific, Newbury Park, CA.

1

TJakopatts, Copenhagen, Denmark. *Sigma Diagnostics, St. Louis, MO. **Leica GmbH, Wetzlar, Germany.

examined in

Protein- Gold Labeling The HEp-2 cells with their bacteria cultures were grown on plastic-coverslips."11 They were fixed in 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 2 hours; transferred to a 0.5 M NH4CI solution in 0.1 M phosphate buffer for 4 hours; dehydrated at gradually decreasing temperatures (down to 35°C); and then embedded in Lowicryl K4M.11'9 Ultrathin sections were cut and sections were floated on drops of a 1/10 dilution of rabbit anti-Aa antibody in PBS buffer supplemented with 0.5% BS A, 0.05% Tween 20 and 0.5 M NaCl for 30 minutes. They were subsequently washed twice in the same buffer (10 minutes) and incubated in a buffer-solution of a 1/50 dilution of 10 nm protein-A-Gold## for 30 minutes. They were subsequently washed twice for 5 minutes in the same buffer and rinsed thoroughly in aqua dest. The sections were treated with uranyl acetate for 6 minutes, lead citrate for 2 minutes, and examined in an

for Fluorescence

To preserve the antigenicity, the fixatives were used at low concentrations and temperature, i.e., 0.3% formaldehyde and 0.2% glutaraldehyde for 1 hour on ice. The specimens then were washed in PBS and, while still wet, incubated with a 1/100 dilution in PBS of rabbit antiAa antibody (kindly provided by Dr. K. Schenk, University of Oslo) for 45 minutes at 37°C in humidified air. Antibodies were removed by washing 3 times 5 minutes with PBS. After drying, a 1/20 dilution of FITC-conjugated swine anti-rabbit Immunoglobulins11 in PBS with Evans blue counterstain 0.5% w/v# was added and incubated for 45 minutes in humidified air. The samples were washed 3 times for 5 minutes in PBS and resuspended in glycerol-PBS and examined in a Leitz Ortholux II microscope** equipped for fluorescence microscopy. With this procedure, only bacteria adhering to the outer surface of the cells will be stained.

and

Transmission Electron Microscopy The specimens were fixed for 4 hours in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3; subsequently rinsed for 10 minutes in 0.15 M phosphate buffer, pH 7.3; and postfixed in 1% osmium tetroxide at 4°C for 2 hours. After fixation small sheets of the cell layer were removed, rapidly dehydrated in a graded series of acetone and embedded in Vestopal-W.** Ultrathin sections were cut§§ and treated with uranyl acetate for 20 minutes, lead citrate for 3 minutes,18 and examined in an electron microscope.t+

Incubation of Specimens for Microscopic Examination After bacterial inoculation, the cell cultures were incubated for 12 hours at 37°C in a humidified atmosphere of 5% C02 in air. The medium was removed and fresh medium without bacteria was added. The cultures were then incubated for another 12 hours. The cell cultures were subsequently washed 3 times with PBS (37°C) to remove non-attached bacteria and supplied with fresh RPMI medium with 1% PBS without antibiotics. They were finally incubated for another 2 hours and washed 3 times with PBS (37°C) before fixation.10

Fixation and

alloy;

"Chemische Werke Lowi, Waldkraiburg, Germany. "Janssen Pharmaceutica, Beerse, Belgium. ***Difco Laboratories, Detroit, MI.

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Figure 1. Indirect immunofluorescence micrograph of the freshly isolated strain of Aa HEp-2 cells (original magnification 202).

at

the

outer

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surface of

Figure 2. Scanning electron micrograph of the FDC Y4 laboratory strain of Aa at the outer surface of a HEp2 cell 12 hours postinfection. The bacteria appear to be captured by microvillies at the HEp-2 cell surface (original magnification x 1,040). Test of HEp-2 Cell Viability After Entrance of Aa HEp-2 cell cultures including intracellular Aa were treated as described for test of "viability of intracellular Aa" (above), and stained with Trypan blue, which revealed that more than 98% of both infected and control cells were viable after incubation for 6 and 24 hours.

RESULTS Fluorescence

Microscopy

Immunofluorescence assays showed strong fluorescence for both the FDC Y4 laboratory strain, and the freshly isolated strain of Aa. Micrographs show that both strains adhere to the outer surface of the HEp-2 cells (Fig. 1). The antibodies

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Figure 3. Transmission electron micrograph of the gold immunolabeled freshly isolated Aa at the outer surface of a HEp-2 cell. The gold markers (10 nm gold particles) are seen as black spots on the bacterial surface (original magnification x 51,000).

Figure 4. do

Transmission electron micrograph ofgold immunolabeled Aa inside the be surrounded by any cellular membrane (original magnification

not appear to

used did not show any cross reactions with Neisseria pharin mixture with the Aa strains.

yngis

Scanning Electron Microscopy

Micrographs showed that both the freshly isolated strain and the FDC Y4 laboratory strain of Aa 12 hours postinfection did adhere to the outer surface of HEp-2 cells. The bacteria appear to be bound to microvilli at the cell surface (Fig. 2). Transmission Electron Microscopy Micrographs showed that both Aa strains did adhere to the outer surface of the HEp-2 cells (Fig. 3). The specimens treated with Aa specific antibodies and gold-labeled protein

HEp-2 cells. x 51,000).

The bacteria

A showed that the freshly isolated strain of Aa was found within the HEp-2 cells (Fig. 4). No cellular membranes surrounding the intracellular Aa were observed. The FDC Y4 strain was confined to the outer surface of the HEp-2 cells only. In one micrograph an intracellular Aa (not carried through gold labeling) was seen to undergo division

(Fig. 5).

DISCUSSION This study has shown that a freshly isolated strain of Aa adheres to and to a limited extent has the ability to enter the epithelial cell line HEp-2 in culture. The first interaction between a pathogenic microorganism and its host entails attachment to the eukaryotic cell surface. This requires an

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Figure 5. Transmission electron micrograph of an intracellular freshly isolated Aa undergoing division (original magnification x 84,000).

interaction between receptors on the cell surface, and adhesins or fimbriae on the bacterial cell surface.21 Freshly isolated Aa strains have been found to possess fimbria which are irreversibly lost after several subcultures in the laboratory.22 24 This loss of fimbriation has been found to be closely associated with the change in colonial morphology; i.e., from rough-surfaced colonies with a starshaped internal structure to smooth-surfaced colonies without any internal structures when grown on agar. The freshly isolated Aa used in this study forms transparent rough-surfaced colonies with a star-shaped internal structure which adheres firmly to the agar.15'24 This strain thus shares properties with other Aa strains shown to be fimbriated. The FDC Y4 laboratory strain of Aa, which is non-fimbriated23 and grows with smooth-surfaced colonies without any internal structures, was confined to the outer surface of the HEp-2 cells. This indicates that fimbria may be important in adhesion and subsequent invasion of host cells by Aa. The mechanisms behind Aa's ability to invade tissue is not known, while other invasive microorganisms like Shigella species have been studied in some detail. The latter belong to the Enterobacteriaceae and invade the mucosal epithelial cells of the colon and it has been used extensively both with the HEp-2 cells and others. No cellular membranes associated with enclosed bacteria can be seen from our micrographs. This is probably secondary to an uptake in a membrane limited vacuole which

subsequently disappears. Shigella species are released into the host cytoplasm from

endocytic vacuoles soon after the entry.25 In the host cytoplasm, Shigella species multiply, kill the cell, and spread to other surface epithelial cells and cause tissue damage, where the bacteria multiply in the ulcérations. Escape of Shigella from the endocytic vacuole is mediated by a virulence plasmid-encoded product, the contact hemolysin, which presumably lyses the host membrane which

encloses the bacterium.26 This is an essential process for Shigella virulence, as intracellular replication does not occur when this activity is disrupted. A similar mode of bacterial release from an endocytic vacuole has been reported for the Gram-positive organism Listeria monocytoThis organism produces listeriolysin, a segenes. creted hemolytic factor, which is required for escape to the cytoplasm from the initial inclusion vacuole. It has been shown that Aa does not hémolyse sheep and rabbit blood cells,16 but we have found that Aa does, indeed, hémolyse human blood (Ingar Olsen, University of

Oslo, unpublished data).

All members of the Enterobacteriaceae entered the cells within a few hours,21 while the entry of our freshly isolated Aa strain was rather slow. This could be indicative of a low potential for entrance into cells, but it may also reflect properties of the cell-line we have chosen. Thus, Neisseria gonorrhoeae invades the HEp-2 cells to a very little extent, while invasion into HecIB cells is substantial.11 It is presently premature to draw firm conclusions from our in vitro experiments, but based on the comparisons with Shigella, they may suggest that some strains of Aa are able

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to invade the

pocket epithelial cells in vivo, and that this could be the port of entry into the periodontal tissue.

13. Bukholm G, Lassen J. Bacterial adhesiveness and invasiveness in cell culture monolayer. Acta Pathol Microbiol Immuno Scand 1982; 90(Sect.

Acknowledgment

14. Slots J. Selective medium for Actinobacillus actinomycetemcomitans. J Clin Microbiol 1982; 15:606. 15. Blix US, Preus HR, Olsen I. Invasive growth oí Actinobacillus actinomycetemcomitans on solid medium (TSBV). Acta Odontol Scand

The authors would like to thank Dr. I. Olsen, Department of Microbiology, University of Oslo, for supplying bacterial strains. REFERENCES 1. Frank RM. Bacterial penetration in the apical pocket wall of advanced human Periodontitis. J Periodont Res 1980; 15:563. 2. Saghe R, Carranza FA, Newman MG, Cheng L, Lewin KJ. Identification of tissue invading bacteria in human periodontal disease. J Periodont Res 1982; 17:452. 3. Carranza FA, Saglie R, Newman MG, Valentin PL. Scanning and transmission electron microscopic study of tissue invading microorganisms in localized juvenile Periodontitis. / Periodontol 1983; 54:598. 4. Gillet R, Johnson NW. Bacterial invasion of the periodontium in a case of juvenile Periodontitis. J Clin Periodontol 1982; 9:93. 5. Saglie R, Newman MG, Carranza FA Jr, Cheng L, Lewin KJ. Immunohistochemical localization oí Actinobacillus actinomycetemcomitans infection in gingival tissue in localized juvenile Periodontitis. Acta Ódontól Lat Am 1984; 1:41. 6. Christersson LA, Albini , Zambón JJ, Wikesjö UME, Genco RJ. Tissue localization of Actinobacillus actinomycetemcomitans in human Periodontitis. I. Light, immunofluorescence and electron microscopic studies. / Periodontol 1987; 58:529. 7. Saglie FR, Carranza FA Jr, Newman MG. The presence of bacteria within the oral epithelium in periodontal disease. I. A scanning and transmission electron microscopic study. / Periodontol 1985; 56:618. 8. Christersson LA, Wikesjö UME, Albini , Zambón JJ, Genco RJ. Tissue localization of Actinobacillus actinomycetemcomitans in human Periodontitis. II. Correlation between immunofluorescence and culture techniques. J Periodontol 1987; 58:540. 9. Devenish JA, Schiemann DA. HeLa cell infection by Yersinia enterocolitica: Evidence of lack of intracellular multiplication and development of a new procedure for quantitative expression of infectivity. Infect Immun 1981; 32:48. 10. Bukholm G, Johansen BV, Namork E, Lassen J. Bacterial adhesiveness and invasiveness in cell culture monolayer. 1. A new combined light optical method evaluated by scanning electron microscopy. Acta Pathol Microbiol Immuno Scand 1982; 90(Sect. B):403. 11. Shaw JH, Hayes F, Brooks GF, Falkow S. Development of a tissue culture model for gonococcal invasion. J Microbiol Serol 1987; 53:485. 12. Sen A, Leon MA, Palchaudhure S. Comparative study of attachment to and invasion of epithelial cell lines by Shigella dysenteriae. Infect Immun 1990; 58:2401.

B):409.

1990; 48:313. 16. Slots J. Salient biochemical characters of Actinobacillus actinomycetemcomitans. Arch Microbiol 1982; 131:60. 17. Preus HR, Olsen I. Possible transmittance of A. actinomycetemcomitans from a dog to a child with rapidly destructive Periodontitis. J Periodont Res 1988; 23:68. 18. Messelt EB, Dahl E. Influence of x-ray irradiation on the ultrastructure of rat submandibular gland striated-duct cells. Acta Odontol Scand 1983; 41:277. 19. Carlemalm E, Garavito RM, Villiger W. Resin development for electron microscopy and an analysis of embedding at low temperature. / Microscopy 1982; 126:123. 20. Miller VI, Falkow S. Evidence for two genetic loci in Yersinia enterocolitica that can promote invasion of epithelial cells. Infect Immun 1988; 56:1242. 21. Finlay BB, Falkow S. Common themes in microbial pathogenicity. Microbiol Rev 1989; 53(2):210. 22. Preus HR, Namork E, Olsen I. Fimbriation of Actinobacillus actinomycetemcomitans. Oral Microbiol Immunol 1988; 3:93. 23. Rosan , Slots J, Lamont RJ, Listgarten MA, Nelson GM. Actinobacillus actinomycetemcomitans fimbriae. Oral Microbiol Immunol 1988; 3:58. 24. Inouye T, Ohta H, Kokeguchi S, Fukui K, Kato K. Colonial variation and fimbriation of Actinobacillus actinomycetemcomitans. FEMS Microbiol Lett 1990; 69:13. 25. Finlay BB, Falkow S. Comparison of the invasion strategies used by Salmonella choleraesuis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: Endosóme acidification is not required for bacterial invasion or intracellular replication. Biochimie 1988; 70:1089. 26. Sansonetti PJ, Mounier J. Multiplication of Shigella flexneri within HeLa cells: Lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infect Immun 1986; 51:461. 27. Kuhn M, Kathariou S, Goebel W. Hemolysin supports survival but not entry of the intracellular bacterium Listeria monocytogenes. infect Immun 1988; 56:79. 28. Portnoy DA, Jacks PS, Hinrichs DJ. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J Exp Med 1988; 167:1459. Send reprint requests to Dr. Inger J. Schytte Blix, Department of Microbiology, Dental Faculty, University of Oslo, POB 1052 Blindern, 0316 Oslo, Norway. Accepted for publication February 20, 1992.

Entrance of Actinobacillus actinomycetemcomitans into HEp-2 cells in vitro.

A strain of actinobacillus actinomycetemcomitans, freshly isolated from a juvenile periodontitis patient, and the FDC Y4 laboratory strain of Aa were ...
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