INFECTION AND IMMUNITY, Sept. 1991, p. 3004-3008 0019-9567/91/093004-05$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 59, No. 9
Protective Effect of Porphyromonas gingivalis Outer Membrane Vesicles against Bactericidal Activity of Human Serum D. GRENIER* AND M. BELANGER
Departement de Sante Buccale, Faculte de Medecine Dentaire, Universite de Montreal, Montreal, Quebec H3C 3J7, Canada Received 5 February 1991/Accepted 11 June 1991
The present study was undertaken to evaluate the effect of Porphyromonas gingivalis outer membrane vesicles the bactericidal activity of human serum. Human serum was pretreated with extracellular vesicles and then incubated with a cell suspension of Capnocytophaga ochracea. After 2 h at 37°C, the percent viability of C. ochracea was determined by cultivation on blood agar plates. At a final concentration of 0.3 mg/ml, outer membrane vesicles completely inhibited the serum bactericidal activity against C. ochracea. Boiling the vesicles prevented this inhibition. However, partial inhibition of the serum lethal action was obtained when a higher concentration (1.5 mg/ml) of boiled vesicles was used, which indicates the involvement of both heat-labile and heat-stable components associated with vesicles. Combining vesicles at a suboptimal concentration (0.1 mg/ml) with a reducing agent brought back inhibition of the bactericidal activity, whereas combining vesicles at an optimal concentration (0.3 mg/ml) with a thiol-blocking reagent caused a restoration of the bactericidal activity. When a purified preparation of P. gingivalis lipopolysaccharides was used instead of vesicles, inhibition of the bactericidal activity was also observed. These results indicate that the lipopolysaccharides and the proteolytic enzyme(s) associated with P. gingivalis outer membrane vesicles are likely to represent the heat-stable and the heat-labile components, respectively. It is possible that outer membrane vesicles released by P. gingivalis protect other bacterial species from complement action, thus favoring the pathogenic process of periodontal disease. on
Porphyromonas gingivalis, a gram-negative anaerobic bacterium, has been found to be a predominant species in the advancing lesions of human periodontal disease (10, 14). Some suspected periodontal pathogens, including P. gingivalis, can release into their environment extracellular outer membrane vesicles that possess several biological activities (9). Indeed, these structures have high proteolytic activity and can attach to oral surfaces and to other bacteria. It is well known that the bactericidal effect of human serum is of utmost importance in the host defense against bacterial infections (20). Exposure of many strains of gramnegative bacteria to low concentrations of human serum results in a loss of viability and sometimes dissolution of the bacterial cells (19). Bacterial resistance to human serum may be conferred by cell surface components such as lipopolysaccharides, capsular polysaccharides, and chromosome- and plasmid-determined outer membrane proteins (19, 20). Previous studies have shown that P. gingivalis elaborates thiol proteases, which are active against a variety of serum constituents including complement factors (12, 15-17), immunoglobulins (7, 8, 16), protease inhibitors (2), and transport proteins (3). P. gingivalis may then have a deleterious effect on the biological activities of serum. The purpose of the present study was to evaluate the effect of P. gingivalis outer membrane vesicles on the bactericidal activity of human serum. An attempt to identify the factors involved in the inhibition of the bactericidal activity was also carried out.
*
MATERIALS AND METHODS Bacteria and growth conditions. P. gingivalis ATCC 33277, Capnocytophaga ochracea 1956c, and Prevotella loescheii ATCC 15930 were used in the present study. Bacteria were grown in brain heart infusion broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with hemin (10 p.g/ml) and vitamin K (1 ,ug/ml). Viable bacterial counts were determined by using the above medium supplemented with 1.7% (wt/vol) agar and 5% (vol/vol) human blood. All cultures were incubated in an anaerobic chamber (N2-H2C02, 80:10:10) at 37°C. Sera. Sera obtained from four healthy human adults were pooled, filter sterilized, and stored at -80°C in aliquots of 1 ml until use in the bactericidal assay. Preparation of P. gingivalis outer membrane vesicles. P. gingivalis outer membrane vesicles were obtained as described by Singh et al. (13). Briefly, a 3-day-old culture (15 liters) of P. gingivalis was concentrated 60 times by passage through an ultrafiltration system (Millipore Corp., Bedford, Mass.) with a membrane molecular weight cutoff of 10,000. The cell suspension was then centrifuged twice at 10,000 x g for 30 min to remove whole bacterial cells. The supernatant (containing vesicles) was dialyzed overnight against 50 mM Tris-hydrochloride buffer (pH 9.5). The vesicles were collected by ultracentrifugation at 90,000 x g for 2 h and then freeze-dried. The lyophilized vesicles were stored at -20°C until use in the bactericidal assay. Serum bactericidal assay. Pooled human serum (150 p.l) was first preincubated at 37°C for 2 h with either 50 mM Tris-hydrochloride pH 7.0 (Tris HCI; 150 Ill) or P. gingivalis vesicles (in Tris-HCl; 150 Rd). Outer membrane vesicles were used at final concentrations ranging from 0.1 to 0.3 mg/ml. Overnight cultures of C. ochracea or P. loescheii
Corresponding author. 3004
VOL. 59,
1991
P. GINGIVALIS INHIBITS SERUM BACTERICIDAL ACTIVITY
were harvested by centrifugation and suspended to an
A660
of 0.1 in Tris-HCl containing 2 mM MgCl2, 0.3 mM CaCI2, and 0.2% gelatin (Tris-HCl-gelatin). The bacterial suspension was then diluted 1:10 in Tris-HCl-gelatin and added (75 ,ul) to the above serum-vesicle mixture along with 188 ,ul of Tris-HCl-gelatin. The assay mixture was then incubated for a further 2 h at 37°C in the anaerobic chamber. The viable bacterial counts were made with 10-fold serially diluted samples spread in duplicate on blood agar plates. After 5 days of incubation at 37°C, the CFU were counted. The percent viability was based on colony counts for heatinactivated (56°C for 30 min) serum. The percent viability was determined by using a shorter preincubation time of the serum-vesicle mixture and preincubation temperatures of 4 and 25°C. The effect of heat treatment of vesicles was also investigated. Outer membrane vesicles of P. gingivalis were pretreated at 60 or 70°C for 30 min or at 100°C for 5 min. The serum bactericidal assay was also carried out as described above, except that vesicles of P. gingivalis were prepared in Tris-HCl supplemented with either 6 mM 2-mercaptoethanol or 6 mM p-chloromercuriphenylsulfonic acid (PCMP). All bactericidal assays were done in triplicate, and the averaged percent viability was calculated. Unless otherwise specified, the serum bactericidal assay was carried out with C. ochracea. Proteolytic activity of P. gingivalis outer membrane vesicles. Extracellular outer membrane vesicles of P. gingivalis were assayed for azocoll-degrading activity. Briefly, 250 ,ul of P. gingivalis vesicles (3 mg/ml) was incubated with 250 ,ul of Tris-HCl and 100 ,ul of either distilled water, 18 mM 2-mercaptoethanol, or 18 mM PCMP at 37°C for 4 h. The assay was also performed with heat-treated (60 or 70°C for 30 min or 100°C for 5 min) vesicles and at incubation temperatures of 4 and 25°C. The A520 was recorded for quantitative determinations. A value of 100 was given to the assay incubated at 37°C without supplement and with no vesicle treatment.
Effect of P. gingivalis lipopolysaccharides on the serum bactericidal activity. The method of Darveau and Hancock (4) was used for the isolation and purification of lipopolysaccharides from P. gingivalis 33277. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the freeze-dried lipopolysaccharide preparation showed the ladderlike profile previously reported for P. gingivalis lipopolysaccharides (5, 13). The effect of purified lipopolysaccharides on the serum bactericidal activity was determined as described above, except that outer membrane vesicles were replaced by lipopolysaccharides. The ability of lipopolysaccharides to bind C. ochracea cells was evaluated by an indirect fluorescent antibody technique. After the serum bactericidal assay, in the presence of lipopolysaccharides, the bacterial cells were recovered by centrifugation (14,000 x g for 5 min), washed twice, and suspended in 250 p.1 of Tris-HCl. A rabbit immunoglobulin G fraction (diluted 1:50) raised against P. gingivalis 33277 whole cells (7) was added (250 ,ul), and the mixture was incubated at room temperature for 2 h. After the cells were washed twice by centrifugation (14,000 x g for 5 min), they were suspended in 250 ,u1 of Tris-HCl. The bound lipopolysaccharides were visualized by examination with a Zeiss Axiophot microscope equipped for epifluorescence (Zeiss, Montreal, Canada) after the cell suspension was reacted with fluorescein-conjugated goat anti-rabbit immunoglobulin G (diluted 1:2,000).
3005
TABLE 1. Effect of extracellular outer membrane vesicles of P. gingivalis on the human serum bactericidal activity against C. ochracea and P. loescheii % Viability Assay
Serum Heat-inactivated serum Serum plus vesiclesa 0.30 0.25 0.20 0.15
C. ochracea
P. loescheii
0 100
0 100
104 112 72 3
108 97 60 0 0
0
0.10
a Final concentrations (milligrams per milliliter) during the preincubation (2 h) of serum and vesicles.
RESULTS Cells of C. ochracea and P. loescheii were markedly sensitive to serum-mediated killing in the assay (Table 1). The viability of both bacterial species was completely abolished after 2 h of incubation in 27% (vol/vol) human serum (final concentration). A complete bactericidal effect was still obtained when the serum was used at a lower concentration (1% [vol/vol]) (data not shown). No loss of viability was evident after exposure of bacteria (approximately 3 x 105 bacteria per ml) to heat-inactivated serum, which indicates that heat-labile serum components were required for the killing of bacteria. The data presented in Table 1 indicate that treatment of human serum with P. gingivalis outer membrane vesicles, at a final concentration of 0.3 mg/ml completely inhibited the bactericidal activity against both C. ochracea and P. Ioescheii. A control assay in which outer membrane vesicles were added to heat-inactivated serum indicated that vesicles themselves have no effect on the test organisms. Further studies concerning the inhibition of the serum bactericidal activity by outer membrane vesicles were done with C. ochracea. The abolishment of the serum bactericidal activity by vesicles was temperature and time dependent. The percent viability of C. ochracea was 0 and 68% when the vesicles (0.3 mg/ml) and serum were preincubated at 4 and 25°C, respectively. To obtain a complete loss of bactericidal activ-
100
M
80
.
60-
40
20 0 0
1
2
3
Time of pre-incubation (h) FIG. 1. Inhibition of human serum bactericidal activity against C. ochracea after preincubation of human serum with P. gingivalis outer membrane vesicles.
3006
GRENIER AND BELANGER
INFECT. IMMUN.
TABLE 2. Effect of reducing and thiol-blocking agents on the vesicle-mediated inhibition of human serum bactericidal activity against C. ochracea % Viability
Assay
0 Serum ................................... ........................... 100 Heat-inactivated serum 0.14 Serum + vesiclesa (0.1 mg/ml) ................................... Serum + vesicles (0.1 mg/ml) + 3 mM ............................. 106 2-mercaptoethanol Serum + vesicles (0.3 mg/ml) ................................... 113 0 Serum + vesicles (0.3 mg/ml) + 3 mM PCMP .............. a Final concentrations during the preincubation (2 h) of serum and vesicles.
ity, the vesicles and serum had to be incubated at 37°C for at least 2 h (Fig. 1). Outer membrane vesicles were treated at different temperatures to determine the heat lability of the vesicle-associated component(s) responsible for the destruction of the serum bactericidal activity. Treatment of the vesicles at 60°C for 30 min resulted in 8.6% viability. Heating the vesicles at either 70°C (30 min) or 100°C (5 min) completely prevented the inhibition of the serum bactericidal activity (0% viability). However, 59.3% viability was obtained when a higher concentration (1.5 mg/ml) of boiled vesicles was used, which suggests the involvement of both heat-labile and heat-stable components associated with the P. gingivalis outer membrane vesicles. Table 2 presents evidence concerning a possible role of the vesicle-associated proteolytic activity in the destruction of the serum bactericidal action against C. ochracea. P. gingivalis outer membrane vesicles, at a final concentration (0.1 mg/ml) that did not cause any significant inhibition of the serum bactericidal activity, were combined with 2-mercaptoethanol (3 mM). The reducing agent restored inhibition of lethal action of human serum. Combining 3 mM PCMP with vesicles at a concentration (0.3 mg/ml) that caused complete inhibition of the serum bactericidal activity resulted in the restoration of the bactericidal action. Either PCMP or 2-mercaptoethanol alone did not modify the serum bactericidal activity. The azocoll-degrading activity of P. gingivalis vesicles was assayed under the same conditions as those used in the serum bactericidal experiments (Table 3). A strong proteolytic activity was associated with untreated vesicles incubated at 37°C. Heat treatment of vesicles reduced this activity to various extents. The degradation of azocoll was
FIG. 2. Binding of purified P. gingivalis lipopolysaccharides to C. ochracea cells as visualized with an indirect fluorescent antibody
technique.
enhanced in the presence of 2-mercaptoethanol and reduced in the presence of PCMP, a thiol-blocking reagent. When purified P. gingivalis lipopolysaccharides were used, instead of outer membrane vesicles, an inhibition of the serum bactericidal activity was also observed. The minimal concentration of lipopolysaccharides required to obtain a complete loss of serum bactericidal activity was found to be 75 jig/ml (final concentration). Inhibition of the bactericidal activity was still obtained when the preincubation step (serum plus lipopolysaccharides) was omitted. Furthermore, the inhibition was not affected by heat treatment of the lipopolysaccharides. When the preincubated mixture (2 h, 37°C) of human serum and lipopolysaccharides was passed through a 0.22-p.m-pore-size membrane filter (to remove a fraction of insoluble lipopolysaccharides) before the bactericidal assay was run in the presence of C. ochracea cells, no inhibition of the serum lethal action was observed (0% viability). This result suggests that the action of lipopolysaccharides is unlikely to be toward the human serum components. The binding of lipopolysaccharides to C. ochracea cells was then evaluated. Bacteria were harvested after the serum bactericidal assay, and bound lipopolysaccharides were detected by an immunofluorescence assay. P. gingivalis lipopolysacchafides showed a high affinity for C. ochracea cells (Fig. 2).
TABLE 3. Azocoll-degrading activity of P. gingivalis
DISCUSSION
outer membrane vesicles
Some suspected periodontal pathogens, including P. gingivalis, are capable of producing extracellular outer membrane vesicles. These structures have been recently well characterized (5, 6, 9) and seem to be the result of a blebbing
Supplement
Incubation temp ('C)
% Proteolytic activity"
37 25
None
None None None None None None 3 mM 2-mercapto-
37 37 37 37
100 58 12 63 13 5 176
None
ethanolb 3 mM PCMPb
37
20
Treatment
None None None
60°C, 30 min 70°C, 30 min 100°C, 5 min
4
a A value of 100% was given to the assay incubated at supplement and with no vesicle treatment. b Final concentration.
37'C
without a
or extrusion from the outer cell membrane. They can either be attached to or released from the cell surface of the bacteria. The extracellular vesicles appear to possess the same biological activities (6, 9, 13) as the outer membrane of the whole cells and a similar composition (5).
Sundqvist and Johansson (18) have shown that strains of P. gingivalis are highly resistant to the lethal action of human serum. They suggested that the resistance was conferred by the structure of the cell envelope. The present study was designed to evaluate the ability of P. gingivalis outer membrane vesicles in protecting oral bacteria against
VOL. 59, 1991
P. GINGIVALIS INHIBITS SERUM BACTERICIDAL ACTIVITY
the bactericidal activity of human serum and to identify possible factors involved in this resistance. The susceptibility of C. ochracea and P. loescheii to human serum, observed in the present study, was previously demonstrated (18, 21); the killing of both bacterial species was shown to be mediated principally via antibody-dependent classical pathway activation. Our data indicate that preincubation of human serum with P. gingivalis outer membrane vesicles can totally prevent the bactericidal activity of human serum against C. ochracea and P. loescheii. This inhibition appears to be mediated by heat-labile and heat-stable components associated with the P. gingivalis outer membrane vesicles. The same components are likely to protect other oral bacterial species against host defense mechanisms involving the complement system. The fact that inhibition of the serum bactericidal activity by outer membrane vesicles was time and temperature dependent suggests a possible role for the vesicle-associated proteolytic activity. Further experiments have shown that inhibition of serum bactericidal activity was increased by a reducing agent and suppressed in the presence of a thiolblocking reagent. In addition, under all conditions where the vesicle-associated proteolytic activity was reduced (low incubation temperature, heat treatment, PCMP treatment), the lethal action of human serum was restored. These data suggest that the vesicle-associated proteolytic activity may be the heat-labile component responsible for the destruction of serum bactericidal activity. This destruction may result from the proteolytic degradation of essential serum factors required for the bactericidal action. Recently, studies concerning the proteolytic activity of P. gingivalis cells have clearly demonstrated the ability of these bacteria to degrade immunoglobulins (7, 8, 16) and selected proteins of the complement system (12, 15-17). A reducing agent-dependent protease from P. gingivalis is currently being purified in our laboratory and will then be assayed in the serum bactericidal assay.
The purified lipopolysaccharides from P. gingivalis were able to inhibit the serum bactericidal activity against C. ochracea. This protective effect may result from the binding of these molecules to C. ochracea cells; the attachment of C3 component or antibody molecules might then be prevented. A similar mechanism of protection against serum bactericidal activity was previously reported by Allen and Scott (1). These authors showed that purified lipopolysaccharides from Escherichia coli could bind to serum-sensitive bacteria and inhibit the bactericidal reaction. In another study, Sansano et al. (11) suggested that increased resistance of a smooth E. coli phenocopy to the lethal action of human serum may be attributable to the blocking of antibody binding sites by the 0 antigen of lipopolysaccharides, thereby preventing activation of the classical pathway of complement. Since outer membrane vesicles from P. gingivalis contain lipopolysaccharides (5, 13), these molecules are likely to represent the heat-stable component responsible for the protection of C. ochracea against serum bactericidal activity. Vesicles could bind (via lipopolysaccharides) to bacteria, allowing the protection of bacteria against the lethal action of serum. However, one should not exclude the possibility that some other heat-resistant components (such as capsular polysaccharides) of vesicles may be involved in the protective effect. The present investigation has shown that lipopolysaccharides and proteolytic enzyme(s) found on outer membrane vesicles of P. gingivalis can mediate the ability of these structures to suppress the bactericidal activity of human
3007
serum. These observations indicate an additional virulence factor associated with P. gingivalis vesicles. Since gingival fluids contain immunoglobulins and complement proteins, it is suggested that vesicles and lipopolysaccharides released by P. gingivalis could protect other bacterial species from complement action, thus favoring the pathogenic process of periodontal disease. ACKNOWLEDGMENTS We thank M. D. McKee (Universitd de Montreal) for critical reading of the manuscript. This work was supported by the Medical Research Council of Canada (grant DG-383-384-385). REFERENCES 1. Allen, R. J., and G. K. Scott. 1980. The effect of purified lipopolysaccharide on the bactericidal reaction of human serum complement. J. Gen. Microbiol. 117:65-72. 2. Carlsson, J., B. F. Herrmann, J. F. Hofling, and G. K. Sundqvist. 1984. Degradation of human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis. Infect. Immun. 43:644-648. 3. Carlsson, J., J. F. Hofling, and G. K. Sundqvist. 1984. Degradation of albumin, haemopexin, haptoglobin and transferrin, by black-pigmented Bacteroides species. J. Med. Microbiol. 18:3946. 4. Darveau, R. P., and R. E. W. Hancock. 1983. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J. Bacteriol. 155:831-838. 5. Deslauriers, M., D. ni Eidhin, L. Lamonde, and C. Mouton. 1990. SDS-PAGE analysis of protein and lipopolysaccharide of extracellular vesicules and Sarkosyl-insoluble membranes from Bacteroides gingivalis. Oral Microbiol. Immunol. 5:1-7. 6. Grenier, D., and D. Mayrand. 1987. Functional characterization of extracellular vesicles produced by Bacteroides gingivalis. Infect. Immun. 55:3131-3136. 7. Grenier, D., D. Mayrand, and B. C. McBride. 1989. Further studies on the degradation of immunoglobulins by black-pigmented Bacteroides. Oral Microbiol. Immunol. 4:12-18. 8. Kilian, M. 1981. Degradation of immunoglobulins Al, A2, and G by suspected principal periodontal pathogens. Infect. Immun. 34:757-765. 9. Mayrand, D., and D. Grenier. 1989. Biological activities of outer membrane vesicles. Can. J. Microbiol. 35:607-613. 10. Okuda, K., and I. Takazoe. 1988. The role of Bacteroides gingivalis in periodontal disease. Adv. Dent. Res. 2:260-268. 11. Sansano, M., A. M. Reynard, and R. K. Cunningham. 1985. Inhibition of serum bactericidal reaction by lipopolysaccharide. Infect. Immun. 48:759-762. 12. Schenkein, H. A. 1988. The effect of periodontal proteolytic Bacteroides species on proteins of the human complement system. J. Periodontal Res. 23:187-192. 13. Singh, U., D. Grenier, and B. C. McBride. 1989. Bacteroides gingivalis vesicles mediate attachment of streptococci to serumcoated hydroxyapatite. Oral Microbiol. Immunol. 4:199-203. 14. Slots, J., and M. A. Listgarten. 1988. Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in human periodontal diseases. J. Clin. Periodontol. 15:85-93. 15. Sundqvist, G., A. Bengtson, and J. Carlsson. 1988. Generation and degradation of the complement fragment C5a in human serum by Bacteroides gingivalis. Oral Microbiol. Immunol. 3:103-107. 16. Sundqvist, G., J. Carlsson, B. Herrmann, and A. Tarnavik. 1985. Degradation of human immunoglobulins G and M and complement factors C3 and C5 by black-pigmented Bacteroides. J. Med. Microbiol. 19:85-94. 17. Sundqvist, G. K., J. Carlsson, B. F. Herrmann, J. F. Hofling, and A. Vaatiinen. 1984. Degradation in vivo of the C3 protein of guinea-pig complement by a pathogenic strain of Bacteroides
3008
GRENIER AND BELANGER
gingivalis. Scand. J. Dent. Res. 92:14-24. 18. Sundqvist, G., and E. Johansson. 1982. Bactericidal effect of pooled human serum on Bacteroides melaninogenicus, Bacteroides asaccharolyticus and Actinobacillus actinomycetemcomitans. Scand. J. Dent. Res. 90:29-36. 19. Taylor, P. W. 1983. Bactericidal and bacteriolytic activity of serum against gram-negative bacteria. Microbiol. Rev. 47:46-83.
INFECT. IMMUN.
20. Taylor, P. W. 1988. Bacterial resistance to complement, p. 107-120. In J. A. Roth (ed.), Virulence mechanisms of bacterial pathogens. American Society for Microbiology, Washington, D.C. 21. Wilson, M. E., R. Burstein, J. T. Jonak-Urbanczyk, and R. J. Genco. 1985. Sensitivity of Capnocytophaga species to bactericidal properties of human serum. Infect. Immun. 50:123-129.
Vol. 59, No. 9
Protective Effect of Porphyromonas gingivalis Outer Membrane Vesicles against Bactericidal Activity of Human Serum D. GRENIER* AND M. BELANGER
Departement de Sante Buccale, Faculte de Medecine Dentaire, Universite de Montreal, Montreal, Quebec H3C 3J7, Canada Received 5 February 1991/Accepted 11 June 1991
The present study was undertaken to evaluate the effect of Porphyromonas gingivalis outer membrane vesicles the bactericidal activity of human serum. Human serum was pretreated with extracellular vesicles and then incubated with a cell suspension of Capnocytophaga ochracea. After 2 h at 37°C, the percent viability of C. ochracea was determined by cultivation on blood agar plates. At a final concentration of 0.3 mg/ml, outer membrane vesicles completely inhibited the serum bactericidal activity against C. ochracea. Boiling the vesicles prevented this inhibition. However, partial inhibition of the serum lethal action was obtained when a higher concentration (1.5 mg/ml) of boiled vesicles was used, which indicates the involvement of both heat-labile and heat-stable components associated with vesicles. Combining vesicles at a suboptimal concentration (0.1 mg/ml) with a reducing agent brought back inhibition of the bactericidal activity, whereas combining vesicles at an optimal concentration (0.3 mg/ml) with a thiol-blocking reagent caused a restoration of the bactericidal activity. When a purified preparation of P. gingivalis lipopolysaccharides was used instead of vesicles, inhibition of the bactericidal activity was also observed. These results indicate that the lipopolysaccharides and the proteolytic enzyme(s) associated with P. gingivalis outer membrane vesicles are likely to represent the heat-stable and the heat-labile components, respectively. It is possible that outer membrane vesicles released by P. gingivalis protect other bacterial species from complement action, thus favoring the pathogenic process of periodontal disease. on
Porphyromonas gingivalis, a gram-negative anaerobic bacterium, has been found to be a predominant species in the advancing lesions of human periodontal disease (10, 14). Some suspected periodontal pathogens, including P. gingivalis, can release into their environment extracellular outer membrane vesicles that possess several biological activities (9). Indeed, these structures have high proteolytic activity and can attach to oral surfaces and to other bacteria. It is well known that the bactericidal effect of human serum is of utmost importance in the host defense against bacterial infections (20). Exposure of many strains of gramnegative bacteria to low concentrations of human serum results in a loss of viability and sometimes dissolution of the bacterial cells (19). Bacterial resistance to human serum may be conferred by cell surface components such as lipopolysaccharides, capsular polysaccharides, and chromosome- and plasmid-determined outer membrane proteins (19, 20). Previous studies have shown that P. gingivalis elaborates thiol proteases, which are active against a variety of serum constituents including complement factors (12, 15-17), immunoglobulins (7, 8, 16), protease inhibitors (2), and transport proteins (3). P. gingivalis may then have a deleterious effect on the biological activities of serum. The purpose of the present study was to evaluate the effect of P. gingivalis outer membrane vesicles on the bactericidal activity of human serum. An attempt to identify the factors involved in the inhibition of the bactericidal activity was also carried out.
*
MATERIALS AND METHODS Bacteria and growth conditions. P. gingivalis ATCC 33277, Capnocytophaga ochracea 1956c, and Prevotella loescheii ATCC 15930 were used in the present study. Bacteria were grown in brain heart infusion broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with hemin (10 p.g/ml) and vitamin K (1 ,ug/ml). Viable bacterial counts were determined by using the above medium supplemented with 1.7% (wt/vol) agar and 5% (vol/vol) human blood. All cultures were incubated in an anaerobic chamber (N2-H2C02, 80:10:10) at 37°C. Sera. Sera obtained from four healthy human adults were pooled, filter sterilized, and stored at -80°C in aliquots of 1 ml until use in the bactericidal assay. Preparation of P. gingivalis outer membrane vesicles. P. gingivalis outer membrane vesicles were obtained as described by Singh et al. (13). Briefly, a 3-day-old culture (15 liters) of P. gingivalis was concentrated 60 times by passage through an ultrafiltration system (Millipore Corp., Bedford, Mass.) with a membrane molecular weight cutoff of 10,000. The cell suspension was then centrifuged twice at 10,000 x g for 30 min to remove whole bacterial cells. The supernatant (containing vesicles) was dialyzed overnight against 50 mM Tris-hydrochloride buffer (pH 9.5). The vesicles were collected by ultracentrifugation at 90,000 x g for 2 h and then freeze-dried. The lyophilized vesicles were stored at -20°C until use in the bactericidal assay. Serum bactericidal assay. Pooled human serum (150 p.l) was first preincubated at 37°C for 2 h with either 50 mM Tris-hydrochloride pH 7.0 (Tris HCI; 150 Ill) or P. gingivalis vesicles (in Tris-HCl; 150 Rd). Outer membrane vesicles were used at final concentrations ranging from 0.1 to 0.3 mg/ml. Overnight cultures of C. ochracea or P. loescheii
Corresponding author. 3004
VOL. 59,
1991
P. GINGIVALIS INHIBITS SERUM BACTERICIDAL ACTIVITY
were harvested by centrifugation and suspended to an
A660
of 0.1 in Tris-HCl containing 2 mM MgCl2, 0.3 mM CaCI2, and 0.2% gelatin (Tris-HCl-gelatin). The bacterial suspension was then diluted 1:10 in Tris-HCl-gelatin and added (75 ,ul) to the above serum-vesicle mixture along with 188 ,ul of Tris-HCl-gelatin. The assay mixture was then incubated for a further 2 h at 37°C in the anaerobic chamber. The viable bacterial counts were made with 10-fold serially diluted samples spread in duplicate on blood agar plates. After 5 days of incubation at 37°C, the CFU were counted. The percent viability was based on colony counts for heatinactivated (56°C for 30 min) serum. The percent viability was determined by using a shorter preincubation time of the serum-vesicle mixture and preincubation temperatures of 4 and 25°C. The effect of heat treatment of vesicles was also investigated. Outer membrane vesicles of P. gingivalis were pretreated at 60 or 70°C for 30 min or at 100°C for 5 min. The serum bactericidal assay was also carried out as described above, except that vesicles of P. gingivalis were prepared in Tris-HCl supplemented with either 6 mM 2-mercaptoethanol or 6 mM p-chloromercuriphenylsulfonic acid (PCMP). All bactericidal assays were done in triplicate, and the averaged percent viability was calculated. Unless otherwise specified, the serum bactericidal assay was carried out with C. ochracea. Proteolytic activity of P. gingivalis outer membrane vesicles. Extracellular outer membrane vesicles of P. gingivalis were assayed for azocoll-degrading activity. Briefly, 250 ,ul of P. gingivalis vesicles (3 mg/ml) was incubated with 250 ,ul of Tris-HCl and 100 ,ul of either distilled water, 18 mM 2-mercaptoethanol, or 18 mM PCMP at 37°C for 4 h. The assay was also performed with heat-treated (60 or 70°C for 30 min or 100°C for 5 min) vesicles and at incubation temperatures of 4 and 25°C. The A520 was recorded for quantitative determinations. A value of 100 was given to the assay incubated at 37°C without supplement and with no vesicle treatment.
Effect of P. gingivalis lipopolysaccharides on the serum bactericidal activity. The method of Darveau and Hancock (4) was used for the isolation and purification of lipopolysaccharides from P. gingivalis 33277. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the freeze-dried lipopolysaccharide preparation showed the ladderlike profile previously reported for P. gingivalis lipopolysaccharides (5, 13). The effect of purified lipopolysaccharides on the serum bactericidal activity was determined as described above, except that outer membrane vesicles were replaced by lipopolysaccharides. The ability of lipopolysaccharides to bind C. ochracea cells was evaluated by an indirect fluorescent antibody technique. After the serum bactericidal assay, in the presence of lipopolysaccharides, the bacterial cells were recovered by centrifugation (14,000 x g for 5 min), washed twice, and suspended in 250 p.1 of Tris-HCl. A rabbit immunoglobulin G fraction (diluted 1:50) raised against P. gingivalis 33277 whole cells (7) was added (250 ,ul), and the mixture was incubated at room temperature for 2 h. After the cells were washed twice by centrifugation (14,000 x g for 5 min), they were suspended in 250 ,u1 of Tris-HCl. The bound lipopolysaccharides were visualized by examination with a Zeiss Axiophot microscope equipped for epifluorescence (Zeiss, Montreal, Canada) after the cell suspension was reacted with fluorescein-conjugated goat anti-rabbit immunoglobulin G (diluted 1:2,000).
3005
TABLE 1. Effect of extracellular outer membrane vesicles of P. gingivalis on the human serum bactericidal activity against C. ochracea and P. loescheii % Viability Assay
Serum Heat-inactivated serum Serum plus vesiclesa 0.30 0.25 0.20 0.15
C. ochracea
P. loescheii
0 100
0 100
104 112 72 3
108 97 60 0 0
0
0.10
a Final concentrations (milligrams per milliliter) during the preincubation (2 h) of serum and vesicles.
RESULTS Cells of C. ochracea and P. loescheii were markedly sensitive to serum-mediated killing in the assay (Table 1). The viability of both bacterial species was completely abolished after 2 h of incubation in 27% (vol/vol) human serum (final concentration). A complete bactericidal effect was still obtained when the serum was used at a lower concentration (1% [vol/vol]) (data not shown). No loss of viability was evident after exposure of bacteria (approximately 3 x 105 bacteria per ml) to heat-inactivated serum, which indicates that heat-labile serum components were required for the killing of bacteria. The data presented in Table 1 indicate that treatment of human serum with P. gingivalis outer membrane vesicles, at a final concentration of 0.3 mg/ml completely inhibited the bactericidal activity against both C. ochracea and P. Ioescheii. A control assay in which outer membrane vesicles were added to heat-inactivated serum indicated that vesicles themselves have no effect on the test organisms. Further studies concerning the inhibition of the serum bactericidal activity by outer membrane vesicles were done with C. ochracea. The abolishment of the serum bactericidal activity by vesicles was temperature and time dependent. The percent viability of C. ochracea was 0 and 68% when the vesicles (0.3 mg/ml) and serum were preincubated at 4 and 25°C, respectively. To obtain a complete loss of bactericidal activ-
100
M
80
.
60-
40
20 0 0
1
2
3
Time of pre-incubation (h) FIG. 1. Inhibition of human serum bactericidal activity against C. ochracea after preincubation of human serum with P. gingivalis outer membrane vesicles.
3006
GRENIER AND BELANGER
INFECT. IMMUN.
TABLE 2. Effect of reducing and thiol-blocking agents on the vesicle-mediated inhibition of human serum bactericidal activity against C. ochracea % Viability
Assay
0 Serum ................................... ........................... 100 Heat-inactivated serum 0.14 Serum + vesiclesa (0.1 mg/ml) ................................... Serum + vesicles (0.1 mg/ml) + 3 mM ............................. 106 2-mercaptoethanol Serum + vesicles (0.3 mg/ml) ................................... 113 0 Serum + vesicles (0.3 mg/ml) + 3 mM PCMP .............. a Final concentrations during the preincubation (2 h) of serum and vesicles.
ity, the vesicles and serum had to be incubated at 37°C for at least 2 h (Fig. 1). Outer membrane vesicles were treated at different temperatures to determine the heat lability of the vesicle-associated component(s) responsible for the destruction of the serum bactericidal activity. Treatment of the vesicles at 60°C for 30 min resulted in 8.6% viability. Heating the vesicles at either 70°C (30 min) or 100°C (5 min) completely prevented the inhibition of the serum bactericidal activity (0% viability). However, 59.3% viability was obtained when a higher concentration (1.5 mg/ml) of boiled vesicles was used, which suggests the involvement of both heat-labile and heat-stable components associated with the P. gingivalis outer membrane vesicles. Table 2 presents evidence concerning a possible role of the vesicle-associated proteolytic activity in the destruction of the serum bactericidal action against C. ochracea. P. gingivalis outer membrane vesicles, at a final concentration (0.1 mg/ml) that did not cause any significant inhibition of the serum bactericidal activity, were combined with 2-mercaptoethanol (3 mM). The reducing agent restored inhibition of lethal action of human serum. Combining 3 mM PCMP with vesicles at a concentration (0.3 mg/ml) that caused complete inhibition of the serum bactericidal activity resulted in the restoration of the bactericidal action. Either PCMP or 2-mercaptoethanol alone did not modify the serum bactericidal activity. The azocoll-degrading activity of P. gingivalis vesicles was assayed under the same conditions as those used in the serum bactericidal experiments (Table 3). A strong proteolytic activity was associated with untreated vesicles incubated at 37°C. Heat treatment of vesicles reduced this activity to various extents. The degradation of azocoll was
FIG. 2. Binding of purified P. gingivalis lipopolysaccharides to C. ochracea cells as visualized with an indirect fluorescent antibody
technique.
enhanced in the presence of 2-mercaptoethanol and reduced in the presence of PCMP, a thiol-blocking reagent. When purified P. gingivalis lipopolysaccharides were used, instead of outer membrane vesicles, an inhibition of the serum bactericidal activity was also observed. The minimal concentration of lipopolysaccharides required to obtain a complete loss of serum bactericidal activity was found to be 75 jig/ml (final concentration). Inhibition of the bactericidal activity was still obtained when the preincubation step (serum plus lipopolysaccharides) was omitted. Furthermore, the inhibition was not affected by heat treatment of the lipopolysaccharides. When the preincubated mixture (2 h, 37°C) of human serum and lipopolysaccharides was passed through a 0.22-p.m-pore-size membrane filter (to remove a fraction of insoluble lipopolysaccharides) before the bactericidal assay was run in the presence of C. ochracea cells, no inhibition of the serum lethal action was observed (0% viability). This result suggests that the action of lipopolysaccharides is unlikely to be toward the human serum components. The binding of lipopolysaccharides to C. ochracea cells was then evaluated. Bacteria were harvested after the serum bactericidal assay, and bound lipopolysaccharides were detected by an immunofluorescence assay. P. gingivalis lipopolysacchafides showed a high affinity for C. ochracea cells (Fig. 2).
TABLE 3. Azocoll-degrading activity of P. gingivalis
DISCUSSION
outer membrane vesicles
Some suspected periodontal pathogens, including P. gingivalis, are capable of producing extracellular outer membrane vesicles. These structures have been recently well characterized (5, 6, 9) and seem to be the result of a blebbing
Supplement
Incubation temp ('C)
% Proteolytic activity"
37 25
None
None None None None None None 3 mM 2-mercapto-
37 37 37 37
100 58 12 63 13 5 176
None
ethanolb 3 mM PCMPb
37
20
Treatment
None None None
60°C, 30 min 70°C, 30 min 100°C, 5 min
4
a A value of 100% was given to the assay incubated at supplement and with no vesicle treatment. b Final concentration.
37'C
without a
or extrusion from the outer cell membrane. They can either be attached to or released from the cell surface of the bacteria. The extracellular vesicles appear to possess the same biological activities (6, 9, 13) as the outer membrane of the whole cells and a similar composition (5).
Sundqvist and Johansson (18) have shown that strains of P. gingivalis are highly resistant to the lethal action of human serum. They suggested that the resistance was conferred by the structure of the cell envelope. The present study was designed to evaluate the ability of P. gingivalis outer membrane vesicles in protecting oral bacteria against
VOL. 59, 1991
P. GINGIVALIS INHIBITS SERUM BACTERICIDAL ACTIVITY
the bactericidal activity of human serum and to identify possible factors involved in this resistance. The susceptibility of C. ochracea and P. loescheii to human serum, observed in the present study, was previously demonstrated (18, 21); the killing of both bacterial species was shown to be mediated principally via antibody-dependent classical pathway activation. Our data indicate that preincubation of human serum with P. gingivalis outer membrane vesicles can totally prevent the bactericidal activity of human serum against C. ochracea and P. loescheii. This inhibition appears to be mediated by heat-labile and heat-stable components associated with the P. gingivalis outer membrane vesicles. The same components are likely to protect other oral bacterial species against host defense mechanisms involving the complement system. The fact that inhibition of the serum bactericidal activity by outer membrane vesicles was time and temperature dependent suggests a possible role for the vesicle-associated proteolytic activity. Further experiments have shown that inhibition of serum bactericidal activity was increased by a reducing agent and suppressed in the presence of a thiolblocking reagent. In addition, under all conditions where the vesicle-associated proteolytic activity was reduced (low incubation temperature, heat treatment, PCMP treatment), the lethal action of human serum was restored. These data suggest that the vesicle-associated proteolytic activity may be the heat-labile component responsible for the destruction of serum bactericidal activity. This destruction may result from the proteolytic degradation of essential serum factors required for the bactericidal action. Recently, studies concerning the proteolytic activity of P. gingivalis cells have clearly demonstrated the ability of these bacteria to degrade immunoglobulins (7, 8, 16) and selected proteins of the complement system (12, 15-17). A reducing agent-dependent protease from P. gingivalis is currently being purified in our laboratory and will then be assayed in the serum bactericidal assay.
The purified lipopolysaccharides from P. gingivalis were able to inhibit the serum bactericidal activity against C. ochracea. This protective effect may result from the binding of these molecules to C. ochracea cells; the attachment of C3 component or antibody molecules might then be prevented. A similar mechanism of protection against serum bactericidal activity was previously reported by Allen and Scott (1). These authors showed that purified lipopolysaccharides from Escherichia coli could bind to serum-sensitive bacteria and inhibit the bactericidal reaction. In another study, Sansano et al. (11) suggested that increased resistance of a smooth E. coli phenocopy to the lethal action of human serum may be attributable to the blocking of antibody binding sites by the 0 antigen of lipopolysaccharides, thereby preventing activation of the classical pathway of complement. Since outer membrane vesicles from P. gingivalis contain lipopolysaccharides (5, 13), these molecules are likely to represent the heat-stable component responsible for the protection of C. ochracea against serum bactericidal activity. Vesicles could bind (via lipopolysaccharides) to bacteria, allowing the protection of bacteria against the lethal action of serum. However, one should not exclude the possibility that some other heat-resistant components (such as capsular polysaccharides) of vesicles may be involved in the protective effect. The present investigation has shown that lipopolysaccharides and proteolytic enzyme(s) found on outer membrane vesicles of P. gingivalis can mediate the ability of these structures to suppress the bactericidal activity of human
3007
serum. These observations indicate an additional virulence factor associated with P. gingivalis vesicles. Since gingival fluids contain immunoglobulins and complement proteins, it is suggested that vesicles and lipopolysaccharides released by P. gingivalis could protect other bacterial species from complement action, thus favoring the pathogenic process of periodontal disease. ACKNOWLEDGMENTS We thank M. D. McKee (Universitd de Montreal) for critical reading of the manuscript. This work was supported by the Medical Research Council of Canada (grant DG-383-384-385). REFERENCES 1. Allen, R. J., and G. K. Scott. 1980. The effect of purified lipopolysaccharide on the bactericidal reaction of human serum complement. J. Gen. Microbiol. 117:65-72. 2. Carlsson, J., B. F. Herrmann, J. F. Hofling, and G. K. Sundqvist. 1984. Degradation of human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis. Infect. Immun. 43:644-648. 3. Carlsson, J., J. F. Hofling, and G. K. Sundqvist. 1984. Degradation of albumin, haemopexin, haptoglobin and transferrin, by black-pigmented Bacteroides species. J. Med. Microbiol. 18:3946. 4. Darveau, R. P., and R. E. W. Hancock. 1983. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J. Bacteriol. 155:831-838. 5. Deslauriers, M., D. ni Eidhin, L. Lamonde, and C. Mouton. 1990. SDS-PAGE analysis of protein and lipopolysaccharide of extracellular vesicules and Sarkosyl-insoluble membranes from Bacteroides gingivalis. Oral Microbiol. Immunol. 5:1-7. 6. Grenier, D., and D. Mayrand. 1987. Functional characterization of extracellular vesicles produced by Bacteroides gingivalis. Infect. Immun. 55:3131-3136. 7. Grenier, D., D. Mayrand, and B. C. McBride. 1989. Further studies on the degradation of immunoglobulins by black-pigmented Bacteroides. Oral Microbiol. Immunol. 4:12-18. 8. Kilian, M. 1981. Degradation of immunoglobulins Al, A2, and G by suspected principal periodontal pathogens. Infect. Immun. 34:757-765. 9. Mayrand, D., and D. Grenier. 1989. Biological activities of outer membrane vesicles. Can. J. Microbiol. 35:607-613. 10. Okuda, K., and I. Takazoe. 1988. The role of Bacteroides gingivalis in periodontal disease. Adv. Dent. Res. 2:260-268. 11. Sansano, M., A. M. Reynard, and R. K. Cunningham. 1985. Inhibition of serum bactericidal reaction by lipopolysaccharide. Infect. Immun. 48:759-762. 12. Schenkein, H. A. 1988. The effect of periodontal proteolytic Bacteroides species on proteins of the human complement system. J. Periodontal Res. 23:187-192. 13. Singh, U., D. Grenier, and B. C. McBride. 1989. Bacteroides gingivalis vesicles mediate attachment of streptococci to serumcoated hydroxyapatite. Oral Microbiol. Immunol. 4:199-203. 14. Slots, J., and M. A. Listgarten. 1988. Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in human periodontal diseases. J. Clin. Periodontol. 15:85-93. 15. Sundqvist, G., A. Bengtson, and J. Carlsson. 1988. Generation and degradation of the complement fragment C5a in human serum by Bacteroides gingivalis. Oral Microbiol. Immunol. 3:103-107. 16. Sundqvist, G., J. Carlsson, B. Herrmann, and A. Tarnavik. 1985. Degradation of human immunoglobulins G and M and complement factors C3 and C5 by black-pigmented Bacteroides. J. Med. Microbiol. 19:85-94. 17. Sundqvist, G. K., J. Carlsson, B. F. Herrmann, J. F. Hofling, and A. Vaatiinen. 1984. Degradation in vivo of the C3 protein of guinea-pig complement by a pathogenic strain of Bacteroides
3008
GRENIER AND BELANGER
gingivalis. Scand. J. Dent. Res. 92:14-24. 18. Sundqvist, G., and E. Johansson. 1982. Bactericidal effect of pooled human serum on Bacteroides melaninogenicus, Bacteroides asaccharolyticus and Actinobacillus actinomycetemcomitans. Scand. J. Dent. Res. 90:29-36. 19. Taylor, P. W. 1983. Bactericidal and bacteriolytic activity of serum against gram-negative bacteria. Microbiol. Rev. 47:46-83.
INFECT. IMMUN.
20. Taylor, P. W. 1988. Bacterial resistance to complement, p. 107-120. In J. A. Roth (ed.), Virulence mechanisms of bacterial pathogens. American Society for Microbiology, Washington, D.C. 21. Wilson, M. E., R. Burstein, J. T. Jonak-Urbanczyk, and R. J. Genco. 1985. Sensitivity of Capnocytophaga species to bactericidal properties of human serum. Infect. Immun. 50:123-129.
