Vol. 58, No. 12
INFECTION AND IMMUNITY, Dec. 1990, p. 4016-4019
0019-9567/90/124016-04$02.00/0 Copyright © 1990, American Society for Microbiology
Hemin Levels in Culture Medium of Porphyromonas (Bacteroides) gingivalis Regulate Both Hemin Binding and Trypsinlike Protease Production ROBERT J. CARMAN,* MEENA D. RAMAKRISHNAN, AND FIONA H. HARPER Medical Research Council Dental Research Unit, Periodontal Diseases Programme, The London Hospital Medical College, 30-32 Newark Street, London El 2AA, England
Received 5 March 1990/Accepted 31 August 1990 Washed cells and Sarkosyl-insoluble outer membrane preparations of the black-pigmented bacteroides Porphyromonas gingivalis W50 bound hemin. The amount of hemin removed from a buffered solution by both cells and outer membranes was significantly larger if bacteria had been grown in broths supplemented with 5 mg of hemin per liter rather than none. Conversely, cells grown without supplemental hemin bound relatively little. However, all preparations bound some hemin. In addition, hemin regulated the production of significantly higher levels of trypsinlike protease by P. gingivalis W50. The nonpigmented variant, W50 BE1, showed no such responses to the levels of hemin in the growth medium.
The gram-negative anaerobe Porphyromonas gingivalis (formerly Bacteroides gingivalis) is a member of the blackpigmented bacteroides. Members of the group are common isolates from the subgingival microflora (9), and, in particular, P. gingivalis, by virtue of its powerful trypsinlike protease (TLPase), is widely believed to contribute to destructive periodontal disease of humans (12). Accumulated hemin (also known variously as protoheme and heme depending upon the oxidation state of the iron atom in the center of the molecule) is the cause of the characteristic black-brown coloration of colonies of the black-pigmented bacteroides when grown on blood agar (18). Hemin consists of the tetrapyrrole molecule protoporphyrin IX, into the center of which iron is ligated. P. gingivalis is unable to synthesize protoporphyrin IX, which it requires as the prosthetic group of cytochrome b (18). The latter serves as an electron sink during the amino acid fermentation typical of this proteolytic bacterium. P. gingivalis W50 BEl (short for W50 beige), a nonpigmented variant of P. gingivalis W50 with uncharacterized genetic defects, is not pigmented on blood agar plates. This change in function is coincident with a significant reduction in virulence in animal models (14), TLPase production (11, 20, 23), cytochrome b formation (20), and capsule (5, 11, 20) and hemagglutinin production (20). Even so, W50 BEl shows no loss of vigor or cell yield compared with the wild-type parental strain (R. J. Carman, unpublished data). Furthermore, the two variants share more than 95% DNA homology (20). Thus, the two strains are genotypically extremely similar and yet have such different phenotypic traits that they might at first glance be regarded as different species. One possible explanation is that although a vigorously growing strain, W50 BEl has lost the ability to respond to environmental levels of hemin, an ability that W50 retains. This study was designed to investigate the effects of high and low hemin in culture medium on the ability of P. gingivalis W50 and W50 BEl washed cells and outer membrane preparations to bind hemin and on the concurrent production of TLPase by whole cells. *
MATERIALS AND METHODS Bacterial strains and media. P. gingivalis W50 and W50 BE1, a nonpigmented variant of the parent, were maintained by weekly subculture on horse blood agar. Cells were grown in BM broth, a peptone-yeast medium (19) containing either the prescribed 5 mg of hemin per liter or no added hemin. Following inoculation with cells from 48-h-old blood agar cultures, the broths were incubated anaerobically (80% N2, 10% H2, 10% C02) for 48 h at 37°C. Washed cells. Cells were harvested by centrifugation (10,000 x g for 20 min at 4°C), resuspended in 100 ml of phosphate-buffered saline (pH 7.2) three times, and then stored at -70°C under an anaerobic headspace until required. Although this undoubtedly led to some loss of viability, this approach allowed us to use a standard assay inoculum in which the levels of already accumulated hemin were constant. No further attempts were made to maintain anaerobiosis; subsequent manipulations were done on the open bench top. Outer membrane preparation (OMP). Cell suspensions were treated with 10 mM EDTA and heated at 60°C for 20 min (10) to protect proteins of interest from proteolytic degradation during outer membrane preparation. Sarkosylinsoluble outer membranes were then prepared (1) and frozen at -70°C until required. Hemin-binding assay. Frozen cells and OMP were thawed and washed twice in sterile phosphate-buffered saline (pH 7.2). After the second wash they were resuspended in fresh phosphate-buffered saline to an optical density of 1.5 optical density units (ODU) for cells at 605 nm in a total volume of 7.76 ml; OMP were diluted to 0.1 ODU. The protein concentration in both suspensions was measured by sodium hydroxide solubilization (22) and the Bio-Rad protein assay (Bio-Rad Laboratories Ltd., Watford, England). For the remainder of the experiment, the suspensions were stored on ice until use. The addition of hemin (final concentration, 30 ,Ig/ml; Sigma Chemical Co. Ltd., Poole, England) from a stock solution in phosphate-buffered saline increased the volume of the reaction mix to 8 ml. The mixture was incubated at 37°C, and at 0, 1, 5, 10, and 15 min and thereafter at 15-min intervals, aliquots (1 ml) were removed and centrifuged (20,000 x g for 60 s). As some membrane
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BLACK-PIGMENTED BACTEROIDES AND HEMIN
TABLE 1. TLPase activity of and hemin uptake by P. gingivalis W50 and W50 BEl grown in either 5 or 0 mg of hemin per liter
Hemin binding to OMP. OMP of P. gingivalis W50 grown in excess hemin adsorbed 12.5 pug of hemin per mg of protein, whereas OMP from cells of the same strain grown in the absence of hemin bound the significantly smaller amount of 9.6 p,g/mg, a difference of 30% (Table 1). OMP from cells of P. gingivalis W50 BEl bound almost the same amounts of hemin whether they had been grown in hemin excess or insufficiency (6.3 and 6.1 ,ug/mg, respectively). TLPase. The levels of TLPase activity of P. gingivalis W50 cells grown in 0 and 5 mg of hemin per liter were 183 and 324 relative fluorescence units/min/,lg of protein, respectively (Table 1). The difference was statistically significant. TLPase activity of W50 BEl was lower than that of its wild-type counterpart in both levels of hemin: 45 and 53 fluorescence units/min/,ug in 0 and 5 mg of hemin, respectively (Table 1).
TLPase activity (SD)' of cells grownin:
Sample
W50 cells W50 OMP
W50 BEl cells W50 BEl OMP
Amt of hemin of protein) (SD) (~j.g/h/mg taken up
in cells grown in:
O mg of hemin/liter
5 mg of hemin/liter
O mg of hemin/liter
183 (21) NDc
324b (28)
3.0 (0.1)
3.8b (0.2)
ND
9.6 (1.1)
12.5b (1.3)
45 (10) ND
53 (12) ND
1.9 (0.2) 6.1 (0.3)
1.8 (0.1) 6.3 (0.2)
5 mg of hemin/liter
a Results are the means of three experiments. Activity is expressed in relative fluorescence units per minute per microgram of protein. SD, Standard deviation. b p < 0.05 versus 0 mg of hemin per liter. c ND, Not done.
fragments may not have been sedimented by 20,000 x g, they may have interfered with the measurement of residual hemin in the assay buffer. Consequently, an unpublished experiment was done. The only difference from that described above was that OMP were spun at 40,000 x g at 4°C for 60 min. The differences between the absorbance of the supernatants generated in the two experiments were negligible. Thus, if present, outer membrane fragments were not affecting the measurement of hemin in the supernatant fluid. Because of the constraints of time, 20,000 x g was used for the remainder of our study. A calibration curve of hemin concentration versus optical density was plotted, and the amount of hemin taken up (micrograms of hemin per milligram of protein) by the different biological preparations was assessed by measuring the fall in the optical density of the supernatant at 400 nm, i.e., close to the wavelength of maximum absorbance by metalloporphyrins. All experiments were done three times, and the results were analyzed by Student's t test. TLPase assay. The TLPase activity of washed cells was quantified by measuring the rate of cleavage of the fluorescently tagged substrate N-benzoyl-L-arginine-7-amido-4-methylcoumarin (3.7 mM; Sigma Chemical Co. Ltd.) at room temperature in 3 ml of 10 mM Tris hydrochloride buffer (pH 7.2) supplemented with 1 mM L-Cys and 10 mM calcium chloride. Following the addition of sample, the rate of increase in fluorescence (excitation, 380 nm; emission, 460 nm) was measured. Activity was expressed in units of relative fluorescence released per minute per microgram of protein. The assay was done three times, and the results were analyzed by Student's t test. RESULTS Hemin binding to whole cells. Washed and frozen cells of P. gingivalis W50 grown in a liquid medium containing no hemin, other than that found inherently in medium constituents, bound 3.0 ,ug of hemin per mg of protein (Table 1). When the same strain was grown in medium supplemented with 5 mg of hemin per liter, cells bound 3.8 ,ug/mg, a statistically significant 27% increase in uptake of the available hemin. P. gingivalis W50 BE1, a weakly pigmented variant of W50, bound 1.9 and 1.8 ,g/mg when grown in 0 and 5 mg of hemin per liter, respectively. This was not a significant difference. In each case, virtually all uptake occurred within the first 15 min of incubation (data not shown).
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DISCUSSION When P. gingivalis W50 was grown in 5 mg of hemin per liter, its abilities to bind hemin and produce TLPase were significantly raised compared with those of cells of the same strain grown in the absence of added hemin (Table 1). W50 BEl was not similarly regulated by environmental hemin. Levels of iron are elevated in the gingival crevicular fluid of patients with periodontal disease (16); the increase is not due to transferrin or lactoferrin (16). However, hemoglobin is found in the crevice during periodontal breakdown, and it is thought to be the origin of the extra iron (2a, 16). Although it may be argued that free hemin and hemoglobin are rapidly bound by hemopexin or albumin (15) and haptoglobin (4, 8), P. gingivalis is quite capable of completely destroying these plasma proteins (2). Thus, in an ecosystem in which the levels of hemin are variable (16), the competitive advantage to P. gingivalis, and possibly other black-pigmented bacteroides, of such a response is profound. When hemin levels in the gingival crevice rise, the molecule will be stored and the period of its bioavailability will consequently be extended, but only for species able to stockpile it. Our data show that hemin uptake by both strains of P. gingivalis was mediated by a factor or factors retained in Sarkosyl-insoluble outer membrane preparations (Table 1). We are currently trying to isolate and identify this factor. That the variant, W50 BEl, bound hemin, albeit at a lower level than its parental wild type, might seem contradictory as it is nonpigmented on blood agar. However, if all the phenotypes regulated by hemin are affected by the same mechanism, when one such phenotype is expressed, so should others be. Therefore, when W50 BEl produces low levels of TLPase, it will also have the ability to bind hemin. With no hemin other than that present in medium constituents (e.g., proteose peptone and Trypticase), there was indeed growth, reduced TLPase production, and a low level of hemin binding (Table 1). We were forced to disregard any possible adverse effects of TLPase on hemin binding, either during cell culture or as a result of incomplete inactivation during the outer membrane preparation. If uptake did occur via a protein, degradation of the binding factor by the TLPase of P. gingivalis may have caused a loss of binding ability by both the parent and, to a lesser extent, the variant. Certainly, trypsin treatment of Shigellaflexneri, to which Congo red, a tetrapyrrole analog, had already been bound, led to the removal of the dye (3). Several species, other than black-pigmented bacteroides, use environmental hemin. Haemophilus influenzae obtains
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INFECT. IMMUN.
CARMAN ET AL.
iron from hemin (27). Similarly, hemin or protoporphyrin IX is an essential growth factors for Bacteroides fragilis (24), and cells grown under conditions of iron limitation have an elevated ability to bind Congo red (7), suggesting that both the metal and tetrapyrrole part of hemin might be required by the organism. Both hemin and hemoglobin can be used as the sole iron source by siderophore-deficient Vibrio cholerae (25). Hemin and Congo red are both bound by strains of S. flexneri and Escherichia coli; the ability to bind hemin is associated with the simultaneous presence of specific virulence factors (26). Binding is via a 101,000-Mr cell surface protein. Binding of protoporphyrin IX, hemin, and Congo red by Aeromonas salmonicida is associated with virulence and a surface protein array, the A layer (6). Yersinia pestis binds both hemin and Congo red, and, as in other species in the family Enterobacteriaceae that also bind hemin, the mechanism is regulated by temperature (17, 21). Heavy binding leads to colored colonies. Clearly, then, P. gingivalis and several other bacteria can bind hemin, using it as an iron supply or for the tetrapyrrole ring or to deprive competing bacteria of hemin, or any combination of these. However, we are unaware of any examples of bacteria, other than black-pigmented bacteroides, whose binding of hemin is enhanced by the presence of increased levels of hemin itself. Some strains of S. flexneri grown in elevated levels of Congo red produce larger amounts of three outer membrane proteins (17), although these proteins are probably not involved directly with tetrapyrrole binding (26). No such studies involving P. gingivalis have been described in the literature. The fact that so many of the virulence properties of P. gingivalis seem to be coexpressed with its ability to become pigmented on blood agar (i.e., to bind hemin) suggests a central role for hemin in the biology of the bacterium and, consequently, in the etiology of destructive periodontal disease. The stockpiling of hemin during episodes of destructive activity in the crevice may represent the evolution of a feast-famine habit. In times when hemin is in short supply, P. gingivalis may draw its energy from the metabolism of such substrates as succinate, in the same way that another porphyromonad, Porphyromonas asaccharolytica CR2A, can (13). When active disease occurs, hemin is accumulated in excess to ensure its availability for the time when the episode is resolved. In conclusion, we propose that in the healthy crevice, where hemin levels are relatively low, black-pigmented bacteroides grow (or possibly just survive) by adopting the habits of nonpigmented variants such as P. gingivalis W50 BEL. When hemin, in the form of erythrocytes, enters the crevice as a result of chronic inflammation, all the phenotypic characteristics of the wild type, which are coordinately regulated by environmental hemin and are therefore expressed only weakly, if at all, by W50 BEl grown on blood agar, are no longer suppressed. In other words, the organism switches to a wild-type metabolism. The effect of this transition on P. gingivalis is the change from a benign, nonpigmented, and nonproteolytic organism to a virulent, pigmented, and proteolytic one.
LITERATURE CITED 1. Carlone, G. M., M. L. Thomas, H. S. Rumschlag, and F. 0. Sottnek. 1986. Rapid microprocedure for isolating detergent-
insoluble outer membrane proteins from Haemophilus species. J. Clin. Microbiol. 24:330-332. 2. 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. 2a.Carman, R. J., M. D. Ramakrishnan, F. H. Harper, and M. A. Curtis. 1990. Bacteroides gingivalis: role of haemin and iron in aetiology of destructive periodontitis, p. 161-167. In S. P. Borriello (ed.), Clinical and molecular aspects of anaerobes. Wrightson Biomedical Publishing, Petersfield, England. 3. Daskaleros, P. A., and S. M. Payne. 1987. Congo red binding phenotype is associated with hemin binding and increased infectivity of Shigella flexneri in the HeLa cell model. Infect. Immun. 55:1393-1398. 4. Eaton, J. W., P. Brandt, J. R. Mahoney, and J. T. Lee. 1982. Haptoglobin: a natural bacteriostat. Science 215:691-693. 5. Haapasalo, M., H. Shah, S. Gharbia, S. Seddon, and K. Lounatmaa. 1989. Surface properties and ultrastructure of Porphyromonas gingivalis W50 and pleiotropic mutants. Scand. J. Dent. Res. 97:355-360. 6. Ishiguro, E. E., T. Ainsworth, T. J. Trust, and W. W. Kay. 1985. Congo red agar, a differential medium for Aeromonas salmonicida, detects the presence of the cell surface protein array involved in virulence. J. Bacteriol. 164:1233-1237. 7. Larkin, M. J., J. McGuigan, and S. Patrick. 1988. Iron limitation and induction of Congo red binding in Bacteroides fragilis NCTC 9343, p. 216-217. In J. M. Hardie and S. P. Borriello (ed.), Anaerobes today. John Wiley & Sons, Chichester, England. 8. Laurell, C. B., and C. Gronvall. 1962. Haptoglobins. Adv. Clin. Chem. 5:135-172. 9. Maiden, M. F. J., R. J. Carman, M. A. Curtis, I. R. Gillett, G. S. Griffiths, J. A. C. Sterne, J. M. A. Wilton, and N. W. Johnson. 1990. Detection of high-risk groups and individuals for periodontal diseases: laboratory markers based on the microbiological analysis of subgingival plaque. J. Clin. Periodontol. 17:1-13. 10. Mansheim, B. J., and D. L. Kasper. 1977. Purification and immunochemical characterization of the outer membrane complex of Bacteroides melaninogenicus subspecies asaccharolyticus. J. Infect. Dis. 135:787-799. 11. Marsh, P. D., A. S. McKee, A. S. McDermid, and A. B. Dowsett. 1989. Ultrastructure and enzyme activity of a virulent and an avirulent variant of Bacteroides gingivalis W50. FEMS Microbiol. Lett. 59:181-186. 12. Mayrand, D., and S. C. Holt. 1988. Biology of asaccharolytic black-pigmented Bacteroides species. Microbiol. Rev. 52:134152. 13. Mayrand, D., and B. C. McBride. 1980. Ecological relationships of bacteria involved in a simple mixed anaerobic infection. Infect. Immun. 27:44-50. 14. McKee, A. S., A. S. McDermid, R. Wait, A. Baskerville, and P. D. Marsh. 1988. Isolation of colonial variants of Bacteroides
15.
16.
17.
18.
ACKNOWLEDGMENTS We are grateful to Mike Curtis, Medical Research Council Dental Research Unit, and Phil Marsh, PHLS Centre for Applied Microbial Research, Salisbury, England, for their advice during this study and for reading our manuscript.
19.
gingivalis W50 with a reduced virulence. J. Med. Microbiol. 27:59-64. Morgan, E. H. 1981. Transferrin, biochemistry, physiology and clinical significance. Mol. Aspects Med. 4:1-123. Mukherjee, S. 1985. The role of crevicular iron in periodontal disease. J. Periodontol. 56:(Suppl.):22-27. Sankaran, K., V. Ramachandran, Y. B. K. Subrahmanyam, S. Rajarathnam, S. Elango, and R. R. Roy. 1989. Congo redmediated regulation of levels of Shigella flexneri 2a membrane proteins. Infect. Immun. 57:2364-2371. Shah, H. N., R. Bonnett, B. Mateen, and R. A. D. Williams. 1979. The porphyrin pigmentation of subspecies of Bacteroides melaninogenicus. Biochem. J. 180:45-50. Shah, H. N., R. A. Nash, J. M. Hardie, D. A. Wheetman, D. A. Geddes, and T. W. MacFarlane. 1985. Detection of acidic end products of metabolism of anaerobic Gram-negative bacteria, p. 317-340, In M. Goodfellow and D. E. Minnikin (ed.), Chemical
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methods in bacterial systematics. Academic Press, Inc. (London), Ltd., London. 20. Shah, H. N., S. V. Seddon, and
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E. Gharbia. 1989. Studies
on
the virulence properties and metabolism of pleiotropic mutants of Porphyromonas gingivalis (Bacteroides gingivalis) W50. Oral Microbiol. Immunol. 4:19-23. 21. Sikkema, D. J., and R. R. Brubaker. 1987. Resistance to pesticin, storage of iron, and invasion of HeLa cells by yersiniae. Infect. Immun. 55:572-578. 22. Simpson, I. A., and 0. Sonne. 1982. A simple, rapid and sensitive method for measuring protein concentration in subcellular membrane fractions prepared by sucrose density ultracentrifugation. Anal. Biochem. 119:424-427. 23. Smalley, J. W., A. J. Birss, H. M. Kay, A. S. McKee, and P. D. Marsh. 1989. The distribution of trypsin-like protease activity in
24.
25. 26.
27.
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cultures of a virulent and an avirulent strain of Bacteroides gingivalis. Oral Microbiol. Immunol. 4:178-181. Sperry, J. F., M. D. Appleman, and T. D. Wilkins. 1977. Requirement of heme for growth of Bacteroides fragilis. Appl. Environ. Microbiol. 34:386-390. Stoebner, J. A., and S. M. Payne. 1988. Iron-regulated hemolysin production and utilization of heme and hemoglobin by Vibrio cholerae. Infect. Immun. 56:2891-2895. Stugard, C. E., P. E. Daskaleros, and S. M. Payne. 1989. A 101-kilodalton heme-binding protein associated with Congo red binding and virulence of Shigella flexneri and enteroinvasive Escherichia coli strains. Infect. Immun. 57:3534-3539. Stull, T. L. 1987. Protein sources of heme for Haemophilus influenzae. Infect. Immun. 55:148-153.
INFECTION AND IMMUNITY, Dec. 1990, p. 4016-4019
0019-9567/90/124016-04$02.00/0 Copyright © 1990, American Society for Microbiology
Hemin Levels in Culture Medium of Porphyromonas (Bacteroides) gingivalis Regulate Both Hemin Binding and Trypsinlike Protease Production ROBERT J. CARMAN,* MEENA D. RAMAKRISHNAN, AND FIONA H. HARPER Medical Research Council Dental Research Unit, Periodontal Diseases Programme, The London Hospital Medical College, 30-32 Newark Street, London El 2AA, England
Received 5 March 1990/Accepted 31 August 1990 Washed cells and Sarkosyl-insoluble outer membrane preparations of the black-pigmented bacteroides Porphyromonas gingivalis W50 bound hemin. The amount of hemin removed from a buffered solution by both cells and outer membranes was significantly larger if bacteria had been grown in broths supplemented with 5 mg of hemin per liter rather than none. Conversely, cells grown without supplemental hemin bound relatively little. However, all preparations bound some hemin. In addition, hemin regulated the production of significantly higher levels of trypsinlike protease by P. gingivalis W50. The nonpigmented variant, W50 BE1, showed no such responses to the levels of hemin in the growth medium.
The gram-negative anaerobe Porphyromonas gingivalis (formerly Bacteroides gingivalis) is a member of the blackpigmented bacteroides. Members of the group are common isolates from the subgingival microflora (9), and, in particular, P. gingivalis, by virtue of its powerful trypsinlike protease (TLPase), is widely believed to contribute to destructive periodontal disease of humans (12). Accumulated hemin (also known variously as protoheme and heme depending upon the oxidation state of the iron atom in the center of the molecule) is the cause of the characteristic black-brown coloration of colonies of the black-pigmented bacteroides when grown on blood agar (18). Hemin consists of the tetrapyrrole molecule protoporphyrin IX, into the center of which iron is ligated. P. gingivalis is unable to synthesize protoporphyrin IX, which it requires as the prosthetic group of cytochrome b (18). The latter serves as an electron sink during the amino acid fermentation typical of this proteolytic bacterium. P. gingivalis W50 BEl (short for W50 beige), a nonpigmented variant of P. gingivalis W50 with uncharacterized genetic defects, is not pigmented on blood agar plates. This change in function is coincident with a significant reduction in virulence in animal models (14), TLPase production (11, 20, 23), cytochrome b formation (20), and capsule (5, 11, 20) and hemagglutinin production (20). Even so, W50 BEl shows no loss of vigor or cell yield compared with the wild-type parental strain (R. J. Carman, unpublished data). Furthermore, the two variants share more than 95% DNA homology (20). Thus, the two strains are genotypically extremely similar and yet have such different phenotypic traits that they might at first glance be regarded as different species. One possible explanation is that although a vigorously growing strain, W50 BEl has lost the ability to respond to environmental levels of hemin, an ability that W50 retains. This study was designed to investigate the effects of high and low hemin in culture medium on the ability of P. gingivalis W50 and W50 BEl washed cells and outer membrane preparations to bind hemin and on the concurrent production of TLPase by whole cells. *
MATERIALS AND METHODS Bacterial strains and media. P. gingivalis W50 and W50 BE1, a nonpigmented variant of the parent, were maintained by weekly subculture on horse blood agar. Cells were grown in BM broth, a peptone-yeast medium (19) containing either the prescribed 5 mg of hemin per liter or no added hemin. Following inoculation with cells from 48-h-old blood agar cultures, the broths were incubated anaerobically (80% N2, 10% H2, 10% C02) for 48 h at 37°C. Washed cells. Cells were harvested by centrifugation (10,000 x g for 20 min at 4°C), resuspended in 100 ml of phosphate-buffered saline (pH 7.2) three times, and then stored at -70°C under an anaerobic headspace until required. Although this undoubtedly led to some loss of viability, this approach allowed us to use a standard assay inoculum in which the levels of already accumulated hemin were constant. No further attempts were made to maintain anaerobiosis; subsequent manipulations were done on the open bench top. Outer membrane preparation (OMP). Cell suspensions were treated with 10 mM EDTA and heated at 60°C for 20 min (10) to protect proteins of interest from proteolytic degradation during outer membrane preparation. Sarkosylinsoluble outer membranes were then prepared (1) and frozen at -70°C until required. Hemin-binding assay. Frozen cells and OMP were thawed and washed twice in sterile phosphate-buffered saline (pH 7.2). After the second wash they were resuspended in fresh phosphate-buffered saline to an optical density of 1.5 optical density units (ODU) for cells at 605 nm in a total volume of 7.76 ml; OMP were diluted to 0.1 ODU. The protein concentration in both suspensions was measured by sodium hydroxide solubilization (22) and the Bio-Rad protein assay (Bio-Rad Laboratories Ltd., Watford, England). For the remainder of the experiment, the suspensions were stored on ice until use. The addition of hemin (final concentration, 30 ,Ig/ml; Sigma Chemical Co. Ltd., Poole, England) from a stock solution in phosphate-buffered saline increased the volume of the reaction mix to 8 ml. The mixture was incubated at 37°C, and at 0, 1, 5, 10, and 15 min and thereafter at 15-min intervals, aliquots (1 ml) were removed and centrifuged (20,000 x g for 60 s). As some membrane
Corresponding author. 4016
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TABLE 1. TLPase activity of and hemin uptake by P. gingivalis W50 and W50 BEl grown in either 5 or 0 mg of hemin per liter
Hemin binding to OMP. OMP of P. gingivalis W50 grown in excess hemin adsorbed 12.5 pug of hemin per mg of protein, whereas OMP from cells of the same strain grown in the absence of hemin bound the significantly smaller amount of 9.6 p,g/mg, a difference of 30% (Table 1). OMP from cells of P. gingivalis W50 BEl bound almost the same amounts of hemin whether they had been grown in hemin excess or insufficiency (6.3 and 6.1 ,ug/mg, respectively). TLPase. The levels of TLPase activity of P. gingivalis W50 cells grown in 0 and 5 mg of hemin per liter were 183 and 324 relative fluorescence units/min/,lg of protein, respectively (Table 1). The difference was statistically significant. TLPase activity of W50 BEl was lower than that of its wild-type counterpart in both levels of hemin: 45 and 53 fluorescence units/min/,ug in 0 and 5 mg of hemin, respectively (Table 1).
TLPase activity (SD)' of cells grownin:
Sample
W50 cells W50 OMP
W50 BEl cells W50 BEl OMP
Amt of hemin of protein) (SD) (~j.g/h/mg taken up
in cells grown in:
O mg of hemin/liter
5 mg of hemin/liter
O mg of hemin/liter
183 (21) NDc
324b (28)
3.0 (0.1)
3.8b (0.2)
ND
9.6 (1.1)
12.5b (1.3)
45 (10) ND
53 (12) ND
1.9 (0.2) 6.1 (0.3)
1.8 (0.1) 6.3 (0.2)
5 mg of hemin/liter
a Results are the means of three experiments. Activity is expressed in relative fluorescence units per minute per microgram of protein. SD, Standard deviation. b p < 0.05 versus 0 mg of hemin per liter. c ND, Not done.
fragments may not have been sedimented by 20,000 x g, they may have interfered with the measurement of residual hemin in the assay buffer. Consequently, an unpublished experiment was done. The only difference from that described above was that OMP were spun at 40,000 x g at 4°C for 60 min. The differences between the absorbance of the supernatants generated in the two experiments were negligible. Thus, if present, outer membrane fragments were not affecting the measurement of hemin in the supernatant fluid. Because of the constraints of time, 20,000 x g was used for the remainder of our study. A calibration curve of hemin concentration versus optical density was plotted, and the amount of hemin taken up (micrograms of hemin per milligram of protein) by the different biological preparations was assessed by measuring the fall in the optical density of the supernatant at 400 nm, i.e., close to the wavelength of maximum absorbance by metalloporphyrins. All experiments were done three times, and the results were analyzed by Student's t test. TLPase assay. The TLPase activity of washed cells was quantified by measuring the rate of cleavage of the fluorescently tagged substrate N-benzoyl-L-arginine-7-amido-4-methylcoumarin (3.7 mM; Sigma Chemical Co. Ltd.) at room temperature in 3 ml of 10 mM Tris hydrochloride buffer (pH 7.2) supplemented with 1 mM L-Cys and 10 mM calcium chloride. Following the addition of sample, the rate of increase in fluorescence (excitation, 380 nm; emission, 460 nm) was measured. Activity was expressed in units of relative fluorescence released per minute per microgram of protein. The assay was done three times, and the results were analyzed by Student's t test. RESULTS Hemin binding to whole cells. Washed and frozen cells of P. gingivalis W50 grown in a liquid medium containing no hemin, other than that found inherently in medium constituents, bound 3.0 ,ug of hemin per mg of protein (Table 1). When the same strain was grown in medium supplemented with 5 mg of hemin per liter, cells bound 3.8 ,ug/mg, a statistically significant 27% increase in uptake of the available hemin. P. gingivalis W50 BE1, a weakly pigmented variant of W50, bound 1.9 and 1.8 ,g/mg when grown in 0 and 5 mg of hemin per liter, respectively. This was not a significant difference. In each case, virtually all uptake occurred within the first 15 min of incubation (data not shown).
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DISCUSSION When P. gingivalis W50 was grown in 5 mg of hemin per liter, its abilities to bind hemin and produce TLPase were significantly raised compared with those of cells of the same strain grown in the absence of added hemin (Table 1). W50 BEl was not similarly regulated by environmental hemin. Levels of iron are elevated in the gingival crevicular fluid of patients with periodontal disease (16); the increase is not due to transferrin or lactoferrin (16). However, hemoglobin is found in the crevice during periodontal breakdown, and it is thought to be the origin of the extra iron (2a, 16). Although it may be argued that free hemin and hemoglobin are rapidly bound by hemopexin or albumin (15) and haptoglobin (4, 8), P. gingivalis is quite capable of completely destroying these plasma proteins (2). Thus, in an ecosystem in which the levels of hemin are variable (16), the competitive advantage to P. gingivalis, and possibly other black-pigmented bacteroides, of such a response is profound. When hemin levels in the gingival crevice rise, the molecule will be stored and the period of its bioavailability will consequently be extended, but only for species able to stockpile it. Our data show that hemin uptake by both strains of P. gingivalis was mediated by a factor or factors retained in Sarkosyl-insoluble outer membrane preparations (Table 1). We are currently trying to isolate and identify this factor. That the variant, W50 BEl, bound hemin, albeit at a lower level than its parental wild type, might seem contradictory as it is nonpigmented on blood agar. However, if all the phenotypes regulated by hemin are affected by the same mechanism, when one such phenotype is expressed, so should others be. Therefore, when W50 BEl produces low levels of TLPase, it will also have the ability to bind hemin. With no hemin other than that present in medium constituents (e.g., proteose peptone and Trypticase), there was indeed growth, reduced TLPase production, and a low level of hemin binding (Table 1). We were forced to disregard any possible adverse effects of TLPase on hemin binding, either during cell culture or as a result of incomplete inactivation during the outer membrane preparation. If uptake did occur via a protein, degradation of the binding factor by the TLPase of P. gingivalis may have caused a loss of binding ability by both the parent and, to a lesser extent, the variant. Certainly, trypsin treatment of Shigellaflexneri, to which Congo red, a tetrapyrrole analog, had already been bound, led to the removal of the dye (3). Several species, other than black-pigmented bacteroides, use environmental hemin. Haemophilus influenzae obtains
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INFECT. IMMUN.
CARMAN ET AL.
iron from hemin (27). Similarly, hemin or protoporphyrin IX is an essential growth factors for Bacteroides fragilis (24), and cells grown under conditions of iron limitation have an elevated ability to bind Congo red (7), suggesting that both the metal and tetrapyrrole part of hemin might be required by the organism. Both hemin and hemoglobin can be used as the sole iron source by siderophore-deficient Vibrio cholerae (25). Hemin and Congo red are both bound by strains of S. flexneri and Escherichia coli; the ability to bind hemin is associated with the simultaneous presence of specific virulence factors (26). Binding is via a 101,000-Mr cell surface protein. Binding of protoporphyrin IX, hemin, and Congo red by Aeromonas salmonicida is associated with virulence and a surface protein array, the A layer (6). Yersinia pestis binds both hemin and Congo red, and, as in other species in the family Enterobacteriaceae that also bind hemin, the mechanism is regulated by temperature (17, 21). Heavy binding leads to colored colonies. Clearly, then, P. gingivalis and several other bacteria can bind hemin, using it as an iron supply or for the tetrapyrrole ring or to deprive competing bacteria of hemin, or any combination of these. However, we are unaware of any examples of bacteria, other than black-pigmented bacteroides, whose binding of hemin is enhanced by the presence of increased levels of hemin itself. Some strains of S. flexneri grown in elevated levels of Congo red produce larger amounts of three outer membrane proteins (17), although these proteins are probably not involved directly with tetrapyrrole binding (26). No such studies involving P. gingivalis have been described in the literature. The fact that so many of the virulence properties of P. gingivalis seem to be coexpressed with its ability to become pigmented on blood agar (i.e., to bind hemin) suggests a central role for hemin in the biology of the bacterium and, consequently, in the etiology of destructive periodontal disease. The stockpiling of hemin during episodes of destructive activity in the crevice may represent the evolution of a feast-famine habit. In times when hemin is in short supply, P. gingivalis may draw its energy from the metabolism of such substrates as succinate, in the same way that another porphyromonad, Porphyromonas asaccharolytica CR2A, can (13). When active disease occurs, hemin is accumulated in excess to ensure its availability for the time when the episode is resolved. In conclusion, we propose that in the healthy crevice, where hemin levels are relatively low, black-pigmented bacteroides grow (or possibly just survive) by adopting the habits of nonpigmented variants such as P. gingivalis W50 BEL. When hemin, in the form of erythrocytes, enters the crevice as a result of chronic inflammation, all the phenotypic characteristics of the wild type, which are coordinately regulated by environmental hemin and are therefore expressed only weakly, if at all, by W50 BEl grown on blood agar, are no longer suppressed. In other words, the organism switches to a wild-type metabolism. The effect of this transition on P. gingivalis is the change from a benign, nonpigmented, and nonproteolytic organism to a virulent, pigmented, and proteolytic one.
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