Oral Mierobiol Imtmttwl 1991: 6: 209-215
Degradation of plasma proteins by the trypsin-iike enzyme of Porphyromonas gingivalis and iniiibition of protease activity by a serine protease inhibitor of human piasma
C. S. Fishburn, J. M. Slaney, R. J. Carman, M. A. Curtis MRC Dental Research Unit, London Hospital Medical College, United Kingdom
Fishburn CS, Slaney JM, Cartnan RJ, Curtis MA. Degradation of plasma proteins by the trypsin-like enzyme o/Porphyromonas gingivalis atrd inhibition of protease Activity by a serine protease inhibitor of hutnan plastna. Oral Mierobiol Itmmmol 1991: 6: 209^215. The interaction between Porphyrotnotuis gingivalis culture supernatant and human serum was examined. Hydrolysis of the major serum proteins was thiol-dependent and correlated with the trypsin-like activity of the sample. Transferrin and igG 'ight chains were less susceptible to degradation than albumin and IgG heavy chains and partially degraded IgG retained antigen-binding capability. Serum inhibited the trypsin-like activity in a fluorimetric assay. The inhibition was shown to be independent of the level of IgG antibody reactive with whole cells of ^ gingivalis. Purified preparations of antithrombin III, a serine protease inhibitor, but not al-antitrypsin nor a2-rnacroglobulin inhibited the trypsin-like activity in the fluorimetric assay.
Porphyrotnotias (Baeteroides) gingivalis munoglobulins (10, 12, 24). Indeed, has been strongly implicated in the aeti- under the appropriate conditions, it apology of adult periodontitis (21, 23). pears that virtually all plasma proteins Cultural studies have demonstrated are susceptible to extensive degradation °oth an increased prevalence and an in- by enzyine(s) produced by P. gittgivalis creased median recovery of this organ- (2). In the subgingival environment, the 'sm in progressing lesions versus quies- extent of plasma protein degradation is cent sites (22) and many studies have determined not only by the numbers demonstrated significantly elevated and activity of protease-producing orSerum immunoglobulin G antibody ganisms but also the flow rate of exu'evels to P. gingivalis in adult perio- date through the crevice. Hence, at in"^ontitis patients compared with healthy flamed sites in the gingiva, where elevControls (8, 9). The precise molecular ated crevicular fluid flow rates would be ^echanisrns by which colonization by anticipated (15), only a limited digestioti ^- gitigivalis can lead to the tissue de- of plasma proteins by P gingivalis may struction characteristic of progressive occur. In such circumstances, the rela•destructive periodontitis are still poorly tive resistance to proteolysis of individUnderstood. However, much attention ual proteins becomes relevant to the •^as been focused upon the proteolytic pathogenic potential of the organism. activities produced by this organism. The purpose of this investigation was *Vhole cells of P. gitigivalis have been to examine the degradation of human shown to degrade plasma proteins in- serum proteins by P. gingivalis under volved in host defence, including iron conditions that permitted estimation of binding proteins (3), complement com- their differing susceptibilities and, in the Ponents (24), anti-proteases (2) and im- case of immunoglobulin G, the effect of
Key words: Porpliyromonas gingivatis; trypsin-iike enzyme; piasma proteolysis: proteaseinhibitor M. A. Curtis, MRC Dental Research Unit, London Hospitai Medical Coilege, 32 Newark Street, London El 2AA, UK Accepted for pubiieation September 4, 1990
limited proteolysis on antigen-binding capacity. During the course of these investigations we observed that serum was able to inhibit the thiol-dependant arginine amidolytic activity of this organism. The likely source of this inhibition was investigated using purified preparations of plasma anti-proteases. Material and methods Preparation of crude protease
P. gingivalis W83, a highly proteolytic strain, was inoculated from a 48-h horse blood agar plate into BM broth, a peptone/yeast medium supplemented with 5 mg haemin/1 (20). The inoculated broths were incubated anaerobically (80% Nj, 10% H,, 10% COj) for 6 d at 37°C. The supernatant fluid was collected after centrifugation (18,000 g, 30 min, 4°C) and then filter-sterilized (0.22-/mi ftlter). The supernatant enzyme activity was then concentrated by precipitation overnight at 4 X with
210
Fishburn et al.
ammonium sulphate to 70% saturation followed by resuspension of the pellet to one-tenth the original volume in 50 mM Tris (hydroxymethyl) methylamine (pH 7.2) and overnight dialysis against the same buffer. The dialysed concentrated culture supernatant (DCCS) was then lyophilized in 5-ml aliquots and stored at — 70°C until required, whereupon it was resuspended in the appropriate chilled buffer and used immediately. Enzyme activity assay and inhibition studies
Trypsin-like enzyme acitivity was assessed by measuring the rate of cleavage of the fluorimetric substrate Nbenzoyl-L-arginine 7-amido-4-methylcoumarin hydrochloride (BAAMC Sigma Chemical, Poole, UK). Assays were routinely performed in 3 ml 50 mM Tris HCI (pH 7.2, 10 mM CaClj, 1 mM cysteine HCI) containing 3.7 mM BAAMC at room temperature. Following addition of the sample, the rate of increase in relative fluorescence (i.e. rate of hydrolysis of BAAMC) was measured in a fluorimeter (SFM25; Kontron Instruments, Zurich, Switzerland; excitation: 380 nm; emission: 460 nm). Enzyme activity was expressed in units of relative fluorescence released per min. All experiments were performed in duplicate on at least 2 separate occasions and the results are presented as mean data. The standard deviations of the means did not exceed ±5% in each case. The effect of serum and purified protease inhibitors on enzyme activity was examined by pre-incubation of DCCS and the inhibitor for 5 min at room temperature followed by measurement of the rate of cleavage of BAAMC. Serum was used at a dilution of 10% in phosphate-buffered saline (PBS), pH 7.2 and the protease inhibitors, a2-macroglobulin (Sigma), al-antitrypsin and anti-thrombin III (both obtained from Dr P. Pemberton, Department of Haematology, Addenbrookes Hospital, Cambridge, UK) at 10% dilutions of their respective plasma concentrations. Albumin adjusted to the total protein concentration of serum was used as a control for competitive inhibition in the assay by serum proteins. The percentage inhibition was calculated relative to the activity of a control incubation of DCCS in the absence of inhibitor.
Preparation of Bacteroides intermedius outer membranes
Baeteroides intertnedius T588 was grown in BM broth for 48 h. Bacterial cells were harvested by centrifugation (10,000 g, 20 min, 4°C) and washed twice in sterile Tris buffer (50 mM, pH 7.2). After the second wash, the cell pellet was resuspended in l/50th of the original culture volume in Tris buffer and stored at -70°C until required. Outer membranes were prepared using the method of Carlone et al. (1). Briefly, the cells were sonicated in HEPES buffer (10 mM, pH 7.2) and centrifuged (10,000 g, 2 min, 4°C) to remove unbroken cells and debris. The crude membranes were then pelleted by centrifugation at 40,000 g for 60 min at 4°C. The outer membranes were obtained following selective solubilization of the inner membranes using sodium lauryl sarcosinate (Sigma - 1 % w/v in HEPES, 10 mM, pH 7.2). Periodontai patient and controi serum sampies
Cases (M = 10) were selected from patients attending a periodontai referral clinic and were defined as having a CPITN (7) score of 4 in one or more sextants, thus exhibiting a history of destructive periodontai disease. Controls {n = 10) were selected from patients attending other clinics for routine dental treatment and were defined as subjects whose worst CPITN code was 2 or less. Cases and controls were matched for age (+ 1 year), ethnic origin, gender and mean oral hygiene scores ( + 0.5 plaque index). Following collection the blood was allowed to clot at room temperature. The serum was then separated within 4 h of venipuncture and stored at — 70°C until required. Eiectrophoresis and Western blotting
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS/PAGE) was carried out according to the method of Laemmh (13) using 10% acrylamide/ bisacrylamide gels (8.0 cm x 5.5 cm x 1.5 mm) on a Midget gel system (LKB, Bromma, Sweden). Gels were run at 30 mA for approximately 1 h and then either stained for protein using 0.2% Coomassie brilliant blue (BDH Chemicals, Poole, UK) or eiectroblotted onto 0.45 ixm nitrocellulose membranes at 400 mA for 2.5 h (26).
The membranes were blocked in 5% BSA/PBS for 30 min at room temperature and then washed twice for 10 min in PBS. Membranes were incubated overnight in primary antisera at the appropriate dilution in 1% BSA/PBS and then washed 6 times for 15 min in 0.1 % Tween-20/PBS. Where necessary, they were incubated in horseradish peroxidase (HRPO)-labelled anti-species antibody for 2 h and then washed as before. The antisera and working dilutions used in this study were as follows: rabbit anti-albumin, 1:5000 (Behringwerke, Marburg, FRG); sheep anti-transferrin, 1:5000 (Unipath, Bedford, UK); sheep anti alpha-1-antitrypsin, 1:40,000 (Unipath); rabbit anti-human kappa light chain, 1:10,000; rabbit anti-human IgA alpha chain, 1:500; HRPO-conjugated swine anti-human IgG gamma chain, 1:1000; HRPO-conjugated swine antihuman IgM mu chain, 1:200; HRPOconjugated swine anti-rabbit IgG, 1:500; HRPO-conjugated swine antisheep IgG, 1:5000 (all Dakopatts Antisera, Glostrup, Denmark). Where rabbit anti-human kappa light chain was used as a secondary antibody, the dilution was 1:200 and the tertiary antibody was HRPO-conjugated swine antirabbit IgG as before. Incubations were carried out at room temperature with agitation. Membranes were given a final 10-min rinse in PBS before being developed in 0.05% (w/v) 3,3-diaminobenzidine tetrahydrochloride with 0.02% (v/v) hydrogen peroxide (BDH) and 0.03% (w/v) nickel chloride. Densitometric scanning of the immunoblots was performed using a laser densitometer (Ultroscan XL, LKB). To investigate the loss of function of human IgG treated with DCCS, the binding of a patient serum IgG antibodies to outer membrane antigens of B. intertnedius on a Western blot was assessed after incubation of the serum with DCCS. Serum and DCCS were incubated in a 1:5 ratio in the presence of 10 mM CaClj and 1 mM L-cys in a final volume of 4.5 ml. A 1:5 ratio of substrate to enzyme had previously been shown to give the optitnum rate of serum protein hydrolysis for a time course experiment at this thiol concentration. Samples (0.5 ml) were withdrawn at time intervals up to 3 h and dispensed into 9.5 ml BSA (2%)/PBS to prevent any further proteolysis of the serum. Outer membrane preparations of S. intertnedittswere run on 10% gels. Western-blotted onto nitrocellulose
Serutn and p. gingivalis
niembranes, cut, anode to cathode, into 4-mm strips and blocked as described. Duplicate strips were incubated for 2 h in each sample of protease-digested serum. After washing, one strip from each time point was incubated in HRPO-conjugated swine anti-human IgG gamma chain, and the other in rabbit anti-human kappa light chain followed by HRPO-conjugated swine antirabbit IgG. Incubations, washes and development in diaminobenzidine were performed as described earlier. Results Demonstration of thioi-dependence
Both the activity of the trypsin-like enzyme, assessed by rate of cleavage of BAAMC, and the degradation of serum proteins by DCCS critically depended On the concentrations of thiol reagents. In the absence of exogenous thiol groups, no cleavage of BAAMC was detected. Introduction of 1 mM L-cys into the assay produced activation of the enzyme to approximately 50% of its maximal activity, which was attained in the presence of 50 mM L-cys. Similarly, in the absence of L-cys, no degradation of serum proteins was observed via SDS/PAGE when DCCS and serum Were incubated at a ratio of 5:1 for 60 min. Addition of increasing amounts of the thiol caused a progressive increase in serum protein degradation (Fig. 1). These findings are consistent with the BAAMC-cleaving activity and serum protein degradation being functions of the same thiol-dependent enzyme. A more striking example of the thiol dependence of the serum protein degradation was obtained when proteolysis Was allowed to proceed in the presence of excess SDS/PAGE reducing sample buffer, which contained 5% 2-mercaptoethanol. DCCS and serum mixtures Were prepared varying in ratio from 1:1 to 5:1 with no added thiol. The mixtures Were then incubated for 3 h at 37'C and then immediately dispensed into excess SDS/PAGE sample buffer that had been preheated to 100°C. A duplicate series was incubated for 3 h at 37°C, dispensed into SDS/PAGE sample buffer at room temperature and then immediately brought to 100°C in an incubator. The resulting protein profiles Were examined via SDS/PAGE. The Samples that had been inactivated after the 3 h incubation by hot sample buffer showed no degradation. Those that had been heat-inactivated by heating from
211
room temperature up to 100°C in the presence of SDS/PAGE sample buffer showed a progressive loss of serum protein with increasing ratio of DCCS:serum (data not shown). Incubation of a DCCS/serum mixture in the presence of individual components of the SDS/PAGE buffer (3% SDS and 0.5 M Tris HCI, pH 7.2) for 10 min followed by inactivation in hot sample buffer had no effect on proteolysis. However, the same procedure in 5% 2rnercaptoethanol produced almost complete degradation of all serum proteins. Thus, to avoid any confusion in later experiments between proteolysis taking place during incubation at 37°C and that taking place as a result of heating in sample buffer, all samples were put directly into hot sample buffer in a 100°C water bath.
run on a 10% polyacrylamide gel lane) and stained with Coomassie Blue (Fig. 2A). Although the gradual degradation of serum proteins was clear, the proteins appeared to exhibit differing susceptibilities towards the protease activity. Bands at the levels of 66 kDa and 55 kDa disappeared with time, whereas those at levels 77 kDa and 25 kDa (arrowed) persisted for up to 30 h. A band at approximately 60 kDa, presumably the product of the degradation of a higher-molecular-weight component (most likely albumin), was generated after 6 h incubation and increased in intensity up to 24 h. By 5 d, no bands were visible. The identity of individual proteins was established by immunoblotting samples taken at different time points and probing the blots with monospecific antisera. The components at 66 kDa and 55 kDa were identified as albumin Susceptibility of different serum proteins and IgG gamma chains, both of which to proteoiysis appeared to be highly susceptible to Samples taken at different time points proteolysis. Similar rates of disappearfrom an incubation of DCCS and serum ance were found for IgM mu chains and (5:1) in the presence of 1 mM L-cys were IgA alpha chains. The relatively resist-
Cysteine (rnM) Fig. 1. Thiol-dcpcndcncc of P. gingivalis culture supernatant trypsin-like enzyme activity (expressed as units of relative fluorescence released per minute in BAAMC assay). Insert shows SDS/PAGE of serum incubated with concentrated supernatant at a ratio 1:5 for 60 min in the presence of: 0 mM cysteine (lane 1): 1 mM (2): 10 niM (3) and 50 mM eysteine (4). Numhers in the right-hand margin refer to migration positions of standard-moleeularweight proteins: ovotransferrin (77,000); albumin (66,000); ovalbumin (45,000): and carbonic anhydrase (29,000).
212
Fishburn et al.
ant proteins at 77 kDa and 25 kDa were identified as transferrin and IgG kappa chains. Although native al-antitrypsin underwent a rapid conversion to a lower-molecular-weight form, this product was stable to further proteolysis and was still detectable irnmunochemically after 5 d of incubation. Western blots of ; = 0 and / = 30 h samples developed using anti-gamma chain, anti-kappa chain, anti-transferrin and anti-a 1-antitrypsin antisera are shown in Fig. 2B. Antigen-binding ability to DCCS-treated serum igG
The observation that kappa chains were relatively resistant to degradation by DCCS led us to examine the antigenbinding ability of serum previously incubated with DCCS. The binding of IgG to B. intermedius outer membrane proteins separated via SDS/PAGE and then transferred to nitrocellulose is shown in Fig. 3. When antibody binding was assessed using anti-gamma chain antiserum, the amount of reactive antibody decreased with increasing tirne of incubation of the serum with DCCS
(Fig. 3b, c). However when anti-kappa chain antiserum was used, the amount of immunoreactivity was undiminished, even in serum which had been incubated for 3 h with DCCS (Fig. 3a and 3c). Serum Inhibition studies
The effect of serum on the thiol-activated enzyme activity of DCCS was examined by measuring the rate of cleavage of the fluorimetric substrate, BAAMC, in the presence of increasing volumes of serum. To establish the level of inhibition produced in the assay as a result of simple competition with serum proteins, parallel assays were carried out using bovine serum albumin (BSA), shown earlier to be susceptible to degradation. A protein assay revealed the average concentration of protein in 20
776645-
29-
1 2
7766-1
3 4
5
6
7 8
457766-
29-
45-
•
•
•
%
•
•
29-
o—'•>•—o 7 7 -
• ^ . •.
6 6 -
; ,
4 5 -
«»,
,
^. ,. r „
'29-
'•' ' \,
..'^••.
:
. ';•
' :'•:
Discusssion
, ,_**:'.,",
. , - : . ; | | | | . ^ . - 3 ,•• .... : : : ' • : - m .
1
2
3
4" 5
6
7
samples of serum to be 74 mg/ml; BSA was made up in PBS to the same concentration. Enzyme activity was expressed as a percentage of the activity in a serum-free control (Fig. 4a). A dosedependent inhibition of BAAMC cleavage was produced by serum; this exceeded the level of inhibition produced by simple competition as a consequence of the introduction of an alternative substrate (in this case BSA) into the assay. To investigate the possibility that the protease inhibition might be the result of anti-protease antibodies in serum, inhibition assays were performed on sera from 10 patients with periodontai disease and corresponding matched controls. Previous whole-cell ELISAs had demonstrated significantly higher levels of IgG antibody towards P. gingivalis in serum from these patients compared with controls (6). The results are expressed as the percentage inhibition of BAAMC cleavage rate per /;1 of 10% serum in the assay and are shown in histogratn forrn in Fig. 5. In 7 of 10 pairs, the control sera exhibited a greater ability to inhibit the protease activity than the patient sera. Although the mean percentage inhibition was greater for control sera than patient, the difference was not significant. The ability of 3 serum protease inhibitors to inhibit the rate of BAAMC cleavage by the thiol-activated enzyme was examined using the individual inhibitors at 10%) of their concentrations reported in whole plasma: a2-macroglobulin, 4.2 mg/ml; al-antitrypsin, 4.0 mg/rnl; antithrombin III, 0.1 mg/rnl. A dose-dependent inhibition was produced by antithrombin III. Neither «2macroglobulin nor al-antitrypsin were able to inhibit the enzyme activity in the concentration range examined (Fig. 4b)-
8
Fig. 2. Degradation of serum proteins by P. gingivalis DCCS over time. A. Coomassie blue stained gel of samples after 0 h (lane 2); 6 h (3); 12 h (4); 24 h (5); and 30 h incubation (6). Lane 1: serum with no DCCS. B. Immunohlots of / = 0 (lanes 1,3,5,7) and / = 30 h samples (lanes 2,4,6,8) using antisera to: IgG gamma chains (lanes 1,2); kappa chains (3,4); transferrin (5,6) and alpha-1 antitrypsin (7,8).
Time(h) Fig. 3. Recognition of B. inlerinedius outer membrane antigens by serum incubated with P gingivalis DCCS for 0 (lane 1), 30 (2), 45 (3), 60 (4), 90 (5), 120 (6), 150 (7) and 180 min (8). A. Immonoblot developed using anti-kappa chain antisera. B. Immunoblot developed using anti-gamma chain antisera. C. Laser densitometry of arrowed bands.
The capacity of P. gingivalis whole cells to degrade human plasma proteins is frequently cited as a major virulence mechanism of the organism. In this investigation, the extent of serum protein proteolysis by culture supernatants of Pgingivalis was shown to correlate with the activity of the thiol-activated, trypsin-like enzyme of this organism. Little information is available on the free thiol concentration in the crevicular environment, although the production of mercaptans by oral Baeteroides during growth in vitro is documented (25) and
Serutn and P. gingivalis 100
X——X
213
lntil>ttlon/ul senm
Ca8e:Control Pair
Fig. 5. Capacity of serum from a ease:eontrol population of adult periodontitis to inhibit the cleavage of BAAMC by P. gingivatis DCCS. Cases (closed bars); controls (open bars).
hibitor(s). The contribution of neutralizing antibody was addressed by comVolume (yl) parison of the inhibitory capacity of Pig. 4. Effect of serum and serum proteinase inhibitors on BAAMC cleavage by concentrated serum samples drawn from a ease-conculture supernatant of P. gingivalis. A. 10% serum (open eircles); bovine serum albumin @ trol study of adult periodontitis (6) in 10% serum total protein concentration (closed eircles). B. ral-antilrypsin (X): a-2 macroglobthe expectation that, if inhibition of the ulin (closed eircles): anlithrombin III (open circles). All inhbitors used at 10% plasma eoneenprotease was a function of antibody, trations. then the patient serum samples would be more effective than the controls. No This investigation demonstrated an significant difference in the inhibition the capacity of mixed subgingival organisms to generate thiol compounds initial selective digestion of the heavy produced by case vs control serum was from plasma proteins has recently been chains of IgG but a retention of func- detected, implying that, if antibody reestablished (19). Plasma protein degra- tionally active fragments from the active with the protease was present, it dation itt vivo therefore may be depend- F(ab)2 region. The complete degrada- was not capable of neutralizing activity. ent not only on the full expression of tion of IgG to low-molecuiar-weight We have concluded, therefore, that an the trypsin-like enzyme by P. gingivalis peptides following incubation with explanation of the serum inhibition but also on the metabolic activities of whole cells of P. gingivalis is well estab- based solely on antibody is unlikely. We therefore addressed the possibility Co-colonizing organisms in subgingival lished (12, 24). Recently, a stepwise digestion of IgG by P. gittgivalis was de- of the presence in plasma of a protease plaque. Of the major serum proteins, albumin scribed in which the heavy chains were inhibitor with specificity for the trypand the immunoglobulin heavy chains initially cleaved to form 33,000 and sin-like enzyme. The main protease inWere the most susceptible to proteolysis. 11,000 Mr polypeptides while the light hibitors in human plasma are al-antiWhereas transferrin and light chains chains remained intact. The antigen- trypsin, a serine protease inhibitor and Were relatively resistant. It is perhaps binding ability of the light chains was a2-macroglobulin, which has a broad significant in terms of the physiology of not examined (10). In this investigation specificity for proteases from all 4 P. gitigivalis that albumin is degraded a 33,000 polypeptide immunoreactive classes. Neither of these anti-proteases more readily than transferrin by the thi- with anti-gamma chain antisera was inhibited the enzyme when examined at ol-activated protease. Although both also detected in the preliminary stages their concentrations in plasma. Howproteins provide amino acids for growth of serum digestion (data not shown). ever, in our investigation of the susceptiof the organism, degradation of albu- Our data suggest that, although the bilities of different serum proteins to min may also result in the liberation antigen-binding capability of IgG anti- hydrolysis we had observed that alof the haemin, which is scavenged and body is not affected by its initial interac- antitrypsin underwent a rapid decrease transported by this protein. The abso- tion with the trypsin-like enzyme, its Fc- in size to a lower-molecular-weight form lute haemin requirement for the growth mediated role in complement activation that remained stable to further degradaof P. gittgivalis and the effects of this and phagocytic cell function is severely tion. A similar observation was made by Nilsson et al. (17) for the interaction tetrapyrrole on the virulence of the or- impaired. ganism are well documented (16). ConThe most interesting observation was of purified Cl inhibitor with P. gingivalversely, the inorganic iron scavenged by the demonstration for the first time that is whole cells. Both of these protease transferrin does not support the growth the trypsin-like enzyme of P. gingivalis inhibitors are members of the serine of the organism in vitro (4). Thus a pref- can be inhibited by a component(s) of protease inhibitors (serpins) of human erential degradation of albumin over human serum. We considered 2 possible plasma: a superfamily of inhibitors with transferrin can be considered of benefit explanations for this phenomenon: the considerable amino acid sequence to the growth of P. gitigivalis in the gin- presence of neutrahzing antibodies and homology (5). The protease specificity gival crevice. the presence of a specific protease in- of the serpins is dictated by the amino 20
40
60
80 100
0
20
40
60 80 100
214
Fishburn et al.
acid residues at the reactive centre of the molecule (Pl-PT position). Binding of the protease to the reactive centre results in the formation of a complex with consequent inhibition of the enzyme. Under appropriate conditions, the complex can dissociate to yield a modified inhibitor of lower molecular weight due to scission of the PI-PI' bond. The decrease in molecular size we had observed for al-antitrypsin suggested that cleavage of the inhibitor may be occurring at or close to the PlPl' position on the molecule, and this led us to examine another member of the serpin family with a more appropriate reactive centre for the inhibition of the enzyme: antithrombin III. In antithrombin III the PI-PI' positions are occupied by arginine-serine. This serpin proved to be inhibitory when tested in the concentration range found in human plasma. It is unlikely that any non-complexed anti-thrombin III is present in serum following clotting and hence the inhibition we observed by serum probably resides in another inhibitor. Consequently we are currently examining the effects of other serpins with arginine residues at their reactive centre on purified preparations of the trypsin-like enzyme of P. gingivalis. There are a number of questions to be addressed in future investigations. First, the observed inhibition by antithrombin III, a serpin and hence, by definition, a specific inhibitor of serine proteases, is not consistent with the view, based largely on its thioi-dependence, that the trypsin-like enzyme of P. gitigivalis is a cysteine protease (18). Second, here we focused solely on culture supernatant enzyme activity. Serpin-inhibition studies of the multiple trypsin-like enzyme activities associated with outer membranes and vesicles of this organism (11) may help to characterize the number and nature of the enzyme activities produced by this important periodontai pathogen. In conclusion, the correlation of trypsin-like enzyme activity with serum protein degradation emphasizes the potential role of this factor in the pathogenicity of P. gingivalis. The differing susceptibilities of individual proteins suggests that even limited proteolysis of plasma proteins entering the gingival crevice may be both beneficial to the organism nutritionally and injurious to the host defences. However, the demonstration that plasma contains a specific inhibitor(s) of this activity may offer
an explanation why colonization by low levels of P. gingivalis can be tolerated by the host in the absence of destructive disease.
Acknowledgements
We thank Dr. P. Pemberton for advice and for providing the antithrombin III and al-antitrypsin preparations and Dr. J. A. C. Sterne for his help with the statistical treatment of the case-control serum data.
References 1. Carlone GM, Thomas ML, Rumschlag HS, Sottnek FO. Rapid microprocedure for isolating detergent insoluble outer membrane proteins from Haemophilus species. J Clin Microbiol 1986: 2: 293-296. 2. Carlsson J, Herrman BF, Hoflung JF, Sundqvist GK. Degradation of the human proteinase inhibitors alpha-1 antitrypsin and aIpha-2 macroglobulin by Bacteroides gingivalis. Infect Immun 1984: 43: 644-648. 3. Carlsson J, Hoflung JG, Sundvist GK. Degradation of albumin, haemopexin, haptoglobin and transferrin by blackpigmented Bacteroides speeies. J Med Microbiol 1984: 18: 39-46. 4. Carman RJ, Ramakrishnan MD, Harper FH, Curtis MA. Bacteroides gingivalis: role of haemin and iron in aetiology of destructive periodontitis. In: Borriello SP, Hardie JM, ed. Anaerobes in medieine and industry. Petersfield, UK: Wrightson Biochemcial (in press). 5. Carrell RW, Pemberton PA, Boswell DR. The serpins: evolution and adaptation in a family of protease inhibitors. Cold Spring Harbour Symposia Quantitative Biol 1987: 52: 527-535. 6. Curtis MA, Slaney JM, Carman RJ, et al. Serum IgG antibody response to antigens of presumed periodontai pathogens: a case-eontrol study using ELISA and Western hlot analysis. Microbial Ecol Health Dis (in press). 7. Cutress TW, Ainamo J, Sardo-Infirri J. The Community Periodontai Index of Treatment Needs (CPITN): procedure for population groups and individuals. Int Dent J 1987: 37: 222-233. 8. Ebersole JL, Taubman MA, Smith DJ, Frey DE. Human immune responses to oral mieroorganisms: patterns of systemic antibody levels to Bacteroides species. Infect Immun 1986: 51: 507513. 9. Geneo RJ, Slots J. Host responses in periodontai diseases. J Dent Res 1984: 63: 441^51.
10. Grenier D, Mayrand D, McBride BC. Further studies on the degradation of immunoglobulins by blaek-pigmented Bacteroides. Oral Microbiol Immunol 1989: 4: 12-18. 11. Grenier D, Chao G, McBride BC. Characterisation of sodium dodecyl sulfate-stable proteases by polyaerylamide gel eleetrophoresis. Infect Immun 1989: 57: 95-99. 12. Kilian M. Degradation of immunoglobulins Al, A2 and G by suspected periodontai pathogens. Infect Immun 1989: 57: 1868-1871. 13. Laemmli UK. Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature 1970: 227: 680-685. 14. Loe H, Silness J. Periodontai disease in pregnancy. I. Prevalence and severityAeta Odontol Scand 1963: 21: 523551. 15. Mann WV. The correlation of gingivitis, pocket depth and exudate from the gingival erevice. J Periodontol 1963: 34: 379-387. 16. McKee AS, MeDermid AS, Baskerville A, Barry-Dowsett A, Ellwood DC, Marsh PD. Effect of hemin on the physiology and virulenee of Bacteroides gingivalis W50. Infeet Immun 1986: 52: 349365. 17. Nilsson T, Carlsson J, Sundqvist G. Inactivation of key factors of the plasma proteinase cascade systems by Bacteroides gingivalis. Infect Immun 1985: 50: 467-471. 18. Ono M, Okuda K, Takazoe 1. Purification and characterisation of a thiolprotease from Bacteroides gingivalis strain 381. Oral Mierobiol Immunol 1987: 2: 77-81. 19. Persson S, Claesson R, Carlsson J. The eapacity of subgingival microbiotas to prodtice volatile sulfur compounds in human serum. Oral Microbiol Immunol 1989: 4: 169-172. 20. Shah HN, Williams RAD, Bowden G H , Hardie JM. Comparison of the biochemical properties of Bacteroides melaninogenicus from human dental plaque and other sites. J Appl Bacteriol 1976: 41: 473-492. 21. Slots J. Importance of black pigmentcd Bacteroides in human periodontai disease. In: Geneo RJ, Mergenhagen SE, ed. Host-parasite interactions in periodontai diseases. Washington, DC: Am Soc Microbiol, 1982: 2 7 ^ 5 . 22. Slots J, Bragd L, Wikstrom M, Dahlen G. The occurrence of Actinohacillus nctinomycetenicomitans, Bacteroides g'"' givalis and Baeteroides intermedius in destructive periodontai disease in adults. J Clin Periodontol 1986: 13: 576-577. 23. Slots J, Listgarten MA. Bacteroides gi"givalis, Bacteroides intermedius and Actinobacilhis actinoinvcetemcotiiitittts i" human periodontai diseases. J Clin Periodontol 1988: 15: 85-93.
Serutn and p. gingi-valis 24. Sundqvist G, Carlsson J, Herrmann B, Tarnvik A. Degradation of human immunoglobulins G and M and complement factors C3 and C5 by black-pigmented Bacteroides. J Med Microbiol 1985: 19: 85-94.
25. Tonzetich J, McBride BC. Charaeterisation of volatile sulphur production by pathogenic and non-pathogenic strains of oral Bactowfev. Arch Oral Biol 1981: 26: 963-969.
215
26. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitroeellulose sheets. Procedure and some applications, Proe Natl Acad Sci USA 1979: 76: 4350^354.
Degradation of plasma proteins by the trypsin-iike enzyme of Porphyromonas gingivalis and iniiibition of protease activity by a serine protease inhibitor of human piasma
C. S. Fishburn, J. M. Slaney, R. J. Carman, M. A. Curtis MRC Dental Research Unit, London Hospital Medical College, United Kingdom
Fishburn CS, Slaney JM, Cartnan RJ, Curtis MA. Degradation of plasma proteins by the trypsin-like enzyme o/Porphyromonas gingivalis atrd inhibition of protease Activity by a serine protease inhibitor of hutnan plastna. Oral Mierobiol Itmmmol 1991: 6: 209^215. The interaction between Porphyrotnotuis gingivalis culture supernatant and human serum was examined. Hydrolysis of the major serum proteins was thiol-dependent and correlated with the trypsin-like activity of the sample. Transferrin and igG 'ight chains were less susceptible to degradation than albumin and IgG heavy chains and partially degraded IgG retained antigen-binding capability. Serum inhibited the trypsin-like activity in a fluorimetric assay. The inhibition was shown to be independent of the level of IgG antibody reactive with whole cells of ^ gingivalis. Purified preparations of antithrombin III, a serine protease inhibitor, but not al-antitrypsin nor a2-rnacroglobulin inhibited the trypsin-like activity in the fluorimetric assay.
Porphyrotnotias (Baeteroides) gingivalis munoglobulins (10, 12, 24). Indeed, has been strongly implicated in the aeti- under the appropriate conditions, it apology of adult periodontitis (21, 23). pears that virtually all plasma proteins Cultural studies have demonstrated are susceptible to extensive degradation °oth an increased prevalence and an in- by enzyine(s) produced by P. gittgivalis creased median recovery of this organ- (2). In the subgingival environment, the 'sm in progressing lesions versus quies- extent of plasma protein degradation is cent sites (22) and many studies have determined not only by the numbers demonstrated significantly elevated and activity of protease-producing orSerum immunoglobulin G antibody ganisms but also the flow rate of exu'evels to P. gingivalis in adult perio- date through the crevice. Hence, at in"^ontitis patients compared with healthy flamed sites in the gingiva, where elevControls (8, 9). The precise molecular ated crevicular fluid flow rates would be ^echanisrns by which colonization by anticipated (15), only a limited digestioti ^- gitigivalis can lead to the tissue de- of plasma proteins by P gingivalis may struction characteristic of progressive occur. In such circumstances, the rela•destructive periodontitis are still poorly tive resistance to proteolysis of individUnderstood. However, much attention ual proteins becomes relevant to the •^as been focused upon the proteolytic pathogenic potential of the organism. activities produced by this organism. The purpose of this investigation was *Vhole cells of P. gitigivalis have been to examine the degradation of human shown to degrade plasma proteins in- serum proteins by P. gingivalis under volved in host defence, including iron conditions that permitted estimation of binding proteins (3), complement com- their differing susceptibilities and, in the Ponents (24), anti-proteases (2) and im- case of immunoglobulin G, the effect of
Key words: Porpliyromonas gingivatis; trypsin-iike enzyme; piasma proteolysis: proteaseinhibitor M. A. Curtis, MRC Dental Research Unit, London Hospitai Medical Coilege, 32 Newark Street, London El 2AA, UK Accepted for pubiieation September 4, 1990
limited proteolysis on antigen-binding capacity. During the course of these investigations we observed that serum was able to inhibit the thiol-dependant arginine amidolytic activity of this organism. The likely source of this inhibition was investigated using purified preparations of plasma anti-proteases. Material and methods Preparation of crude protease
P. gingivalis W83, a highly proteolytic strain, was inoculated from a 48-h horse blood agar plate into BM broth, a peptone/yeast medium supplemented with 5 mg haemin/1 (20). The inoculated broths were incubated anaerobically (80% Nj, 10% H,, 10% COj) for 6 d at 37°C. The supernatant fluid was collected after centrifugation (18,000 g, 30 min, 4°C) and then filter-sterilized (0.22-/mi ftlter). The supernatant enzyme activity was then concentrated by precipitation overnight at 4 X with
210
Fishburn et al.
ammonium sulphate to 70% saturation followed by resuspension of the pellet to one-tenth the original volume in 50 mM Tris (hydroxymethyl) methylamine (pH 7.2) and overnight dialysis against the same buffer. The dialysed concentrated culture supernatant (DCCS) was then lyophilized in 5-ml aliquots and stored at — 70°C until required, whereupon it was resuspended in the appropriate chilled buffer and used immediately. Enzyme activity assay and inhibition studies
Trypsin-like enzyme acitivity was assessed by measuring the rate of cleavage of the fluorimetric substrate Nbenzoyl-L-arginine 7-amido-4-methylcoumarin hydrochloride (BAAMC Sigma Chemical, Poole, UK). Assays were routinely performed in 3 ml 50 mM Tris HCI (pH 7.2, 10 mM CaClj, 1 mM cysteine HCI) containing 3.7 mM BAAMC at room temperature. Following addition of the sample, the rate of increase in relative fluorescence (i.e. rate of hydrolysis of BAAMC) was measured in a fluorimeter (SFM25; Kontron Instruments, Zurich, Switzerland; excitation: 380 nm; emission: 460 nm). Enzyme activity was expressed in units of relative fluorescence released per min. All experiments were performed in duplicate on at least 2 separate occasions and the results are presented as mean data. The standard deviations of the means did not exceed ±5% in each case. The effect of serum and purified protease inhibitors on enzyme activity was examined by pre-incubation of DCCS and the inhibitor for 5 min at room temperature followed by measurement of the rate of cleavage of BAAMC. Serum was used at a dilution of 10% in phosphate-buffered saline (PBS), pH 7.2 and the protease inhibitors, a2-macroglobulin (Sigma), al-antitrypsin and anti-thrombin III (both obtained from Dr P. Pemberton, Department of Haematology, Addenbrookes Hospital, Cambridge, UK) at 10% dilutions of their respective plasma concentrations. Albumin adjusted to the total protein concentration of serum was used as a control for competitive inhibition in the assay by serum proteins. The percentage inhibition was calculated relative to the activity of a control incubation of DCCS in the absence of inhibitor.
Preparation of Bacteroides intermedius outer membranes
Baeteroides intertnedius T588 was grown in BM broth for 48 h. Bacterial cells were harvested by centrifugation (10,000 g, 20 min, 4°C) and washed twice in sterile Tris buffer (50 mM, pH 7.2). After the second wash, the cell pellet was resuspended in l/50th of the original culture volume in Tris buffer and stored at -70°C until required. Outer membranes were prepared using the method of Carlone et al. (1). Briefly, the cells were sonicated in HEPES buffer (10 mM, pH 7.2) and centrifuged (10,000 g, 2 min, 4°C) to remove unbroken cells and debris. The crude membranes were then pelleted by centrifugation at 40,000 g for 60 min at 4°C. The outer membranes were obtained following selective solubilization of the inner membranes using sodium lauryl sarcosinate (Sigma - 1 % w/v in HEPES, 10 mM, pH 7.2). Periodontai patient and controi serum sampies
Cases (M = 10) were selected from patients attending a periodontai referral clinic and were defined as having a CPITN (7) score of 4 in one or more sextants, thus exhibiting a history of destructive periodontai disease. Controls {n = 10) were selected from patients attending other clinics for routine dental treatment and were defined as subjects whose worst CPITN code was 2 or less. Cases and controls were matched for age (+ 1 year), ethnic origin, gender and mean oral hygiene scores ( + 0.5 plaque index). Following collection the blood was allowed to clot at room temperature. The serum was then separated within 4 h of venipuncture and stored at — 70°C until required. Eiectrophoresis and Western blotting
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS/PAGE) was carried out according to the method of Laemmh (13) using 10% acrylamide/ bisacrylamide gels (8.0 cm x 5.5 cm x 1.5 mm) on a Midget gel system (LKB, Bromma, Sweden). Gels were run at 30 mA for approximately 1 h and then either stained for protein using 0.2% Coomassie brilliant blue (BDH Chemicals, Poole, UK) or eiectroblotted onto 0.45 ixm nitrocellulose membranes at 400 mA for 2.5 h (26).
The membranes were blocked in 5% BSA/PBS for 30 min at room temperature and then washed twice for 10 min in PBS. Membranes were incubated overnight in primary antisera at the appropriate dilution in 1% BSA/PBS and then washed 6 times for 15 min in 0.1 % Tween-20/PBS. Where necessary, they were incubated in horseradish peroxidase (HRPO)-labelled anti-species antibody for 2 h and then washed as before. The antisera and working dilutions used in this study were as follows: rabbit anti-albumin, 1:5000 (Behringwerke, Marburg, FRG); sheep anti-transferrin, 1:5000 (Unipath, Bedford, UK); sheep anti alpha-1-antitrypsin, 1:40,000 (Unipath); rabbit anti-human kappa light chain, 1:10,000; rabbit anti-human IgA alpha chain, 1:500; HRPO-conjugated swine anti-human IgG gamma chain, 1:1000; HRPO-conjugated swine antihuman IgM mu chain, 1:200; HRPOconjugated swine anti-rabbit IgG, 1:500; HRPO-conjugated swine antisheep IgG, 1:5000 (all Dakopatts Antisera, Glostrup, Denmark). Where rabbit anti-human kappa light chain was used as a secondary antibody, the dilution was 1:200 and the tertiary antibody was HRPO-conjugated swine antirabbit IgG as before. Incubations were carried out at room temperature with agitation. Membranes were given a final 10-min rinse in PBS before being developed in 0.05% (w/v) 3,3-diaminobenzidine tetrahydrochloride with 0.02% (v/v) hydrogen peroxide (BDH) and 0.03% (w/v) nickel chloride. Densitometric scanning of the immunoblots was performed using a laser densitometer (Ultroscan XL, LKB). To investigate the loss of function of human IgG treated with DCCS, the binding of a patient serum IgG antibodies to outer membrane antigens of B. intertnedius on a Western blot was assessed after incubation of the serum with DCCS. Serum and DCCS were incubated in a 1:5 ratio in the presence of 10 mM CaClj and 1 mM L-cys in a final volume of 4.5 ml. A 1:5 ratio of substrate to enzyme had previously been shown to give the optitnum rate of serum protein hydrolysis for a time course experiment at this thiol concentration. Samples (0.5 ml) were withdrawn at time intervals up to 3 h and dispensed into 9.5 ml BSA (2%)/PBS to prevent any further proteolysis of the serum. Outer membrane preparations of S. intertnedittswere run on 10% gels. Western-blotted onto nitrocellulose
Serutn and p. gingivalis
niembranes, cut, anode to cathode, into 4-mm strips and blocked as described. Duplicate strips were incubated for 2 h in each sample of protease-digested serum. After washing, one strip from each time point was incubated in HRPO-conjugated swine anti-human IgG gamma chain, and the other in rabbit anti-human kappa light chain followed by HRPO-conjugated swine antirabbit IgG. Incubations, washes and development in diaminobenzidine were performed as described earlier. Results Demonstration of thioi-dependence
Both the activity of the trypsin-like enzyme, assessed by rate of cleavage of BAAMC, and the degradation of serum proteins by DCCS critically depended On the concentrations of thiol reagents. In the absence of exogenous thiol groups, no cleavage of BAAMC was detected. Introduction of 1 mM L-cys into the assay produced activation of the enzyme to approximately 50% of its maximal activity, which was attained in the presence of 50 mM L-cys. Similarly, in the absence of L-cys, no degradation of serum proteins was observed via SDS/PAGE when DCCS and serum Were incubated at a ratio of 5:1 for 60 min. Addition of increasing amounts of the thiol caused a progressive increase in serum protein degradation (Fig. 1). These findings are consistent with the BAAMC-cleaving activity and serum protein degradation being functions of the same thiol-dependent enzyme. A more striking example of the thiol dependence of the serum protein degradation was obtained when proteolysis Was allowed to proceed in the presence of excess SDS/PAGE reducing sample buffer, which contained 5% 2-mercaptoethanol. DCCS and serum mixtures Were prepared varying in ratio from 1:1 to 5:1 with no added thiol. The mixtures Were then incubated for 3 h at 37'C and then immediately dispensed into excess SDS/PAGE sample buffer that had been preheated to 100°C. A duplicate series was incubated for 3 h at 37°C, dispensed into SDS/PAGE sample buffer at room temperature and then immediately brought to 100°C in an incubator. The resulting protein profiles Were examined via SDS/PAGE. The Samples that had been inactivated after the 3 h incubation by hot sample buffer showed no degradation. Those that had been heat-inactivated by heating from
211
room temperature up to 100°C in the presence of SDS/PAGE sample buffer showed a progressive loss of serum protein with increasing ratio of DCCS:serum (data not shown). Incubation of a DCCS/serum mixture in the presence of individual components of the SDS/PAGE buffer (3% SDS and 0.5 M Tris HCI, pH 7.2) for 10 min followed by inactivation in hot sample buffer had no effect on proteolysis. However, the same procedure in 5% 2rnercaptoethanol produced almost complete degradation of all serum proteins. Thus, to avoid any confusion in later experiments between proteolysis taking place during incubation at 37°C and that taking place as a result of heating in sample buffer, all samples were put directly into hot sample buffer in a 100°C water bath.
run on a 10% polyacrylamide gel lane) and stained with Coomassie Blue (Fig. 2A). Although the gradual degradation of serum proteins was clear, the proteins appeared to exhibit differing susceptibilities towards the protease activity. Bands at the levels of 66 kDa and 55 kDa disappeared with time, whereas those at levels 77 kDa and 25 kDa (arrowed) persisted for up to 30 h. A band at approximately 60 kDa, presumably the product of the degradation of a higher-molecular-weight component (most likely albumin), was generated after 6 h incubation and increased in intensity up to 24 h. By 5 d, no bands were visible. The identity of individual proteins was established by immunoblotting samples taken at different time points and probing the blots with monospecific antisera. The components at 66 kDa and 55 kDa were identified as albumin Susceptibility of different serum proteins and IgG gamma chains, both of which to proteoiysis appeared to be highly susceptible to Samples taken at different time points proteolysis. Similar rates of disappearfrom an incubation of DCCS and serum ance were found for IgM mu chains and (5:1) in the presence of 1 mM L-cys were IgA alpha chains. The relatively resist-
Cysteine (rnM) Fig. 1. Thiol-dcpcndcncc of P. gingivalis culture supernatant trypsin-like enzyme activity (expressed as units of relative fluorescence released per minute in BAAMC assay). Insert shows SDS/PAGE of serum incubated with concentrated supernatant at a ratio 1:5 for 60 min in the presence of: 0 mM cysteine (lane 1): 1 mM (2): 10 niM (3) and 50 mM eysteine (4). Numhers in the right-hand margin refer to migration positions of standard-moleeularweight proteins: ovotransferrin (77,000); albumin (66,000); ovalbumin (45,000): and carbonic anhydrase (29,000).
212
Fishburn et al.
ant proteins at 77 kDa and 25 kDa were identified as transferrin and IgG kappa chains. Although native al-antitrypsin underwent a rapid conversion to a lower-molecular-weight form, this product was stable to further proteolysis and was still detectable irnmunochemically after 5 d of incubation. Western blots of ; = 0 and / = 30 h samples developed using anti-gamma chain, anti-kappa chain, anti-transferrin and anti-a 1-antitrypsin antisera are shown in Fig. 2B. Antigen-binding ability to DCCS-treated serum igG
The observation that kappa chains were relatively resistant to degradation by DCCS led us to examine the antigenbinding ability of serum previously incubated with DCCS. The binding of IgG to B. intermedius outer membrane proteins separated via SDS/PAGE and then transferred to nitrocellulose is shown in Fig. 3. When antibody binding was assessed using anti-gamma chain antiserum, the amount of reactive antibody decreased with increasing tirne of incubation of the serum with DCCS
(Fig. 3b, c). However when anti-kappa chain antiserum was used, the amount of immunoreactivity was undiminished, even in serum which had been incubated for 3 h with DCCS (Fig. 3a and 3c). Serum Inhibition studies
The effect of serum on the thiol-activated enzyme activity of DCCS was examined by measuring the rate of cleavage of the fluorimetric substrate, BAAMC, in the presence of increasing volumes of serum. To establish the level of inhibition produced in the assay as a result of simple competition with serum proteins, parallel assays were carried out using bovine serum albumin (BSA), shown earlier to be susceptible to degradation. A protein assay revealed the average concentration of protein in 20
776645-
29-
1 2
7766-1
3 4
5
6
7 8
457766-
29-
45-
•
•
•
%
•
•
29-
o—'•>•—o 7 7 -
• ^ . •.
6 6 -
; ,
4 5 -
«»,
,
^. ,. r „
'29-
'•' ' \,
..'^••.
:
. ';•
' :'•:
Discusssion
, ,_**:'.,",
. , - : . ; | | | | . ^ . - 3 ,•• .... : : : ' • : - m .
1
2
3
4" 5
6
7
samples of serum to be 74 mg/ml; BSA was made up in PBS to the same concentration. Enzyme activity was expressed as a percentage of the activity in a serum-free control (Fig. 4a). A dosedependent inhibition of BAAMC cleavage was produced by serum; this exceeded the level of inhibition produced by simple competition as a consequence of the introduction of an alternative substrate (in this case BSA) into the assay. To investigate the possibility that the protease inhibition might be the result of anti-protease antibodies in serum, inhibition assays were performed on sera from 10 patients with periodontai disease and corresponding matched controls. Previous whole-cell ELISAs had demonstrated significantly higher levels of IgG antibody towards P. gingivalis in serum from these patients compared with controls (6). The results are expressed as the percentage inhibition of BAAMC cleavage rate per /;1 of 10% serum in the assay and are shown in histogratn forrn in Fig. 5. In 7 of 10 pairs, the control sera exhibited a greater ability to inhibit the protease activity than the patient sera. Although the mean percentage inhibition was greater for control sera than patient, the difference was not significant. The ability of 3 serum protease inhibitors to inhibit the rate of BAAMC cleavage by the thiol-activated enzyme was examined using the individual inhibitors at 10%) of their concentrations reported in whole plasma: a2-macroglobulin, 4.2 mg/ml; al-antitrypsin, 4.0 mg/rnl; antithrombin III, 0.1 mg/rnl. A dose-dependent inhibition was produced by antithrombin III. Neither «2macroglobulin nor al-antitrypsin were able to inhibit the enzyme activity in the concentration range examined (Fig. 4b)-
8
Fig. 2. Degradation of serum proteins by P. gingivalis DCCS over time. A. Coomassie blue stained gel of samples after 0 h (lane 2); 6 h (3); 12 h (4); 24 h (5); and 30 h incubation (6). Lane 1: serum with no DCCS. B. Immunohlots of / = 0 (lanes 1,3,5,7) and / = 30 h samples (lanes 2,4,6,8) using antisera to: IgG gamma chains (lanes 1,2); kappa chains (3,4); transferrin (5,6) and alpha-1 antitrypsin (7,8).
Time(h) Fig. 3. Recognition of B. inlerinedius outer membrane antigens by serum incubated with P gingivalis DCCS for 0 (lane 1), 30 (2), 45 (3), 60 (4), 90 (5), 120 (6), 150 (7) and 180 min (8). A. Immonoblot developed using anti-kappa chain antisera. B. Immunoblot developed using anti-gamma chain antisera. C. Laser densitometry of arrowed bands.
The capacity of P. gingivalis whole cells to degrade human plasma proteins is frequently cited as a major virulence mechanism of the organism. In this investigation, the extent of serum protein proteolysis by culture supernatants of Pgingivalis was shown to correlate with the activity of the thiol-activated, trypsin-like enzyme of this organism. Little information is available on the free thiol concentration in the crevicular environment, although the production of mercaptans by oral Baeteroides during growth in vitro is documented (25) and
Serutn and P. gingivalis 100
X——X
213
lntil>ttlon/ul senm
Ca8e:Control Pair
Fig. 5. Capacity of serum from a ease:eontrol population of adult periodontitis to inhibit the cleavage of BAAMC by P. gingivatis DCCS. Cases (closed bars); controls (open bars).
hibitor(s). The contribution of neutralizing antibody was addressed by comVolume (yl) parison of the inhibitory capacity of Pig. 4. Effect of serum and serum proteinase inhibitors on BAAMC cleavage by concentrated serum samples drawn from a ease-conculture supernatant of P. gingivalis. A. 10% serum (open eircles); bovine serum albumin @ trol study of adult periodontitis (6) in 10% serum total protein concentration (closed eircles). B. ral-antilrypsin (X): a-2 macroglobthe expectation that, if inhibition of the ulin (closed eircles): anlithrombin III (open circles). All inhbitors used at 10% plasma eoneenprotease was a function of antibody, trations. then the patient serum samples would be more effective than the controls. No This investigation demonstrated an significant difference in the inhibition the capacity of mixed subgingival organisms to generate thiol compounds initial selective digestion of the heavy produced by case vs control serum was from plasma proteins has recently been chains of IgG but a retention of func- detected, implying that, if antibody reestablished (19). Plasma protein degra- tionally active fragments from the active with the protease was present, it dation itt vivo therefore may be depend- F(ab)2 region. The complete degrada- was not capable of neutralizing activity. ent not only on the full expression of tion of IgG to low-molecuiar-weight We have concluded, therefore, that an the trypsin-like enzyme by P. gingivalis peptides following incubation with explanation of the serum inhibition but also on the metabolic activities of whole cells of P. gingivalis is well estab- based solely on antibody is unlikely. We therefore addressed the possibility Co-colonizing organisms in subgingival lished (12, 24). Recently, a stepwise digestion of IgG by P. gittgivalis was de- of the presence in plasma of a protease plaque. Of the major serum proteins, albumin scribed in which the heavy chains were inhibitor with specificity for the trypand the immunoglobulin heavy chains initially cleaved to form 33,000 and sin-like enzyme. The main protease inWere the most susceptible to proteolysis. 11,000 Mr polypeptides while the light hibitors in human plasma are al-antiWhereas transferrin and light chains chains remained intact. The antigen- trypsin, a serine protease inhibitor and Were relatively resistant. It is perhaps binding ability of the light chains was a2-macroglobulin, which has a broad significant in terms of the physiology of not examined (10). In this investigation specificity for proteases from all 4 P. gitigivalis that albumin is degraded a 33,000 polypeptide immunoreactive classes. Neither of these anti-proteases more readily than transferrin by the thi- with anti-gamma chain antisera was inhibited the enzyme when examined at ol-activated protease. Although both also detected in the preliminary stages their concentrations in plasma. Howproteins provide amino acids for growth of serum digestion (data not shown). ever, in our investigation of the susceptiof the organism, degradation of albu- Our data suggest that, although the bilities of different serum proteins to min may also result in the liberation antigen-binding capability of IgG anti- hydrolysis we had observed that alof the haemin, which is scavenged and body is not affected by its initial interac- antitrypsin underwent a rapid decrease transported by this protein. The abso- tion with the trypsin-like enzyme, its Fc- in size to a lower-molecular-weight form lute haemin requirement for the growth mediated role in complement activation that remained stable to further degradaof P. gittgivalis and the effects of this and phagocytic cell function is severely tion. A similar observation was made by Nilsson et al. (17) for the interaction tetrapyrrole on the virulence of the or- impaired. ganism are well documented (16). ConThe most interesting observation was of purified Cl inhibitor with P. gingivalversely, the inorganic iron scavenged by the demonstration for the first time that is whole cells. Both of these protease transferrin does not support the growth the trypsin-like enzyme of P. gingivalis inhibitors are members of the serine of the organism in vitro (4). Thus a pref- can be inhibited by a component(s) of protease inhibitors (serpins) of human erential degradation of albumin over human serum. We considered 2 possible plasma: a superfamily of inhibitors with transferrin can be considered of benefit explanations for this phenomenon: the considerable amino acid sequence to the growth of P. gitigivalis in the gin- presence of neutrahzing antibodies and homology (5). The protease specificity gival crevice. the presence of a specific protease in- of the serpins is dictated by the amino 20
40
60
80 100
0
20
40
60 80 100
214
Fishburn et al.
acid residues at the reactive centre of the molecule (Pl-PT position). Binding of the protease to the reactive centre results in the formation of a complex with consequent inhibition of the enzyme. Under appropriate conditions, the complex can dissociate to yield a modified inhibitor of lower molecular weight due to scission of the PI-PI' bond. The decrease in molecular size we had observed for al-antitrypsin suggested that cleavage of the inhibitor may be occurring at or close to the PlPl' position on the molecule, and this led us to examine another member of the serpin family with a more appropriate reactive centre for the inhibition of the enzyme: antithrombin III. In antithrombin III the PI-PI' positions are occupied by arginine-serine. This serpin proved to be inhibitory when tested in the concentration range found in human plasma. It is unlikely that any non-complexed anti-thrombin III is present in serum following clotting and hence the inhibition we observed by serum probably resides in another inhibitor. Consequently we are currently examining the effects of other serpins with arginine residues at their reactive centre on purified preparations of the trypsin-like enzyme of P. gingivalis. There are a number of questions to be addressed in future investigations. First, the observed inhibition by antithrombin III, a serpin and hence, by definition, a specific inhibitor of serine proteases, is not consistent with the view, based largely on its thioi-dependence, that the trypsin-like enzyme of P. gitigivalis is a cysteine protease (18). Second, here we focused solely on culture supernatant enzyme activity. Serpin-inhibition studies of the multiple trypsin-like enzyme activities associated with outer membranes and vesicles of this organism (11) may help to characterize the number and nature of the enzyme activities produced by this important periodontai pathogen. In conclusion, the correlation of trypsin-like enzyme activity with serum protein degradation emphasizes the potential role of this factor in the pathogenicity of P. gingivalis. The differing susceptibilities of individual proteins suggests that even limited proteolysis of plasma proteins entering the gingival crevice may be both beneficial to the organism nutritionally and injurious to the host defences. However, the demonstration that plasma contains a specific inhibitor(s) of this activity may offer
an explanation why colonization by low levels of P. gingivalis can be tolerated by the host in the absence of destructive disease.
Acknowledgements
We thank Dr. P. Pemberton for advice and for providing the antithrombin III and al-antitrypsin preparations and Dr. J. A. C. Sterne for his help with the statistical treatment of the case-control serum data.
References 1. Carlone GM, Thomas ML, Rumschlag HS, Sottnek FO. Rapid microprocedure for isolating detergent insoluble outer membrane proteins from Haemophilus species. J Clin Microbiol 1986: 2: 293-296. 2. Carlsson J, Herrman BF, Hoflung JF, Sundqvist GK. Degradation of the human proteinase inhibitors alpha-1 antitrypsin and aIpha-2 macroglobulin by Bacteroides gingivalis. Infect Immun 1984: 43: 644-648. 3. Carlsson J, Hoflung JG, Sundvist GK. Degradation of albumin, haemopexin, haptoglobin and transferrin by blackpigmented Bacteroides speeies. J Med Microbiol 1984: 18: 39-46. 4. Carman RJ, Ramakrishnan MD, Harper FH, Curtis MA. Bacteroides gingivalis: role of haemin and iron in aetiology of destructive periodontitis. In: Borriello SP, Hardie JM, ed. Anaerobes in medieine and industry. Petersfield, UK: Wrightson Biochemcial (in press). 5. Carrell RW, Pemberton PA, Boswell DR. The serpins: evolution and adaptation in a family of protease inhibitors. Cold Spring Harbour Symposia Quantitative Biol 1987: 52: 527-535. 6. Curtis MA, Slaney JM, Carman RJ, et al. Serum IgG antibody response to antigens of presumed periodontai pathogens: a case-eontrol study using ELISA and Western hlot analysis. Microbial Ecol Health Dis (in press). 7. Cutress TW, Ainamo J, Sardo-Infirri J. The Community Periodontai Index of Treatment Needs (CPITN): procedure for population groups and individuals. Int Dent J 1987: 37: 222-233. 8. Ebersole JL, Taubman MA, Smith DJ, Frey DE. Human immune responses to oral mieroorganisms: patterns of systemic antibody levels to Bacteroides species. Infect Immun 1986: 51: 507513. 9. Geneo RJ, Slots J. Host responses in periodontai diseases. J Dent Res 1984: 63: 441^51.
10. Grenier D, Mayrand D, McBride BC. Further studies on the degradation of immunoglobulins by blaek-pigmented Bacteroides. Oral Microbiol Immunol 1989: 4: 12-18. 11. Grenier D, Chao G, McBride BC. Characterisation of sodium dodecyl sulfate-stable proteases by polyaerylamide gel eleetrophoresis. Infect Immun 1989: 57: 95-99. 12. Kilian M. Degradation of immunoglobulins Al, A2 and G by suspected periodontai pathogens. Infect Immun 1989: 57: 1868-1871. 13. Laemmli UK. Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature 1970: 227: 680-685. 14. Loe H, Silness J. Periodontai disease in pregnancy. I. Prevalence and severityAeta Odontol Scand 1963: 21: 523551. 15. Mann WV. The correlation of gingivitis, pocket depth and exudate from the gingival erevice. J Periodontol 1963: 34: 379-387. 16. McKee AS, MeDermid AS, Baskerville A, Barry-Dowsett A, Ellwood DC, Marsh PD. Effect of hemin on the physiology and virulenee of Bacteroides gingivalis W50. Infeet Immun 1986: 52: 349365. 17. Nilsson T, Carlsson J, Sundqvist G. Inactivation of key factors of the plasma proteinase cascade systems by Bacteroides gingivalis. Infect Immun 1985: 50: 467-471. 18. Ono M, Okuda K, Takazoe 1. Purification and characterisation of a thiolprotease from Bacteroides gingivalis strain 381. Oral Mierobiol Immunol 1987: 2: 77-81. 19. Persson S, Claesson R, Carlsson J. The eapacity of subgingival microbiotas to prodtice volatile sulfur compounds in human serum. Oral Microbiol Immunol 1989: 4: 169-172. 20. Shah HN, Williams RAD, Bowden G H , Hardie JM. Comparison of the biochemical properties of Bacteroides melaninogenicus from human dental plaque and other sites. J Appl Bacteriol 1976: 41: 473-492. 21. Slots J. Importance of black pigmentcd Bacteroides in human periodontai disease. In: Geneo RJ, Mergenhagen SE, ed. Host-parasite interactions in periodontai diseases. Washington, DC: Am Soc Microbiol, 1982: 2 7 ^ 5 . 22. Slots J, Bragd L, Wikstrom M, Dahlen G. The occurrence of Actinohacillus nctinomycetenicomitans, Bacteroides g'"' givalis and Baeteroides intermedius in destructive periodontai disease in adults. J Clin Periodontol 1986: 13: 576-577. 23. Slots J, Listgarten MA. Bacteroides gi"givalis, Bacteroides intermedius and Actinobacilhis actinoinvcetemcotiiitittts i" human periodontai diseases. J Clin Periodontol 1988: 15: 85-93.
Serutn and p. gingi-valis 24. Sundqvist G, Carlsson J, Herrmann B, Tarnvik A. Degradation of human immunoglobulins G and M and complement factors C3 and C5 by black-pigmented Bacteroides. J Med Microbiol 1985: 19: 85-94.
25. Tonzetich J, McBride BC. Charaeterisation of volatile sulphur production by pathogenic and non-pathogenic strains of oral Bactowfev. Arch Oral Biol 1981: 26: 963-969.
215
26. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitroeellulose sheets. Procedure and some applications, Proe Natl Acad Sci USA 1979: 76: 4350^354.
