Oral Mierobiol Immunol 1991: 6: 216-220
Complement factor D-like activity of Porphyromonas gingivaiis W83
Harvey A. Schenkein Department of Periodontics, School of Dentistry, Virginia Commonwealtfi University, Richmond, Virginia, USA
Sehenkein HA. Complement faetor D-like aetivity o/Porphyromonas gingivalis W83. Oral Mierobiol Immunol 1991: 6: 216-220. Porpliyromonas gitigivalis is a proteolytic gram-negative anaerobic bacterium that is frequently isolated from lesions of human periodontai disease. Previous studies have shown that P. gingivalis strain W83 inactivates C3 in pooled normal human serum (NHS) by a mechanism that is inhibitable by EDTA, yet it degrades purified complement proteins by a mechanism that is not EDTA-inhibitable. Furthermore, during complement activation, only a small number of C3 molecules accumulate on the surface of this organism unless the bacteria are treated with the protease inhibitor TLCK prior to complement activation. The hypothesis was tested that P. gitigivalis W83 contains protease activity mimicking that of complement factor D, thus enabling it to activate C3 in serum without significant C3 accumulation on the cell surface. It was first noted that incubation of P. gingivalis W83 in absorbed human serum that was depleted of faetor D resulted in C3 consumption that was reversed in the presence of the protease inhibitor TLCK. To directly demonstrate that factor B-dependent C3 consumption occurs in the absence of factor D, P. gingivalis W83 was incubated with purified C3 or a mixture of C3 and B. Although some proteolysis of C3 was noted, increased C3 consumption was noted in mixtures containing both C3 and B. This increment in C3 consumption was inhibited by both EDTA and TLCK. Furthermore, the addition of purified factor H to this mixture inhibited the increment in C3 consumption, indicating that a C3 convertase was probably formed. Following incubation of P. gingivalis W83 in a mixture of C3 + B containing '"I-C3, cleavage of '-H-C3 and formation of C3b was detectable by SDS-PAGE and autoradiography; C3 cleavage was inhibited by addition of H. This demonstrates the activity of a functional C3-cleaving enzyme in the absence of factor D that, like the alternative pathway C3 convertase, depends on factor B and Mg++ and is inhibited by factor H. Thus, P. gingivalis proteases that are inhibitable by TLCK substitute for factor D and initiate C3 consumption in serum, using factor B of the alternative complement pathway.
Accepted for publication November 8, 1990
Porphyromonas gingivalis, a gram-nega- ence the interaction of this organism tive anaerobic bacterium, is thought to with the complement system via exbe an important pathogen in various pression of its proteolytic eapacity. It forms of periodontai disease (14). In ad- has been demonstrated, for example, dition to its presence in lesions of this that some strains of P. gittgivalis will disease and its capacity to induce perio- proteolytically degrade serum compledontitis in experimental animal models, ment and individual complement prostrains of this organism have been teins. found to display many pathogenic Some strains of P. gingivalis have mechanisms of particular relevance to been described to be "invasive", as they the destruction of soft and hard connec- cause abscess formation and death tive tissues. Of particular relevance is when introduced into animal models (8, the proteolytic capacity of this species; 9, 19). We have examined one such numerous proteases of various speci- strain {P. gingivalis W83) and discovered ficities have been described and purified that, despite its capacity to activate (1,2, 18,21). serum complement (as demonstrated by Previous studies from our laboratory EDTA-inhibitable depletion of fluid and others (12, 15) have demonstrated phase hemolytic activity despite the failthat strains of P. gingivalis may influ- ure of EDTA to inhibit its proteolytic
activity against purified complement proteins), little C3 accumulates on its surface. Furthermore, treatment of the bacteria with the serine protease inhibitor TLCK prior to incubation with serum permits the accumulation of C3 on the surface in the form of C3b and iC3b (13). Thus, this organism may protect itself from the antibacterial effects of complement by failing to accumulate or by proteolytically removing complement-derived opsonins during complement activation. In addition, since little C3 is detectable on its surface during incubation with serum, it appears that this organism may activate the alternative complement pathway via an atypical activation mechanism. In the studies described here, we
Key words: Porptiyromonas gingivatis] complement: periodontai disease: factor D Harvey A. Schenkein, Virginia Commonwealth University, School of Dentistry, MCV Station Box 566, Richmond, VA 23298, USA
Complement activation by P. gingivalis examined alternative pathway activation by P. gitigivalis W83. This was of interest since activation of the alternative complement pathway results in accumulation of C3 fragments, in particular C3b and iC3b, on the surface of the activating particle. C3b deposition in turn permits formation of a particlebound C3 convertase. Factor D, a serine esterase found in a proteolytically active forrn in serum, participates in this process by cleavage of its substrate factor B when factor B is associated with C3b in solution or on the activating surface (6). Cleavage of B to form the Bb fragment results in the formation of the alternative pathway convertase C3bBb. Thus, D is required for activation of the alternative complement pathway. The surface of the activating particle participates in alternative pathway activation by providing protection from dissociation or decay of the C3 convertase and allows accelerated accumulation of C3b and thus the C3 convertase on its surface. It was thus of interest to determine how P. gingivalis W83 activates the alternative pathway without accumulating C3b at the cell surface. Since factor D is an active serine esterase with specificity for C3b-bound factor B, we hypothesized that P. gitigivalis W83 proteases might mimic the activity of factor 0 and thus participate in alternative pathway activation by this organism by bypassing the usual requirement for deposition of C3b and subsequent association of C3b with factor B on the activating surface prior to its cleavage by factor D. We found that P. gingivalis W83 activates the alternative pathway in serum by a mechanism independent of factor 0 (but dependent on factor B and Mg++) and that a bacterial protease or proteases inhibitable by the serine esterase inhibitor TCLK may substitute for factor D during activation.
NaCl) that had been deaerated by boiling. Bacteria were quantified after washing by determining the OD(,so of suspensions of the washed bacteria and comparing this with previously determined standards of known concentrations. Complement components
Purified C3 was prepared as described by Tack et al. (17) with the addition of an immunoadsorption step to remove traces of C5, H, and IgG; purity was verified by polyacrylamide gel eleetrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and by hemolytic assay. Factor B was purified as described by Kerr (4); faetor H was isolated as described in (20). SDS-PAGE was performed by the method of Laemmli (5) using 10% separation gels. Hemolytic titrations for quantification of C3 (11) were performed as described previously. Preserved sheep erythrocytes and antisheep erythrocyte hemolysin were purchased from Diamedix, Miami, FL. Guinea pig Cl (10), human C4 (3), human C5 (16), and human C2 (4) were prepared as previously described. C3 was radioiodinated to a specific activity of 0.3-0.5 /uCi/ftg using Iodobeads (Pierce Biochemicals, Rockford, IL) according the manufacturer's instructions. Unbound iodine was removed by passing the preparation through a column containing Sephadex G25. The resulting '-'I-C3 preparations retained 85% or more of their original hemolytic activity, and were quantified by single radial immunodiffusion (7). The protease inhibitor TLCK was purchased from Sigma Chemical (St. Louis, MO).
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yield a final concentration of 0.01 M EDTA (EDTA serum). To measure the inactivation of C3 hemolytic function, bacteria were washed and pelleted by centrifugation, and serum reagent or mixtures of purified serum proteins were added for the indicated period of time. Following incubation, the reaction mixtures were centrifuged and the supernatants were removed and placed on ice or frozen until assayed. For determination of the effect of protease inhibitor on C3 consumption, bacterial suspensions were first suspended at 1 X 10'' cells/ml in VBS containing the indicated concentration of inhibitor for 30 min at 37°C, Serum mixtures were then added and procedures were carried out as indicated above. Demonstration of C3 fragments
To demonstrate the C3 fragments formed upon incubation with P. gingivalis W83, '-^I-C3 was added to supernatants of reaction mixtures, from which bacteria were removed by centrifugation. Following incubation, aliquots of the mixtures were removed and analyzed by SDS-PAGE in 4-15% gradient separation gels (5) followed by autoradiography. Statistical analysis
Experimental data were analyzed for significance using Student's /-test. Results C3 consumption by P. gingivaiis W83 in human serum and D-deficient serum
Pooled human serum (NHS) was first extensively absorbed with P. gingivalis as described above to remove specific Bacterial incubations antibodies. Incubation of P. gingivalis Bacteria were suspended at a final con- W83 with absorbed Mg-EGTA'^-NHS centration of 5 X 10* particles/ml in the significantly depleted C3 hemolytic acMaterial and methods appropriate serum reagent (final serum tivity; the depletioti of C3 function was Bacterial strains dilution 1:3). The buffers for these ex- inhibited by EDTA (Fig. 1), a result P. gingivalis strain W83 and Baeteroides periments contained either the chelating consistent with the capacity to activate intertneditts 8944 were obtained from agent EGTA (to remove Ca++ and the alternative complement pathway. A Dr. L. V. H. Moore, Virginia Polytech- therefore inhibit the classical comple- portion of the P. gingivalis-ahsorbed nic Institute and State University, ment pathway) or EDTA (to remove serum was depleted of factor D by gel Blacksburg, VA. They were cultured an- both Ca++ and Mg"*'"' and thus inhibit filtration. The factor D-depleted serum aerobically for 18 h in brain-heart in- all complement activity). Diluent buf- demonstrated no hemolytic activity in fusion medium (Carr-Scarborough fers were either VBS containing suf- the rabbit CH50 assay, whereas unMierobiologicals, Stone Mountain, ficient EGTA and MgCU to yield a final treated absorbed serum regularly proGA), and then washed 3 times in ver- concentration of 0.01 M EGTA and duced a titer of 50-60 RCH50 units/ml. onal-buffered saline (VBS, 0.01 M ver- 0.008 M Mg++ (Mg-EGTA serum) or Incubation of the factor D-depleted onal buffer, pH 7.5, containing 0.13 M VBS containing sufficient EDTA to serum with B. intertnedius 8944, a pro-
218
Sehenkein 100 -
hibited by EDTA but proceeds in the absence of factor D.
80 -
s
Effect of TLOK on C3 consumption 60 -
TLCK is a serine esterase inhibitor that we have shown inhibits the ability of P. "M gitigivalis W83 proteases to block C3 40 0} EC accumulation on the surface of this strain (13). To assess the capacity of 20 TLCK to influence C3 consumption in serum, P. gingivalis W83 was -first treated with the inhibitor (10 mM) for NHS-EDTA NHS-EGTA RD-EDTA RD-EGTA RD-TLCK 30 min prior to addition of serum. When P. gingivalis W83 was first treated Fig. 1. Consumption of C3 in NHS and factor D-deficient serum (RD) hy P gingivalis W83. with TLCK prior to incubation in factor Sera at a dilution of 1 :3 in Mg-EGTA buffer were incubated with bacteria (5 x lO'/ml) in the D-depleted serum, substantial inhipresence or absence of 10 mM EDTA for 20 min at 37°C. RD-TLCK indicates that sera were bition of C3 consumption was noted diluted in Mg-EGTA buffer containing 10 mM TLCK. Following incubation, bacteria were (Fig. 1), indicating that a bacterial proremoved by centrifugation and C3 titers were determined by hemolytic assay. The results are expressed as percentage residual titer vs matched controls incubated in the absence of bacteria. tease may be required for C3 consumpOne standard deviation (SD) is shown by the error bars for 3 replicate experiments. tion in the absence of factor D. Thus, it appears that activation of the alternative complement pathway by P teolytic organism previously shown to way hemolytic activity of the factor D- gitigivalis W83 occurs in the absence of activate the alternative complement depleted serum reagent, C3 consump- factor D and depends on B and Mg^^pathway, resulted in no C3 consump- tion occurred during incubation with P. Therefore, P. gitigivalis W83 proteases tion, further verifying its lack of alterna- gingivalis W83 (Fig. 1). This activity re- may provide D-like activity in alternative pathway activity. In addition, a por- quired the presence of divalent cations, tive pathway activation. tion of the absorbed serum pool was since little C3 consumption occurred in heated to 50°C to remove factor B activ- the presence of EDTA. Additionally, inity; this reagent also displayed no hemo- cubation of P. gingivalis W83 in B-de- Demonstration of B-dependent C3 lytic activity against rabbit erythrocytes pleted serurn failed to deplete C3 activ- consumption with purified proteins unless reconstituted with factor B. ity. These experiments indicated that C3 To demonstrate that B-dependent C3 Despite the lack of alternative path- consumption by P. gittgivalis W83 is in- consumption by P. gingivalis W83 occurs, experiments using mixtures of purified components were carried out. Since P. gingivalis W83 readily proteo100 H lyzes purified solutions of complement proteins (12), it was necessary to dilute the complement reagents in buffer containing BSA (20 mg/ml), to simulate the total protein concentration of the diluted serurn. This resulted in approxi0 ,g 60 mately 80% inhibition of proteolytic C3 destruction by P. gitigivalis W83 during 20 min incubation at 37"C (Fig. 2). 40 When a mixture of B (70 fig/m\) and C3 (400 /
Complement factor D-like activity of Porphyromonas gingivaiis W83
Harvey A. Schenkein Department of Periodontics, School of Dentistry, Virginia Commonwealtfi University, Richmond, Virginia, USA
Sehenkein HA. Complement faetor D-like aetivity o/Porphyromonas gingivalis W83. Oral Mierobiol Immunol 1991: 6: 216-220. Porpliyromonas gitigivalis is a proteolytic gram-negative anaerobic bacterium that is frequently isolated from lesions of human periodontai disease. Previous studies have shown that P. gingivalis strain W83 inactivates C3 in pooled normal human serum (NHS) by a mechanism that is inhibitable by EDTA, yet it degrades purified complement proteins by a mechanism that is not EDTA-inhibitable. Furthermore, during complement activation, only a small number of C3 molecules accumulate on the surface of this organism unless the bacteria are treated with the protease inhibitor TLCK prior to complement activation. The hypothesis was tested that P. gitigivalis W83 contains protease activity mimicking that of complement factor D, thus enabling it to activate C3 in serum without significant C3 accumulation on the cell surface. It was first noted that incubation of P. gingivalis W83 in absorbed human serum that was depleted of faetor D resulted in C3 consumption that was reversed in the presence of the protease inhibitor TLCK. To directly demonstrate that factor B-dependent C3 consumption occurs in the absence of factor D, P. gingivalis W83 was incubated with purified C3 or a mixture of C3 and B. Although some proteolysis of C3 was noted, increased C3 consumption was noted in mixtures containing both C3 and B. This increment in C3 consumption was inhibited by both EDTA and TLCK. Furthermore, the addition of purified factor H to this mixture inhibited the increment in C3 consumption, indicating that a C3 convertase was probably formed. Following incubation of P. gingivalis W83 in a mixture of C3 + B containing '"I-C3, cleavage of '-H-C3 and formation of C3b was detectable by SDS-PAGE and autoradiography; C3 cleavage was inhibited by addition of H. This demonstrates the activity of a functional C3-cleaving enzyme in the absence of factor D that, like the alternative pathway C3 convertase, depends on factor B and Mg++ and is inhibited by factor H. Thus, P. gingivalis proteases that are inhibitable by TLCK substitute for factor D and initiate C3 consumption in serum, using factor B of the alternative complement pathway.
Accepted for publication November 8, 1990
Porphyromonas gingivalis, a gram-nega- ence the interaction of this organism tive anaerobic bacterium, is thought to with the complement system via exbe an important pathogen in various pression of its proteolytic eapacity. It forms of periodontai disease (14). In ad- has been demonstrated, for example, dition to its presence in lesions of this that some strains of P. gittgivalis will disease and its capacity to induce perio- proteolytically degrade serum compledontitis in experimental animal models, ment and individual complement prostrains of this organism have been teins. found to display many pathogenic Some strains of P. gingivalis have mechanisms of particular relevance to been described to be "invasive", as they the destruction of soft and hard connec- cause abscess formation and death tive tissues. Of particular relevance is when introduced into animal models (8, the proteolytic capacity of this species; 9, 19). We have examined one such numerous proteases of various speci- strain {P. gingivalis W83) and discovered ficities have been described and purified that, despite its capacity to activate (1,2, 18,21). serum complement (as demonstrated by Previous studies from our laboratory EDTA-inhibitable depletion of fluid and others (12, 15) have demonstrated phase hemolytic activity despite the failthat strains of P. gingivalis may influ- ure of EDTA to inhibit its proteolytic
activity against purified complement proteins), little C3 accumulates on its surface. Furthermore, treatment of the bacteria with the serine protease inhibitor TLCK prior to incubation with serum permits the accumulation of C3 on the surface in the form of C3b and iC3b (13). Thus, this organism may protect itself from the antibacterial effects of complement by failing to accumulate or by proteolytically removing complement-derived opsonins during complement activation. In addition, since little C3 is detectable on its surface during incubation with serum, it appears that this organism may activate the alternative complement pathway via an atypical activation mechanism. In the studies described here, we
Key words: Porptiyromonas gingivatis] complement: periodontai disease: factor D Harvey A. Schenkein, Virginia Commonwealth University, School of Dentistry, MCV Station Box 566, Richmond, VA 23298, USA
Complement activation by P. gingivalis examined alternative pathway activation by P. gitigivalis W83. This was of interest since activation of the alternative complement pathway results in accumulation of C3 fragments, in particular C3b and iC3b, on the surface of the activating particle. C3b deposition in turn permits formation of a particlebound C3 convertase. Factor D, a serine esterase found in a proteolytically active forrn in serum, participates in this process by cleavage of its substrate factor B when factor B is associated with C3b in solution or on the activating surface (6). Cleavage of B to form the Bb fragment results in the formation of the alternative pathway convertase C3bBb. Thus, D is required for activation of the alternative complement pathway. The surface of the activating particle participates in alternative pathway activation by providing protection from dissociation or decay of the C3 convertase and allows accelerated accumulation of C3b and thus the C3 convertase on its surface. It was thus of interest to determine how P. gingivalis W83 activates the alternative pathway without accumulating C3b at the cell surface. Since factor D is an active serine esterase with specificity for C3b-bound factor B, we hypothesized that P. gitigivalis W83 proteases might mimic the activity of factor 0 and thus participate in alternative pathway activation by this organism by bypassing the usual requirement for deposition of C3b and subsequent association of C3b with factor B on the activating surface prior to its cleavage by factor D. We found that P. gingivalis W83 activates the alternative pathway in serum by a mechanism independent of factor 0 (but dependent on factor B and Mg++) and that a bacterial protease or proteases inhibitable by the serine esterase inhibitor TCLK may substitute for factor D during activation.
NaCl) that had been deaerated by boiling. Bacteria were quantified after washing by determining the OD(,so of suspensions of the washed bacteria and comparing this with previously determined standards of known concentrations. Complement components
Purified C3 was prepared as described by Tack et al. (17) with the addition of an immunoadsorption step to remove traces of C5, H, and IgG; purity was verified by polyacrylamide gel eleetrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and by hemolytic assay. Factor B was purified as described by Kerr (4); faetor H was isolated as described in (20). SDS-PAGE was performed by the method of Laemmli (5) using 10% separation gels. Hemolytic titrations for quantification of C3 (11) were performed as described previously. Preserved sheep erythrocytes and antisheep erythrocyte hemolysin were purchased from Diamedix, Miami, FL. Guinea pig Cl (10), human C4 (3), human C5 (16), and human C2 (4) were prepared as previously described. C3 was radioiodinated to a specific activity of 0.3-0.5 /uCi/ftg using Iodobeads (Pierce Biochemicals, Rockford, IL) according the manufacturer's instructions. Unbound iodine was removed by passing the preparation through a column containing Sephadex G25. The resulting '-'I-C3 preparations retained 85% or more of their original hemolytic activity, and were quantified by single radial immunodiffusion (7). The protease inhibitor TLCK was purchased from Sigma Chemical (St. Louis, MO).
217
yield a final concentration of 0.01 M EDTA (EDTA serum). To measure the inactivation of C3 hemolytic function, bacteria were washed and pelleted by centrifugation, and serum reagent or mixtures of purified serum proteins were added for the indicated period of time. Following incubation, the reaction mixtures were centrifuged and the supernatants were removed and placed on ice or frozen until assayed. For determination of the effect of protease inhibitor on C3 consumption, bacterial suspensions were first suspended at 1 X 10'' cells/ml in VBS containing the indicated concentration of inhibitor for 30 min at 37°C, Serum mixtures were then added and procedures were carried out as indicated above. Demonstration of C3 fragments
To demonstrate the C3 fragments formed upon incubation with P. gingivalis W83, '-^I-C3 was added to supernatants of reaction mixtures, from which bacteria were removed by centrifugation. Following incubation, aliquots of the mixtures were removed and analyzed by SDS-PAGE in 4-15% gradient separation gels (5) followed by autoradiography. Statistical analysis
Experimental data were analyzed for significance using Student's /-test. Results C3 consumption by P. gingivaiis W83 in human serum and D-deficient serum
Pooled human serum (NHS) was first extensively absorbed with P. gingivalis as described above to remove specific Bacterial incubations antibodies. Incubation of P. gingivalis Bacteria were suspended at a final con- W83 with absorbed Mg-EGTA'^-NHS centration of 5 X 10* particles/ml in the significantly depleted C3 hemolytic acMaterial and methods appropriate serum reagent (final serum tivity; the depletioti of C3 function was Bacterial strains dilution 1:3). The buffers for these ex- inhibited by EDTA (Fig. 1), a result P. gingivalis strain W83 and Baeteroides periments contained either the chelating consistent with the capacity to activate intertneditts 8944 were obtained from agent EGTA (to remove Ca++ and the alternative complement pathway. A Dr. L. V. H. Moore, Virginia Polytech- therefore inhibit the classical comple- portion of the P. gingivalis-ahsorbed nic Institute and State University, ment pathway) or EDTA (to remove serum was depleted of factor D by gel Blacksburg, VA. They were cultured an- both Ca++ and Mg"*'"' and thus inhibit filtration. The factor D-depleted serum aerobically for 18 h in brain-heart in- all complement activity). Diluent buf- demonstrated no hemolytic activity in fusion medium (Carr-Scarborough fers were either VBS containing suf- the rabbit CH50 assay, whereas unMierobiologicals, Stone Mountain, ficient EGTA and MgCU to yield a final treated absorbed serum regularly proGA), and then washed 3 times in ver- concentration of 0.01 M EGTA and duced a titer of 50-60 RCH50 units/ml. onal-buffered saline (VBS, 0.01 M ver- 0.008 M Mg++ (Mg-EGTA serum) or Incubation of the factor D-depleted onal buffer, pH 7.5, containing 0.13 M VBS containing sufficient EDTA to serum with B. intertnedius 8944, a pro-
218
Sehenkein 100 -
hibited by EDTA but proceeds in the absence of factor D.
80 -
s
Effect of TLOK on C3 consumption 60 -
TLCK is a serine esterase inhibitor that we have shown inhibits the ability of P. "M gitigivalis W83 proteases to block C3 40 0} EC accumulation on the surface of this strain (13). To assess the capacity of 20 TLCK to influence C3 consumption in serum, P. gingivalis W83 was -first treated with the inhibitor (10 mM) for NHS-EDTA NHS-EGTA RD-EDTA RD-EGTA RD-TLCK 30 min prior to addition of serum. When P. gingivalis W83 was first treated Fig. 1. Consumption of C3 in NHS and factor D-deficient serum (RD) hy P gingivalis W83. with TLCK prior to incubation in factor Sera at a dilution of 1 :3 in Mg-EGTA buffer were incubated with bacteria (5 x lO'/ml) in the D-depleted serum, substantial inhipresence or absence of 10 mM EDTA for 20 min at 37°C. RD-TLCK indicates that sera were bition of C3 consumption was noted diluted in Mg-EGTA buffer containing 10 mM TLCK. Following incubation, bacteria were (Fig. 1), indicating that a bacterial proremoved by centrifugation and C3 titers were determined by hemolytic assay. The results are expressed as percentage residual titer vs matched controls incubated in the absence of bacteria. tease may be required for C3 consumpOne standard deviation (SD) is shown by the error bars for 3 replicate experiments. tion in the absence of factor D. Thus, it appears that activation of the alternative complement pathway by P teolytic organism previously shown to way hemolytic activity of the factor D- gitigivalis W83 occurs in the absence of activate the alternative complement depleted serum reagent, C3 consump- factor D and depends on B and Mg^^pathway, resulted in no C3 consump- tion occurred during incubation with P. Therefore, P. gitigivalis W83 proteases tion, further verifying its lack of alterna- gingivalis W83 (Fig. 1). This activity re- may provide D-like activity in alternative pathway activity. In addition, a por- quired the presence of divalent cations, tive pathway activation. tion of the absorbed serum pool was since little C3 consumption occurred in heated to 50°C to remove factor B activ- the presence of EDTA. Additionally, inity; this reagent also displayed no hemo- cubation of P. gingivalis W83 in B-de- Demonstration of B-dependent C3 lytic activity against rabbit erythrocytes pleted serurn failed to deplete C3 activ- consumption with purified proteins unless reconstituted with factor B. ity. These experiments indicated that C3 To demonstrate that B-dependent C3 Despite the lack of alternative path- consumption by P. gittgivalis W83 is in- consumption by P. gingivalis W83 occurs, experiments using mixtures of purified components were carried out. Since P. gingivalis W83 readily proteo100 H lyzes purified solutions of complement proteins (12), it was necessary to dilute the complement reagents in buffer containing BSA (20 mg/ml), to simulate the total protein concentration of the diluted serurn. This resulted in approxi0 ,g 60 mately 80% inhibition of proteolytic C3 destruction by P. gitigivalis W83 during 20 min incubation at 37"C (Fig. 2). 40 When a mixture of B (70 fig/m\) and C3 (400 /
