Eur J Oral Sci 1998; 106: 863–871 Copyright © Eur J Oral Sci 1998Printed in UK. All rights reserved EUROPEAN JOURNAL OF

ORAL SCIENCESISSN 0909-8836

Anders Johansson, Sotirios KalfasDepartment of Oral Biology, Faculty ofCharacterization of theOdontology, University of Umea, Sweden

proteinase-dependentcytotoxicity ofPorphyromonas gingivalisJohansson A, Kalfas S: Characterization of the proteinase-dependent cytotoxicityof Porphyromonas gingivalis. Eur J Oral Sci 1998; 106: 863–871. © Eur J OralSci, 1998

Cytotoxicity of proteins from Porphyromonas gingivalis towards gingivalfibroblasts and epithelial cells was studied by methods measuring vital dyeuptake, DNA synthesis, release of cytoplasmic components, and apoptosis.Microscopic examination of the cells was also performed for detection ofmorphological changes. Experiments were made with dialyzed culturesupernatants of P. gingivalis as well as supernatant fractions obtained byisoelectric focusing. The main cell damage observed was rounding of the cellsand detachment from each other and the underlying surface. The cell-damaging Anders Johansson, Department of Oral

Biology, University of Umea, S-902 85activity correlated with the cystein-dependent proteolytic activity of the variousUmea, Swedensupernatant preparations as well as with the occurrence of gelatinolytic proteinTelefax: +46-90770580bands with molecular weights previously reported for the cysteine proteinasesE-mail: anders.johansson@oralbio.umu.segingipains R and K of P. gingivalis. The activity was abolished by heat

treatment or by the addition of cysteine proteinase inhibitors. The results Key words: gingival fibroblasts; gingivalepithelial cells; cytotoxicity;indicate that the main cytotoxic effect towards fibroblasts and epithelial cells isPorphyromonas gingivalis; gingipainsdegradation of the intercellular matrix. The gingipains released in P. gingivalis

culture supernatants are the responsible factors for this degradation. Accepted for publication March 1998

Porphyromonas gingivalis, a periodontitis- duces a collagenase (22) and the serine proteaseglycylprolyl peptidase (23).associated bacterium (1–3), produces proteolytic

enzymes which are considered potential virulence Proteolytic enzymes as well as metabolic endproducts such as organic acids and ammonia fromfactors (4–6). These enzymes are suggested to cause

tissue degradation directly (7, 8) or indirectly by P. gingivalis exhibit cytotoxic effects in vitro againstfibroblast and epithelial cells (8, 24–26). The cyto-induction and activation of tissue proteinases (4,

9, 10). They can also degrade certain defense toxic effects reported include cell rounding, celldetachment from underlying surface, cell death,proteins and may facilitate evasion of host defense

mechanisms (11–14). decreased dye uptake and metabolic activity (4, 8,25–27). In one study (8), the morphologicalThere are nearly 40 apparently distinct proteo-

lytic enzymes and enzyme isoforms purified from changes were attributed to proteolytic enzymes.These changes did not correlate to cell death deter-P. gingivalis (15). Most of them are cysteine pro-

teinases that account for more than 95% of the mined by exclucion of the dye trypan blue. Sincethe mechanisms of the cytotoxic effects were poorlyproteolytic activity. They show trypsin-like activity,

occur in multiple forms, and are classified as gingi- defined in these studies, the specific factors respons-ible for the above effects still remain unknown (27).pain R and gingipain K since they cleave arginyl

and lysyl bonds, respectively (16, 17). Both gingi- The purpose of this study was to characterize theprotein-dependent cytotoxic effect of P. gingivalispains have been described in the literature under

other names such as gingivain (18, 19), porphypain culture supernatants directed towards gingivaltissue cells and carcinoma cell lines, and to evaluate(20) and argingipain (21). P. gingivalis also pro-

864 Johansson & Kalfas

the involvement of P. gingivalis proteinases in theEukaryotic cell culturescytotoxic mechanisms.The target cells used were human gingivalfibroblasts, gingival epithelial cells, human oralMaterial and methodsepidermoid carcinoma cells ( KB-line, CCL 17;

Bacterial preparations Flow Laboratories, Glasgow, UK) and humanP. gingivalis strains ATCC 33277, 381 and W50 cervical carcinoma cells (HeLa, CCL2, Flowwere grown anaerobically in BM broth (28), at Laboratories) grown at 37°C in the presence of37°C for 64 h. Inocula of broth-adapted cells were 5% CO2 .used at 0.1% concentration for the cultures. The For primary fibroblast cultures, gingival tissueoptical density at 600 nm of each culture as well from a patient with periodontal disease was mincedas the N-a-benzoyl-DL-arginine p-nitroaniline to pieces of approximately 1 mm3 each, transferred(BApNA)-hydrolyzing activity (29) of cell free to tissue culture flasks (Flow Laboratories) and leftculture were determined after 16, 40 and 64 h of for 30 min at 37°C to settle. To each flask, 5 mlincubation. At the end of the incubation, the Eagle minimum essential medium (E-MEM) sup-cells were removed by centrifugation (10,000×g, plemented with 20,000 U/l penicillin, 100 mg/l strep-10 min), and the culture supernatants were stored tomycin, 0.2 mg/l L-glutamine and 10% fetal calfat −80°C until used. For some experiments, the serum were added and the gingival tissue explantssupernatants were heated for 30 min at 100°C. were incubated until outgrowth of fibroblasts was

To remove salts and metabolites of low molecular obtained. Further preparation of fibroblasts andweight, the supernatants were dialyzed against maintenance of the cultures were performed asdeionized water at 4°C, using a membrane with a described previously (16).nominal cut-off of 3,500 Da (Spectrum Medical To obtain primary cultures of gingival epithelialIndustries, Houston, TX, USA). Protein separation cells, the pieces of gingival tissue were cultured inwas achieved by isoelectric focusing in a Rotofor keratinocyte growth medium which selectively sup-(Bio Rad Laboratories, Melville, NY, USA). ports growth of epithelial cells. HeLa cells wereForty-six ml of dialyzed supernatant were mixed grown in E-MEM supplemented as above. For KBwith 2.9 ml glycerol, 24 g urea and 1 ml Bio-lyte, cell cultures, the medium was enriched with non-pH range 3.5–10 and the sample was electrofocused essential amino acids (10 ml/l from a stock solu-at constant power (12 W ) for approximately 4 h tion). The growth medium was changed every 48thuntil the current and voltage values reached steady- h. The primary cell cultures were in their third tostate levels. One ml of each fraction was dialyzed eighth passage when they were used in the cyto-against 3×100 ml of 0.1 M phosphate buffer toxicity experiments.(pH 7.3) with 5 mM cysteine at 4°C for 24 h toremove urea, ampholytes and glycerol from the

Cytotoxicity assayssamples. The gingipain R and gingipain K activitiesof the various preparations were determined by the To determine the cytotoxic effect of various

P. gingivalis preparations (final concentration inhydrolysis of the synthetic substrates BAPNA andN-a-benzoyl-L-lysine p-nitroanilide (Z-lys-pNA) the assay mixture 10% v/v), several assays were

used. Changes in cell morphology and detachmentrespectively, substrate concentration 1 mM, in thepresence of 5 mM L-cysteine and 50 mM Tris-HCl from the underlying surface were examined by light

microscopy at 400×magnification. For estimationbuffer pH 7.4 (30). Hydrolysis of Azocoll(5 mg/ml ) was determined in the presence of 5 mM of the uninjured cells attached to the surface of the

culture well, the neutral red uptake assay (35) wasL-cysteine, 200 mM borate buffer pH 7.4 and150 mM NaCl (31). used. For this purpose, fresh confluent cell layers

in a 96-well microtiter plate were incubated at 37°CThe protein profile of each preparation wasanalyzed by sodium dodecyl sulfate-polyacrylamide for up to 4 h in culture medium (E-MEM with

antibiotics and glutamine or keratinocyte growthgel electrophoresis (SDS-PAGE) in a discontinuousbuffer system (32). Prior to electrophoresis, the medium) with 5 mM L-cysteine and various con-

centrations of bacterial culture preparations. Thesamples were mixed with an equal volume of samplebuffer (50 mM Tris-buffer, pH 7.4, containing 2% medium was replaced with 0.1 ml E-MEM-medium

containing 10% fetal calf serum and 40 mg/ml neut-SDS, 0.03% bromphenol blue, 20% sucrose and 5%mercaptoethanol ) and heat-treated in boiling water ral red and incubated at 37°C for 3 h. The uptake

of neutral red by the cells was measured at 540 nmfor 5 min. Protein bands in the gel were visualizedwith Coomassie brilliant blue R-250 or silver stain after fixation and solubilization of the cells in 50%

ethanol with 1% acetic acid (35).(14, 33). The gelatinolytic proteins in unheatedsamples were detected by gel zymography (34). To examine the viability of the cells detached

P. gingivalis cytotoxicity 865

upon incubation with P. gingivalis preparations, a cysteine. The cells were then rinsed once withE-MEM containing 10% fetal calf serum and incub-modified neutral red uptake assay was used. The

detached cells were harvested by centrifugation ated for 20 h in this medium. Cells exposed toultraviolet light (UVB, 302 nm) for 30 min served(500×g for 5 min) in Eppendorf tubes, re-

incubated in the presence of the dye, washed once as the positive control. Sample preparation anddetection of DNA were done following the manu-with the fixative solution, and solubilized before

measuring the absorbance of the solution at facturer’s instructions.Experiments were also performed in the presence540 nm.

Effects on the DNA synthesis of the cells were of various proteinase inhibitors (Table 2). Allexperiments were done in duplicate unless stateddetermined by the incorporation of [3H]thymidine

(36). Confluent cell layers in microtiter plates otherwise.were exposed for 4 h to various concentrationsof P. gingivalis supernatants in the culture

Chemicalsmedium (0.1 ml/well ) supplemented with 2 mCi[3H]thymidine/ml. [3H]thymidine-containing DNA E-MEM, L-glutamine, and fetal calf serum were

purchased from Flow Laboratories, Glasgow, UK.was retained in the cell pellet after precipitation by3 washes with 5% trichloracetic acid. The pellet Keratinocyte growth medium was obtained from

Promocell, Heidelberg, Germany. Radioactivewas dissolved in 0.1 ml 1 M NaOH, neutralizedwith 0.1 ml glacial acetic acid, and mixed with 5 ml compounds were purchased from Amersham Life

Science, Little Chalfont, UK. Proteinase inhibitorsscintillation liquid (Ready SafeTM ; Beckman,Bromma, Sweden). The radioactivity of the samples and N-a-benzoyl-DL-arginine p-nitroaniline

(BApNA) were obtained from Boeringerwas measured in a b-counter (Beckman). Wellswithout cells served as controls for background Mannheim Biochemicals, Mannheim, Germany,

while Bio-lyte and material for SDS-were from Bio-radioctivity, which was considered equivalent ofmaximum damage in DNA syntesis. Rad Laboratories, Richmond, CA, USA. N-a-

benzoyl-L-lysine p-nitroanilide (Z-lys-pNA) andCell membrane damage, which allows leakage ofintracellular components, was determined by meas- the apoptosis ELISA kit were from Calbiochem,

Cambridge, MA, USA. Bovine serum albumin,uring the release of cytoplasmatic lactate dehydro-genase (37) as well as by the release of the uptaken antibiotics, non-essential amino acids, Azocoll and

other compounds were obtained from Sigma[3H]thymidine (38) or neutral red dye (39) afterincubation with certain preparations of extracellu- Chemical Co., St. Louis, MO, USA. Human recom-

binant cystatin C was kindly provided by Dr.lar compounds from P. gingivalis. For the latterassays, confluent cell layers were labeled in a 24-well A. Grubb (Lund University, Lund, Sweden).microtiter plate by incubating for 3 h, in E-MEMsupplemented with either 2 mCi [3H]thymidine/ml Resultsor 40 mg/ml neutral red. The cells were washed(3×0.2 ml ) with E-MEM to remove excess of the In BM broth, a strain-dependent variation was

found in the growth kinetics of P. gingivalis. Strainsmarkers. Cells were exposed to P. gingivalis prod-ucts for 4 h ([H3 ]-labelled cells 6 h). Release of the ATCC 33277 and 381 reached stationary phase of

growth within 40 h, while strain W50 continuedmarkers in the supernatant obtained after centrifu-gation (150×g, 5 min) was estimated enzymatically growing during the 3rd day and reached a higher

optical density value than the other two strains( lactate dehydrogenase assay), through radio-activity measurment ([3H ]thymidine release), or (Fig. 1). The proteolytic activity (BApNA hydro-

lysis) of the supernatants increased with increasingoptically by the increase of absorbance at 540 nm(neutral red release) as described above. The release cell density in the culture (Fig. 1).

The protein fraction from a 64-h culture super-of the intracellular components was compared withthe concentrations of the same compounds found natant of P. gingivalis strain ATCC 33277 was

selected for characterization of cytotoxicity againstin the attached cells of the negative controls, i.e.,cells incubated without P. gingivalis products and various cell layers. Microscopic examination

revealed cells uprounded and detached from thethose in the positive controls, i.e., cells treated with0.1% v/v solution of Triton X-100. culture surface. Quantitative results obtained by

the neutral red uptake assay showed that less thanInduction of programmed cell death, apoptosis,by P. gingivalis proteins was assayed with a com- 5% of the cells remained attached after a 4-h

incubation (Table 1). The effect was concentration-mercially available ELISA for detection of DNAfragmentation. Confluent cell layers in a 24-well and time-dependent, and it could be enhanced by

the presence of 5 mM cysteine. Cysteine concentra-microtiter plate were exposed for 4 h to P. gingivalispreparations in E-MEM supplemented with 5 mM tions higher than 10 mM were toxic to the target

866 Johansson & Kalfas

various extent while inhibitors for metallo-proteinases (EDTA) as well as for serine(PefablocA) and aspartic acid (PepstatinA) pro-teinases showed no effect. EDTA was only testedat 1 mM, since concentrations �5 mM exerted acytotoxic effect.

Activities of BApNA hydrolysis and cell layerdisruption were mainly concentrated in 3 fractions(pH range 5.1–6.0) out of the 20 fractions obtainedafter isoelectric focusing (Fig. 2). These activitiescorrelated with the occurrence of a protein band atthe ~45 k position on SDS-of these samples(Fig. 2).

Proteolytic activity specific for gingipain K(Z-Lys-pNA hydrolysis) as well as Azocoll degrada-tion were also detected in the culture supernatants(Table 3). The degradation of Azocoll was inhibited

Fig. 1. Growth kinetics of P. gingivalis strains ATCC 33277 by TLCK or leupeptin (Table 3) and enhanced by(rhombes), 381 (squares) and W50 (triangles) in BM-broth and the presence of cysteine (not shown). Z-Lys-pNABApNA-hydrolyzing activity in the supernatants of the cultures. hydrolysis was inhibited only by TLCK (Table 3).

Contrary to strain ATCC 33277, the culturesupernatant of strain W50 caused no degradationcells and therefore not used, though they furtherof Z-Lys-pNA (Table 4). Leupeptin alone couldpotentiated the BApNA-hydrolyzing activity of thetotally inhibit the cytotoxic effect of the lattersamples. Culture supernatants of the other twosupernatant, but not the one caused by ATCCP. gingivalis strains evoked similar effects irrespect-33277, especially not the effect towards the KB-cellsive of the target cells tested. The detached cells(Table 4).retained their ability to accumulate neutral red in

SDS-and gel zymography of the culture supernat-their lysosomes, and could re-attach to the cultureant revealed the occurrence of several gelatinolyticdish and grow upon addition of new growthprotein bands in the area 75–180 k (Fig. 3). Themedium (data not shown). None of the other assaysstrongest activity was found at the position 180 k,revealed significant effects on either cell membraneshown with the 1580 diluted sample. A densepermeability or DNA synthesis and fragmentationprotein band was observed at the corresponding(Table 1).position in SDS-of the non-heated sample. WeakerSeveral proteinase inhibitors that decreased theprotein bands were also found at other positions.proteolytic activity (BApNA-hydrolysis) could alsoUpon heat treatment under reducing conditions,inhibit the disruption of the cell layer as measuredthese protein bands disappeared, and new bandsby the neutral red uptake (Table 2). Presence of

leupeptin or TLCK protected the cell layer to appeared in the area 45–70 k ( lane 5 in Fig. 3).

Table 1

Effects of dialyzed culture supernatant of P. gingivalis ATCC 33277 on layers of gingival and carcinoma cells. The effect is expressedin percent of the maximum cell damage (100%) found in the controls described in material and methods. The values of the negative

controls (untreated cells) were withdrawn. Mean±SD and the number (n) of experiments are shown

Gingival cells Carcinoma cells

Fibroblasts Epithelial cells HeLa cells KB-cells

Cytotoxicity assay n x:±SD n x:±SD n x:±SD n x:±SD

Neutral red uptake 3 95±6 3 97±3 3 96±2 3 100±9Neutral red release 4 2±2 ND 4 3±2 5 1±23H-thymidine incorporation 7 −1±8 7 9±17 5 −13±7 4 4±43H-thymidine release 4 19±2 ND 5 6±3 5 14±3Lactate dehydrogenase release 4 −4±1 4 −5±2 4 −2±2 4 −7±2Apoptosis 2 −5±9 3 2±10 3 −5±6 3 4±9

ND: not determined

P. gingivalis cytotoxicity 867

Table 2

BApNA-hydrolysis and neutral red uptake by fibroblasts and epithelial cells, in the presence of proteinase inhibitors and culturesupernatant from P. gingivalis ATCC 33277. The activities are expressed in percent of the activity values recorded in control mixtures

without proteinase inhibitors. Mean±SD of four experiments are given

Neutral red uptakeBApNA

Inhibitors Concentration hydrolysis Fibroblasts Epithelial cells

Serine proteinasesPefablocA 1.0 mM 91±10 94±2 94±3

Serine and cysteine proteinasesTLCK 1.0 mM 8±7 0±7 38±8Leupeptin 1.0 mM 2±4 47±10 75±3

MetalloproteinasesPhosphoramidon 1.0 mM 92±10 98±4 97±3

Aspartic acid proteinasesPepstatin 0.1 mM 100±8 98±6 100±1

Biological compoundsa2-macrogrobulin 0.1 mg/ml 88±9 84±9 95±2

1.0 mg/ml 66±2 −2±13 −6±10Cystatin C 0.1 mg/ml 88±4 84±4 96±2

1.0 mg/ml 29±2 4±9 0±4Bovine serum albumin 1.0 mg/ml 99±4 95±7 98±1

Table 3 DiscussionDegradation of BApNA, Z-Lys-pNA and Azocoll by dialyzed

The present results indicate that the main cellculture supernatant from P. gingivalis ATCC 33277 in thedamage caused by proteins released in cultures ofpresence or absence of the protease inhibitors leupeptin and

TLCK. The activity is expressed as the increase in optical density P. gingivalis is morphological alterations (cell(DOD

nm) over time. Mean±SD of four experiments are shown rounding) and detachment of the target cells from

each other and from the surface of the culture dish.DOD414/min

This reversible damage is probably caused by the(×10−3) DOD540/h hydrolysis of cell surface proteins that mediate(×10−3)Mixture BApNA Z-Lys-pNA Azocoll intercellular adhesion as well as attachment. The

effect is thus similar to the one caused by trypsinCulture supernatant 295±9 29±4 28±2 treatment of cell cultures to release single cells fromCulture supernatant+

cell layers (40).leupeptin 3±3 23±3 3±1The bacterial proteins responsible for thisCulture supernatant+TLCK 3±4 1±1 3±1

damage seem to be the cysteine proteinases

Table 4

Relative activities of gingipain R (BApNA-hydrolysis) and gingipain K (Z-lys-pNA hydrolysis) in culture supernatants fromP. gingivalis ATCC 33277 and W50, and effect of these supernatants on the neutral red uptake by gingival fibroblasts and KB cells.The activities were determined in the absence or presence (1 mM) of the proteinase inhibitors leupeptin and TLCK. The activity in the

absence of the inhibitors is considered as 100%. Mean±SD of three experiments are given

Hydrolysis Neutral red uptake by

BAPNA Z-lys-PNA Fibroblasts KB cells

P. gingivalis ATCC 33277culture supernatant 100±2 100±16 100±1 100±1culture supernatant+TLCK 19±1 0±0 10±8 0±5culture supernatant+leupeptin 19±1 100±8 38±8 88±3

P. gingivalis W50culture supernatant 100±2 * 100±2 100±1culture supernatant+TLCK 6±1 0±6 0±8culture supernatant+leupeptin 5±1 4±6 0±2

*: No detectable enzyme activity.

868 Johansson & Kalfas

Fig. 3. Profiles of proteins (SDS-PAGE) and gelatinolyticenzymes (zymogram) in culture supernatant from P. gingivalisATCC 33277. Lanes 1, 2 and 3 were loaded with supernatantdiluted 1580, 1520 and 155, respectively. Lane 4 containedundiluted sample and lane 5 was loaded with heat-treated(100°C, 5 min) undiluted sample. Filled arrowheads indicatethe positions of the gelatinolytic bands detected in the zymog-ram ( lanes 1–3) and the corresponding protein bands in thenon-heated sample ( lane 4). Open arrowheads show the posi-tions of the main protein bands that appeared in the heatedsample ( lane 5).

that TLCK, which can inhibit both gingipains Rand K, was a better inhibitor of the cell layerdamage than leupeptin when the culture supernat-ant from strain ATCC 33277 was tested. The partialinhibition of the damage caused by leupeptin maytherefore depend on the activity of gingipain K, aleupeptin-insensitive protease. Support for theinvolvement of gingipain K in the intercellularFig. 2. Inhibitory effect on neutral red uptake by gingival

fibroblasts and KB-cells (expressed in per cent of the activity matrix degradation is also derived from the experi-of nontreated cells), BApNA hydrolytic activity (expressed as ments with P. gingivalis strain W50 (Table 4). Thethe increase in absorbance at 414 nm per min) and protein culture supernatant of W50 showed no activity ofprofiles in SDS-PAGE, found with culture supernatant from

gingipain K, and its cell-damaging effect was com-P. gingivalis strain ATCC 33277 (crude) and in 10 supernatantfractions obtained by isoelectric focusing. The asterisk indicates pletely abolished by leupeptin alone.the protein enrinched in the proteolytic fractions. P. gingivalis proteinases are known to degrade

matrix proteins that favor cell adhesion, such asfibronectin (7, 43, 44) and collagen (22, 45, 46).gingipain R and gingipain K. This conclusion is

based on: (a) the damaging process exhibited Beside the gingipains, a serine protease, glycylpro-pyl peptidase (23), and collagenases (22, 46) canenzyme kinetics and was enhanced by reducing

agents such as cysteine; (b) the culture supernatant be produced by strains of P. gingivalis. This studygave no support for a serine protease-dependentshowed enzymatic activities specific for arginyl and

lysyl bonds and contained gelatinolytic proteins cell damaging activity. In agreement with previousfindings (22, 45), the proteolytic activity detectedwith apparent molecular weights in the range

75–180 k (45–70 k in heated samples), character- with the substrate Azocoll appeared to depend onP. gingivalis enzymes with characteristics of cysteineistics of gingipains (15, 16); and (c) inhibitors for

the cysteine proteinases abolished the cell-damaging proteinases, i.e., sensitivity to the inhibitors TLCKand leupeptin and activation by cysteine. On theeffect of the culture supernatant.

Leupeptin and TLCK affect the activity of gingi- other hand, no collagenase activity independent ofreducing agents and insensitive to TLCK waspains differently (16). The present results showed

P. gingivalis cytotoxicity 869

detected. The latter collagenase was reported to be suggest that gingipains may contribute to anincreased tissue permeability by a direct degrada-the product of P. gingivalis gene prtC (46).

Recent studies indicate that P. gingivalis pro- tion of the intercellular proteins and destruction ofthe tissue connectivity. A similar though indirectteinases are able to activate matrix metallo-

proteinases (47, 48). These enzymes might also effect could be the result of the activation of thekallikrein-kinin system by gingipains as proposedhave caused dissolution of the cell layer. However,

the present experiments were performed with by I et al. (41).In the periodontal area, certain defense moleculeswashed cell layers, and the extracellular concentra-

tion of metalloproteinases should therefore have such as specific antibodies and proteinase inhibitorscan counteract the effect of proteinases. The pro-been low. Degradation of extracellular proteins by

the direct action of gingipains is thus the most teinase inhibitors a2-macroglobulin and cystatinshave been found in the gingival crevicular fluid andprobable cause of the morphological cell changes,

this observation being in agreement with a previous are suggested to play an important role in thecontrol of proteolysis at inflammatory sitessuggestion (49).

Other authors (8, 25) have reported a non- (57–59). Destructive periodontal inflammation cor-relates with an increased consumption of a2-macro-proteinase-dependent cell death of fibroblast and

epithelial cells determined by the trypan blue exclu- globulin and with a high conversion of a2-macroglobulin to the complexed form with pro-sion method and caused by P. gingivalis products.Extracellular vesicles of P. gingivalis were found to teinases (60, 61). Gingipain R is efficiently inhibited

at a 151 molar ratio by a2-macroglobulin, whereascontain compounds cytotoxic to neutrophils (50),while some researchers failed to detect leukotoxic the activity of gingipain K cannot be eliminated

(62). The present results indicate that both a2-activity in P. gingivalis strains (51, 52). In thepresent study, no support was found for similar macroglobulin and cystatin C can counteract the

cell layer detachment. It is therefore possible thattoxic effects towards the cells examined. Oneexplanation may be that such bacterial products these biological inhibitors to some extent protect

the periodontal tissues from being damaged by theare too small to be retained in the dialyzed culturesupernatant used in the toxicity assay. Indeed, gingipains.cytotoxic metabolic end products of low molecular

Acknowledgements – Financial support was obtained from theweight have earlier been found in P. gingivalisPatent Revenue Fund for Research in Dental Prevention, thecultures (24, 53, 54). Another explanation couldSwedish Dental Society, and the Swedish Medical Researchbe that the incubation time required to obtainCouncil (grant K98–24X-12684–01A).

cytotoxic effects is much longer (24–72 h, as per-formed earlier) than the time used in the presentstudy. In the previous studies, activation of the Referencesgingipains was not attempted by reducing agents,

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