INFECTION AND IMMUNITY, May 2002, p. 2512–2518 0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.5.2512–2518.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 5
Hydrolysis of Epithelial Junctional Proteins by Porphyromonas gingivalis Gingipains Jannet Katz,1* Qiu-Bo Yang,2 Ping Zhang,2 Jan Potempa,3,4 James Travis,4 Suzanne M. Michalek,1,2 and Daniel F. Balkovetz5,6,7 Department of Oral Biology,1 Department of Microbiology,2 Department of Medicine,5 and Department of Cell Biology,6 University of Alabama at Birmingham, Birmingham, Alabama 35294; Veterans Administration Medical Center, Birmingham, Alabama 352337; Institute of Microbiology and Immunology, Jagiellonian University, Cracow, Poland3; and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 306024 Received 24 September 2001/Returned for modification 12 December 2001/Accepted 3 January 2002
Porphyromonas gingivalis has been implicated as an etiologic agent of adult periodontitis. We have previously shown that P. gingivalis can degrade the epithelial cell-cell junction complexes, thus suggesting that this bacterium can invade the underlying connective tissues via a paracellular pathway. However, the precise mechanism(s) involved in this process has not been elucidated. The purpose of this study was to determine if the arginine- and lysine-specific gingipains of P. gingivalis (i.e., HRgpA and RgpB, and Kgp, respectively) were responsible for the degradation of E-cadherin, the cell-cell adhesion protein in the adherens junctions. In addition, we compared the degradative abilities of the whole gingipains HRgpA and Kgp to those of their catalytic domains alone. In these studies, immunoprecipitated E-cadherin as well as monolayers of polarized Madin-Darby canine kidney (MDCK) epithelial cell cultures were incubated with the gingipains and hydrolysis of E-cadherin was assessed by Western blot analysis. Incubation of P. gingivalis cells with immunoprecipitated E-cadherin resulted in degradation, whereas prior exposure of P. gingivalis cells to leupeptin and especially acetyl-Leu-Val-Lys-aldehyde (which are arginine- and lysine-specific inhibitors, respectively) reduced this activity. Furthermore, incubation of E-cadherin immunoprecipitates with the different gingipains resulted in an effective and similar hydrolysis of the protein. However, when monolayers of MDCK cells were exposed to the gingipains, Kgp was most effective in hydrolyzing the E-cadherin molecules in the adherens junction. Kgp was more effective than its catalytic domain in degrading E-cadherin at 500 nM but not at a lower concentration (250 nM). These results suggest that the hemagglutinin domain of Kgp plays a role in degradation and that there is a critical threshold concentration for this activity. Taken together, these results provide evidence that the gingipains, especially Kgp, are involved in the degradation of the adherens junction of epithelial cells, which may be important in the invasion of periodontal connective tissue by P. gingivalis. changes (26). Internalization of P. gingivalis by epithelial cells has been shown to result in rearrangement of the actin cytoskeleton (2). This process may be similar to that reported for another periodontal pathogen, Actinobacillus actinomycetemcomitans, where invasion involves an actin-dependent process (4, 45). P. gingivalis may also gain access to the underlying tissues via a paracellular pathway. P. gingivalis cells and culture supernatant have been shown to break down the epithelial transmembrane proteins E-cadherin and occludin, which form, in part, the adherens and tight junctions, respectively (20). This is not unique to P. gingivalis, since the enteropathogen Bacteroides fragilis has been shown to specifically degrade these interconnecting epithelial proteins via a secreted toxin (30, 49). This destruction of the transmembrane proteins renders the epithelial cells and the underlying connective tissues susceptible to infection. The P. gingivalis virulence factors which could potentially degrade epithelial junctional proteins are powerful proteases whose proteolytic activity has been recognized for a number of years (24). Recently, protease-encoding genes have been isolated and cloned, which has facilitated the characterization of their specificity and activity (26). The best-characterized proteases of P. gingivalis are the gingipains, a group of cysteine proteases with specificity for arginine- or lysine-containing
Human adult periodontitis is a chronic, infectious, inflammatory process involving the structures supporting the teeth. It is presently accepted that periodontal disease is initiated and perpetuated by specific gram-negative bacteria, among which the black-pigmented anaerobe Porphyromonas gingivalis has been implicated in the pathogenesis of the disease (6, 12, 14, 18, 23). P. gingivalis is commonly isolated from sites of periodontal disease, and patients with periodontitis have serum antibodies specific to this periodontal pathogen (28, 29, 31). Furthermore, in vitro studies have shown that P. gingivalis can invade and multiply in primary cultures of gingival epithelial cells and in oral epithelial cell lines (11, 25, 41, 42). These findings provided evidence that P. gingivalis has the ability to invade host tissues; however, the precise mechanism(s) by which this pathogen accesses the underlying connective tissue has not been fully defined. Two possible mechanisms by which P. gingivalis could invade tissue is via the transcellular route or a paracellular pathway. P. gingivalis has been shown to actively internalize within epithelial cells, which results in calcium ion fluxes and other cellular
* Corresponding author. Mailing address: Departments of Microbiology and Oral Biology, University of Alabama at Birmingham, 845 19th St. South, BBRB 258/5, Birmingham, AL 35294-2170. Phone: (205) 9343470. Fax: (205) 934-1426. E-mail:
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peptide bonds (15, 24, 34, 38). Three genes, rgpA, rgpB, and kgp, have been cloned. The genes rgpA and kgp encode a prepropeptide, a catalytic domain, and a hemagglutinin domain, and the initial polyprotein seems to be subject to posttranslational processing. The gingipain RgpB lacks the hemagglutinin domain (24, 38). A number of relevant functions have been attributed to these P. gingivalis proteases, such as hemagglutination, cytokine regulation, antibody degradation, alterations on cytokine receptors, and degradation of epithelial cell-cell junctional complexes (5, 9, 16, 20, 43, 50). Interestingly, studies have shown that the adhesin domains of P. gingivalis HRgpA and Kgp share homology with domains encoded by the hemagglutinin hagA gene (1, 8, 17). It is thought that the adhesin domain of gingipains plays an important role in the adhesion of the protease to host tissue, although a direct role for it in host tissue binding has not been demonstrated. The purpose of this study was to determine the involvement of the gingipains HRgpA, Kgp, and/or RgpB in E-cadherin hydrolysis. The ability of the catalytic domains of HRgpA and Kgp to hydrolyze E-cadherin was also examined in order to determine the importance of the protease adhesin domain in this process. MATERIALS AND METHODS Bacteria. P. gingivalis ATCC 33277 was used in these studies. The bacteria were cultured and maintained on enriched Trypticase soy agar plates, consisting of Trypticase soy agar supplemented with yeast extract (1%), 5% defribinated sheep blood, hemin (5 mg/liter), and menadione (1 mg/liter), at 37°C in an anaerobic atmosphere of 10% H2, 5% CO2, and 85% N2 (46, 47). For the preparation of P. gingivalis for in vitro studies, cultures were grown in basal anaerobic broth (47) at 37°C under anaerobic conditions (19, 21, 47). The bacteria were harvested and washed in sterile Dulbecco’s phosphate-buffered saline containing Mg2⫹ and Ca2⫹ (PBS⫹) (6,000 ⫻ g for 20 min). The number of bacteria in the suspension was determined by reading the optical density at 580 nm and extrapolating from a standard curve. The bacteria were then centrifuged and resuspended in antibiotic-free minimal essential medium containing Earle’s balanced salt solution (Cellgro; Mediatech, Inc., Washington, D.C.) supplemented with 5% fetal calf serum (HyClone, Logan, Utah). Cell culture. Type II Madin-Darby canine kidney (MDCK) cells were used between passages 5 and 15. Cells were cultured in minimal essential medium containing Earle’s balanced salt solution supplemented with 5% fetal calf serum, 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 0.25 g of amphotericin B per ml. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air. For all of our experiments, MDCK cells were seeded at confluency on Transwell filter units (Costar, Cambridge, Mass.) with 0.4-mdiameter pores. Cell monolayers were used for experiments after 3 days of culture with daily changes in medium. Proteases and inhibitors. HRgpA, RgpB, and Kgp were purified as previously described (34, 37). This procedure also resulted in the purification of the catalytic domains. Briefly, during a gel filtration of the acetone-precipitated, cell-free culture fluid on the Sephadex G-150 column, the catalytic domains of RgpA and Kgp were eluted together with RgpB in the 50-kDa protein peak. The peak fractions were pooled, concentrated, and extensively dialyzed against 50 mM bis-Tris–1 mM CaCl2, pH 6.5. This pool was used for the purification of RgpB. The first step in the procedure involved ion-exchange chromatography on a DE-52 cellulose column equilibrated with 50 mM bis-Tris–1 mM CaCl2, pH 6.5 (37). During this purification step, the catalytic domains of RgpA and Kgp did not bind to DE-52 cellulose and passed through the column slightly retarded. The RgpB was eluted with 100 mM NaCl. The fractions constituting the column wash with equilibration buffer (V0) and containing activity against Na-benzoylL-arginine-p-nitroanilide and Na-benzyloxycarbonyl-L-lysine-p-nitroanilide were pooled, dialyzed against 20 mM Tris–1 mM CaCl2 (pH 7.6), and applied to a MonoQ column (Pharmacia-Amersham fast-flow protein liquid chromatography system) equilibrated with the same buffer. The column was developed with 50 ml of an NaCl gradient from 0 to 0.2 M at the flow rate of 1 ml/min. Using these conditions, the activities of the catalytic domains of RgpA and Kgp were resolved, with the former eluting at the lower ionic strength. Final purification was
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achieved by rechromatography on the MonoQ column using the same conditions. The amount of active enzyme in each purified proteinase preparation (HRgpA, RgpB and its catalytic domain, and Kgp and its catalytic domain) was determined by active-site titration with D-phenylalanylpropylarginylchloromethyl ketone and benzyloxycarbonyl-Phe-Lys-2,4,6-trimethylbenzoyloxymethylketone, respectively (39). The concentration of active gingipains was calculated from the amount of inhibitor needed for complete inactivation of the proteinase. The knowledge of the precise molar concentration of active proteinases used in the experiments allowed direct comparison of the catalytic efficiencies of the various gingipains in E-cadherin degradation. The enzymes were activated in buffer (0.2 M HEPES, 1 mM CaCl2, pH 8.0) with a 10 mM final cysteine concentration for 10 to 20 min at 37°C. The protease inhibitors used in some experiments were acetyl-Leu-Val-Lys-aldehyde (AcLeu), which specifically inhibits the lysine-specific protease Kgp; leupeptin, which inhibits the active site of the arginine-specific enzymes HRgpA and RgpB; and L-trans-epoxysuccinyl-Leu-4-guanidinobutylamide (E-64), which inhibits PrtT and periodontain. PrtT and periodontain are cysteine endopeptidases with homology to the streptococcal pyogenic exotoxin B (27). These protease inhibitors were obtained from Bachem (Torrance, Calif.). Cell lysate preparation and immunoprecipitation of E-cadherin. MDCK cell cultures were used to prepare cell lysates as previously described (20). Nonadherent cells were harvested from the apical compartment of Transwell plates by centrifugation. The adherent cells on the filters were exposed to 0.2 ml of 20 mM Tris-HCl (pH 7.4)–150 mM NaCl–0.1% sodium dodecyl sulfate–1% Triton X-100–1% deoxycholic acid–5 mM EDTA (radioimmunoprecipitation assay [RIPA] buffer) containing inhibitors of proteases (2 mM phenylmethylsulfonyl fluoride, 50 g of pepstatin per ml, 50 g of chymostatin per ml, and 10 g of antipain per ml) for 15 min on ice. The adherent cells were scraped from the filter with a rubber policeman. The nonadherent cells from the apical compartment were then added to the RIPA buffer containing adherent cells from the respective filters. The total cell lysates were sedimented at 4°C in a Microfuge at maximum speed for 10 min to remove insoluble DNA. The protein concentration of each cell lysate was determined by using the bicinchoninic acid protein determination assay (Pierce Chemical Co., Rockford, Ill.). For the immunoprecipitation of E-cadherin, MDCK cells were cultured in 10-cm-diameter tissue culture petri dishes until confluent. Confluent monolayers were rinsed once in ice-cold PBS⫹ and lysed in 2 ml of RIPA buffer. RIPA buffer-soluble cell lysates were collected by centrifugation at 4°C in a Microfuge. The soluble cell lysates were rotated for 4 h at 4°C with 10 l of mouse anti-Ecadherin monoclonal antibody (Transduction Laboratories, Lexington, Ky.) and 1 ml of a 10% slurry of rabbit anti-mouse immunoglobulin G (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) coupled to protein A-Sepharose beads. E-cadherin-containing immunoprecipitate beads were washed three times with PBS⫹, resuspended in 500 l of PBS⫹, and stored in aliquots at ⫺20°C. Assessment of E-cadherin degradation by P. gingivalis or purified gingipains. P. gingivalis cells (1010/ml) were incubated with 100 M or 1 mM concentrations of the protease inhibitors Ac-Leu, leupeptin, and E-64 at room temperature for 20 min. Suspensions of P. gingivalis incubated with or without inhibitors or PBS⫹ as a control (5 l) were then added to immunoprecipitates of E-cadherin (20 l) and incubated for 10 min in a 37°C water bath. The reaction was stopped by the addition of 8 l of 4⫻ Laemmli buffer containing 100 mM dithiothreitol and boiling for 5 min. The samples were stored frozen (⫺20°C) until analyzed by Western blotting. In other experiments, purified HRgpA, RgpB, and Kgp were activated and 5 l of various concentrations of each protease (1, 10, and 100 nM) was incubated at 37°C with 20 l of immunoprecipitated E-cadherin for 10 min. In addition, the catalytic domains of HRgpA and Kgp (100 or 250 nM) were incubated with E-cadherin immunoprecipitates, and their degradative abilities were compared to those of the whole proteases. Hydrolysis of E-cadherin was analyzed by Western blotting. Assessment of MDCK cell-associated E-cadherin degradation by purified gingipains. Cultures of MDCK cells were plated at confluency on Transwell filters as described above. Purified and activated HRgpA, RgpB, and Kgp (100, 250, and 500 nM) or the catalytic domains of HRgpA and Kgp (100 or 250 nM) were added to the basolateral compartment of the wells, whereas medium was added to the apical compartment. After incubation at 37°C for 2, 4, 6, 8, and 24 h, cell lysates were prepared as described above. The reaction was stopped by the addition of 4⫻ Laemmli buffer containing 100 mM dithiothreitol and boiling. These samples were stored frozen until analyzed for E-cadherin degradation by Western blot analysis. Electrophoresis and Western blotting. Cell lysates or immunoprecipitates of E-cadherin incubated with or without purified P. gingivalis proteases were elec-
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FIG. 1. Effect of Ac-Leu, E-64, and leupeptin on P. gingivalis degradation of E-cadherin. The immunoblot analysis was performed as described in Materials and Methods.
trophoresed in sodium dodecyl sulfate-polyacrylamide minigels and transferred to Immobilon P filters (Millipore Corp., Bedford, Mass.). The filters were blocked for 60 min at room temperature with PBS⫺ (PBS⫹ without Ca2⫹ or Mg2⫹)–5% milk–0.1% Tween 20 (block solution) and probed with an E-cadherin monoclonal antibody (1:500) (Transduction Laboratories). The filters were then washed five times for 5 min each with PBS⫺ containing 0.1% Tween 20 (wash solution) and probed with horseradish peroxidase-labeled goat anti-mouse immunoglobulin G (1:25,000; Jackson ImmunoResearch Laboratories, Inc.) diluted in block solution for 60 min. Filters were washed five times for 5 min each with wash solution. All filters were visualized on Kodak X-OMAT AR film with an enhanced chemiluminescence kit (ECL; Amersham Corp., Piscataway, N.J.). Autoradiographs were scanned and saved as Adobe Photoshop files with a UMAX PowerLook II scanner.
RESULTS Inhibition of P. gingivalis degradation of E-cadherin. The cysteine proteases HRgpA, RgpB, and Kgp of P. gingivalis, which are known as gingipains, have been shown to participate in a number of processes of potential importance for the pathogenesis of periodontal disease (10, 22). Therefore, we wanted to determine the contribution of these proteases to the ability of P. gingivalis to degrade the E-cadherin adherens junctions of epithelial cells by utilizing the well known MDCK epithelial cell line (20). In an initial series of experiments, P. gingivalis cells were incubated with or without optimal amounts of the inhibitors Ac-Leu, leupeptin, and E-64, followed by incubation with immunoprecipitates of E-cadherin. Incubation of immunoprecipitated E-cadherin with P. gingivalis resulted in an almost complete degradation of E-cadherin (Fig. 1). The greatest inhibition of E-cadherin degradation by P. gingivalis occurred with Ac-Leu, which specifically inhibits the lysinespecific protease Kgp. Partial inhibition was also seen with
leupeptin, an inhibitor of the active site of the arginine-specific enzymes HRgpA and RgpB. No inhibition was observed with E-64. These results suggest that the gingipains associated with P. gingivalis cells, HRgpA, RgpB, and especially Kgp, contribute to the degradation of the adherens junction protein Ecadherin. Effect of purified gingipains on E-cadherin. In order to further establish that the observed degradation was indeed due to the P. gingivalis gingipains, we next examined the proteolytic effect of purified HRgpA, RgpB, and Kgp on immunoprecipitates of E-cadherin. The activated gingipains were incubated with immunoprecipitated E-cadherin for 10 min, which had been shown in a kinetic study to be optimal for degradation (data not shown). The gingipains HRgpA, RgpB, and Kgp were capable of degrading E-cadherin, and complete breakdown of the protein was seen with 10 and 100 nM Kgp, HRgpA, or RgpB (Fig. 2). A protease concentration of as low as 1 nM resulted in breakdown of E-cadherin. In order to determine the importance of the hemagglutinin domain for the catalytic activity of the proteases, we next tested the purified catalytic domains of HRgpA and Kgp for their ability to hydrolyze E-cadherin. We speculated that the whole HRgpA or Kgp proteases would be more efficient than the catalytic domains only. However, no difference was seen in the ability of the catalytic domains and the complete HRgpA and Kgp proteases to degrade E-cadherin (Fig. 3). These results indicate that the hemagglutinin domains of the gingipains are not required for the ability of the catalytic domains to degrade E-cadherin. Effect of purified gingipains on E-cadherin in epithelial cell
FIG. 2. Effect of purified Kgp, HRgpA, and RgpB on immunoprecipitated E-cadherin. The immunoblot analysis was performed as described in Materials and Methods.
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FIG. 3. Comparison between whole protease and the protease catalytic domain (cat) in degrading immunoprecipitates of E-cadherin. Aliquots of immunoprecipitated E-cadherin were incubated with 250 nM HRgpA or Kgp or their catalytic domains. The immunoblot analysis was performed as described in Materials and Methods.
monolayers. Epithelial cells throughout the body are polarized, which plays a significant role in resistance to infection (13). MDCK cells grown on permeant filters form a well-polarized cell monolayer, basically reconstituting a simple epithelial tissue. Essential for this process is E-cadherin-mediated cell-cell adhesion, the major structural component of the adherens junction. Using this in vitro model of simple epithelium, we next assessed the ability of the purified gingipains to degrade E-cadherin molecules in monolayers of MDCK cells. MDCK cell monolayers were exposed on their basolateral surface to the gingipains for various periods of time. Western blot analysis of cell lysates showed that Kgp caused significant degradation of E-cadherin (Fig. 4). Incubation of MDCK cell monolayers with HRgpA or RgpB resulted in essentially no degradation, even at a concentration of 500 nM. Since Kgp was able to hydrolyze the E-cadherin involved in forming the adherens junction among MDCK cells, we next wanted to evaluate the effectiveness of the catalytic domains of HRgpA and Kgp alone in this process. Although we have shown that 500 nM Kgp completely degraded the E-cadherin within 2 h of incubation in MDCK cells (Fig. 4), we decided to
FIG. 4. Effect of basolateral exposure of MDCK cell monolayers to purified proteases on E-cadherin after 2, 4, 6, 8, and 24 h. The preparation of cell lysates and the immunoblot analysis were performed as described in Materials and Methods.
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test lower concentrations of the gingipains in an effort to see differences in the degradative properties of the whole protease versus the catalytic domain over time. We selected concentrations of 100 and 250 nM to test because previous observations showed that 100 nM Kgp was enough to cause some hydrolysis of the adherens junctions of MDCK cells (data not shown). Western blot analysis showed that Kgp and the catalytic domain of Kgp were able to degrade the E-cadherin adherens junctions of MDCK cells (Fig. 5). At a lower concentration of Kgp (100 nM), the catalytic domain alone was similar to or slightly more effective than the whole molecule in the degradation of E-cadherin (Fig. 5A). When a 250 nM concentration of Kgp catalytic domain was assessed, partial hydrolysis of E-cadherin was evident at 2, 4, 8, and 24 h, while complete degradation occurred at 6 h (Fig. 5B). Complete hydrolysis of E-cadherin by the Kgp whole molecule was seen at 2, 4, and 6 h, whereas only partial or no degradation of E-cadherin was seen at 8 and 24 h. HRgpA and its catalytic domain showed minimal but similar degradation of E-cadherin at 100 and 250 nM, although the whole HRgpA molecule seemed to be slightly more effective than the catalytic domain (Fig. 5). The finding that the Kgp whole molecule appeared to be more effective than the catalytic domain in degrading the adherens junction E-cadherin of MDCK cells at 250 nM suggests a role for the hemagglutinin/adhesin domain of this protease in this degradative activity. Furthermore, the results indicate that 6 h is a critical time for Kgp hydrolysis of E-cadherin in MDCK cells. Taken together, these findings indicate that Kgp is the major gingipain involved in the degradation of the E-cadherin adherens junction under the conditions tested. DISCUSSION P. gingivalis has been shown to invade gingival epithelial cells (40) and to replicate in cultures of primary gingival epithelial cells and oral epithelial cell lines in vitro (11, 25, 41, 42). These studies and others (2, 26) on P. gingivalis invasion indicate that this process occurs via an intracellular pathway. Recently, our laboratory has provided evidence that P. gingivalis can hydrolyze the interconnecting adherens junction E-cadherin molecules in an in vitro model of simple epithelium using monolayers of polarized MDCK cells (20). These observations lend support to the concept that P. gingivalis can also gain access to the underlying connective tissue by a paracellular pathway. However, the precise mechanism(s) accounting for this type of invasion was not defined. P. gingivalis produces a battery of proteases with powerful proteolytic activities (24). Among these proteases are the well characterized gingipains HRgpA, RgpB, and Kgp (36). In the present study, evidence is provided that the major proteolytic activity involved in the degradation of E-cadherin and the breakdown of the adherens junction of the epithelium was derived from the lysine-specific protease Kgp. It was initially thought from our results with P. gingivalis cells that the activity attributed to Kgp hydrolysis of E-cadherin was perhaps due to a lesser concentration of arginine-specific proteases produced by P. gingivalis and not due to substrate availability, since E-cadherin has an equal availability of lysine- and arginine-specific sites. This possibility gained support from the finding that when equal amounts of HRgpA, RgpB, or Kgp were incubated with immunoprecipitated
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FIG. 5. Effect of basolateral exposure of MDCK cell monolayers to the catalytic domains and the whole molecules of purified Kgp and HRgpA at 100 nM (A) and 250 nM (B) on E-cadherin after 2, 4, 6, 8, and 24 h. The preparation of cell lysates and the immunoblot analysis were performed as described in Materials and Methods.
E-cadherin, similar amounts of degradation were seen, whereas a different degradation pattern was seen when MDCK cell monolayers were exposed to the purified gingipains; i.e., Kgp, but not HRgpA and RgpB, caused hydrolysis of E-cadherin. It is possible that the difference in the gingipain activities observed with immunoprecipitates and with the cell monolayers could be explained by the possibility that binding of the protease to a substrate such as epithelial cells requires certain signals or motifs (7, 33, 48) which are not required with E-cadherin immunoprecipitates. It is also possible that higher concentrations of HRgpA and RgpB than of Kgp are needed for the degradation of the adherens junction E-cadherin in MDCK cells. Concentrations higher than 500 nM were not tested because the amounts of proteases required to do such experiments are impractical. Alternatively, the kinetic activities of the arginine- and lysine-specific proteases may differ in that the arginine-specific proteases may be capable of degrading E-cadherin under experimental conditions similar to those for Kgp but with an extended experimental exposure time. However, under the experimental conditions used in these studies, the purified gingipains stay active for only 24 h (unpublished findings). The gingipains HRgpA and Kgp contain a catalytic domain and a hemagglutinin/adhesin domain. RgpB consists of a catalytic domain which is homologous to that of HRgpA, but it lacks the C-terminal region containing the hemagglutinin/adhesin domain (24, 34, 36). Studies by Pike et al. (35) demonstrated the potential of the hemagglutinin domain to adhere to fibrinogen, fibronectin, and laminin. Clinical studies by Booth and Lehner (3) showed that a monoclonal antibody which recognized epitopes of the hemagglutinin domain could prevent P. gingivalis recolonization of gingival tissue. These studies indicate the importance of the hemagglutinin/adhesin domains of gingipains in adhering to host tissues. In our study, no difference was seen in the ability of the whole HRgpA and Kgp proteases or their catalytic domains to hydrolyze E-cadherin immunoprecipitates, suggesting that the hemagglutinin/adhesin domain is not necessary for simple proteolysis of purified E-cadherin molecules. This was not the case when we evaluated the breakdown of adherens junction E-cadherin molecules of MDCK cell mono-
layers. In this system, minimum degradation was seen with either the catalytic domain only or the whole HRgpA protease. Conversely, the Kgp catalytic domain and the whole Kgp molecule were able to significantly degrade E-cadherin. Furthermore, the whole Kgp molecule completely hydrolyzed E-cadherin at 2, 4, and 6 h, whereas complete degradation with the catalytic domain alone was observed only at 6 h (Fig. 5B). These results indicate that the whole Kgp molecule is more efficient than its catalytic domain alone in degrading E-cadherin molecules in MDCK cell monolayers, thus suggesting a role for the hemagglutinin/adhesin domain in this process. Perhaps this region adheres to a host tissue or substrate which allows the catalytic domain to be more effective. It is interesting that after complete degradation of E-cadherin by Kgp during the first 6 h of incubation with MDCK cell monolayers, E-cadherin was detected at 8 and 24 h (Fig. 5). It is known that MDCK cells are constantly producing E-cadherin molecules at a high synthetic rate (44). Therefore, it is possible that the proteases are exhausted over time, whereas E-cadherin synthesis continues. Although a difference was seen in the activities of Kgp and its catalytic domain when a concentration of 250 nM was used, a lower concentration (100 nM) revealed essentially no difference in the degradative abilities of the Kgp catalytic domain alone and the whole molecule. In fact, the catalytic domain was perhaps slightly more effective than the whole molecule in degrading the E-cadherin of MDCK cells. Therefore, it is possible that a certain threshold level of enzyme is required for hydrolysis of E-cadherin in MDCK cell monolayers. In our previous studies (20), we observed that 1010 P. gingivalis cells were the minimum number of bacterial cells needed for hydrolysis of the adherens junction E-cadherin molecules in MDCK cell monolayers, which suggested that a certain threshold level of bacteria or bacterial products was necessary for degradation to occur. Although it is not known how much Kgp is produced by 1010 P. gingivalis bacterial cells, based on the conditions set in this study, it seems that a concentration of at least 100 nM is necessary. The degradation of interepithelial junctional proteins is not
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unique to the P. gingivalis gingipains. A zinc-dependent metalloproteinase toxin (BFT) derived from B. fragilis, the most commonly isolated anaerobe of the human colonic microflora implicated in diarrheal disease, has also been shown to degrade the zonula adherens E-cadherin protein (30, 49). Since P. gingivalis was at one time classified as a Bacteroides species (31, 32), it is tempting to speculate that the ability to cleave epithelial junctional proteins is a commonality among these seemingly related species. A variety of functions have been shown for the gingipains (5, 9, 16, 43, 50), but to our knowledge, this study is the first to provide evidence for differences in the catalytic abilities of the arginine-specific and lysine-specific gingipains and between the whole Kgp molecule and its catalytic domain alone. Furthermore, this study provides evidence that Kgp is the major gingipain involved in the degradation of adherent junction Ecadherin. This process could play an important role in periodontal disease pathogenesis. ACKNOWLEDGMENTS These studies were supported by Public Health Service grants DE 10607, DE 13269, and DE 08228; a grant from the University of Alabama at Birmingham Research Center for Oral Biology; a grant from the Medical Research Service of the Department of Veterans Affairs; and grant 6 PO4A 047 17 from the State Committee of Scientific Research (KBN, Poland) (to J.P.). D.F.B. is a recipient of a Veterans Affairs Career Development Award. REFERENCES 1. Barkocy-Gallagher, G. A., N. Han, J. M. Patti, J. Whitlock, A. ProgulskeFox, and M. S. Lantz. 1996. Analysis of the prtP gene encoding porphypain, a cysteine protease of Porphyromonas gingivalis. J. Bacteriol. 178:2734–2741. 2. Belton, C. M., K. T. Izutsu, P. C. Goodwin, Y. Park, and R. J. Lamont. 1999. Fluorescence image analysis of the association between Porphyromonas gingivalis and gingival epithelial cells. Cell. Microbiol. 1:215–223. 3. Booth, V., and T. Lehner. 1997. Characterization of the Porphyromonas gingivalis antigen recognized by a monoclonal antibody which prevents colonization by this organism. J. Periodontal Res. 32:54–60. 4. Brissette, C. A., and P. M. Fives-Taylor. 1999. Actinobacillus actinomycetemcomitans may utilize either actin-dependent or actin-independent mechanisms of invasion. Oral Microbiol. Immunol. 14:137–142. 5. Calkins, C. C., K. Platt, J. Potempa, and J. Travis. 1998. Inactivation of tumor necrosis factor alpha by proteinases (gingipains) from the periodontal pathogen, Porphyromonas gingivalis. Implications of immune evasion. J. Biol. Chem. 273:6611–6614. 6. Chen, P. B., L. B. Davern, and A. Aguirre. 1991. Experimental Porphyromonas gingivalis infection in nonimmune athymic BALB/c mice. Infect. Immun. 59:4706–4709. 7. Curtis, M. A., J. Aduse-Opoku, J. M. Slaney, M. Rangaraja, V. Booth, J. Cridland, and P. Shephard. 1996. Characterization of an adherence and antigenic determinant of the Arg1 protease of Porphyromonas gingivalis which is present on multiple gene products. Infect. Immun. 64:2532–2539. 8. Curtis, M. A., H. K. Kuramitsu, M. Lantz, F. L. Macrina, K. Nakayama, J. Potempa, E. C. Reynolds, and J. Aduse-Opoku. 1999. Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis. J. Periodontal Res. 34:464–472. 9. Darveau, R. P., C. M. Belton, R. A. Reife, and R. J. Lamont. 1998. Local chemokine paralysis, a novel pathogenic mechanism for Porphyromonas gingivalis. Infect. Immun. 66:1660–1665. 10. DeCarlo, A. A., M. Paramaesvaran, P. L. W. E. Yun, C. Collyer, and N. Hunter. 1999. Porphyrin-mediated binding to hemoglobin by the HA2 domain of cysteine proteinases (gingipains) and hemagglutinins from the periodontal pathogen Porphyromonas gingivalis. J. Bacteriol. 181:3784–3791. 11. Duncan, M. J., S. Nakao, Z. Skobe, and H. Xie. 1993. Interactions of Porphyromonas gingivalis with epithelial cells. Infect. Immun. 61:2260–2265. 12. Dzink, J. L., A. D. Haffajee, and S. S. Socransky. 1988. The predominant cultivable microbiota of active and inactive periodontal lesions. J. Clin. Periodontol. 15:316–323. 13. Fleiszig, S. M., D. J. Evans, N. Do, V. Vallas, S. Shin, and K. Mostov. 1997. Epithelial cell polarity affects susceptibility to Pseudomonas aeruginosa invasion and cytotoxicity. Infect. Immun. 65:2861–2867. 14. Genco, C. A., C. W. Cutler, D. Kapczynski, K. Maloney, and R. R. Arnold. 1991. A novel mouse model to study the virulence of and host response to Porphyromonas (Bacteroides) gingivalis. Infect. Immun. 59:1255–1263.
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Editor: R. N. Moore
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