From the Department of Biochemistry and Molecular
Biology, University of Georgia, Athens, Georgia 30602 and the
¶ Department of Microbiology and Immunology, Institute of
Molecular Biology, Jagiellonian University, 31-120 Krakow, Poland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Porphyromonas gingivalis is one of
the major pathogens associated with adult periodontitis, a major
chronic inflammatory disease. Potent proteinases elaborated by these
bacteria aid directly and indirectly in both the development of the
pathophysiology of the disease and in host defense evasion. For these
reasons they are considered key virulence factors. To investigate
whether possible immune evasion mechanisms involve the dysregulation of
the host cytokine network, we examined the ability of P. gingivalis cysteine proteinases, including Arg-specific
gingipains HRGP and RGP2 and Lys-specific KGP, to degrade the
proinflammatory cytokine tumor necrosis factor- (TNF-
). All three
gingipains rapidly degraded TNF-
as exhibited by immunoblot
analysis. Moreover, all biological activity was significantly reduced
over extended incubation periods with the proteinases tested, whereas
the host neutrophil proteinases were ineffective. These results
indicate that the gingipain proteinases elaborated by P. gingivalis are capable of disrupting the cytokine network at the
site of infection through the degradation of the proinflammatory
cytokine TNF-
, suggesting the removal of one of several mediators
important to the function of polymorphonuclear leukocytes. Such a
mechanism is likely to be utilized by other infective organisms not
only for survival but also for growth and proliferation.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adult periodontitis occurs as a result of a bacterial infection that, if left untreated, may result in tooth loss (1, 2). It is commonly characterized as a chronic inflammatory disease, exemplified by inflamed gingival tissues, increased crevicular fluid, and massive polymorphonuclear leukocyte (PMN)1 infiltration of the gingival tissues, all of which contribute to both connective tissue loss and alveolar bone loss (3, 4). The development of adult periodontitis is now believed to be a result of the uncontrolled activity of two members of a family of cysteine proteinases synthesized and secreted by the organism Porphyromonas gingivalis, an opportunistic pathogen that colonizes beneath the gum line in infected individuals. The two proteinase types are referred to as Arg-gingipains (HRGP and RGP2) and Lys-gingipain (KGP) because of their specificity for cleavage after arginyl and lysyl residues, respectively. HRGP differs from RGP2 in that the protein contains an additional adhesion domain accounting for its higher molecular mass in comparison to RGP2 (100 and 50 kDa, respectively). Significantly, the enzymatic activity of the gingipains is not susceptible to inhibition by human plasma proteinase inhibitors (5). For this reason one family member (RGP) can rapidly activate proteinases involved in the complement (6-8), kallikrein/kinin (9-11), and coagulation/fibrinolytic pathways (12, 13), nearly all of which require cleavage after arginyl-X residues, whereas the other (KGP) degrades fibrinogen (13). In addition, these enzymes can also inactivate many of the host plasma proteinase inhibitors, essentially dysregulating each of the cascade pathways.
Because P. gingivalis cannot extensively degrade proteins, it is now believed that this organism receives its amino acid/nitrogen requirements through the fragmentation of host proteins by proteinases produced by PMNs, including those found in crevicular fluid and, more importantly, gingival connective tissues. This apparently occurs through the release and/or activation of chemotactic factors such as C5a (6) and IL-8,2 which cause PMNs to migrate toward the bacterium. However, cleavage of chemotactic receptors from the PMN surface by RGPs and KGP inhibits complete migration and phagocytosis (8), with the result being the death and disintegration of these cells and the release of their potent proteolytic and oxidative enzymes, including elastase, cathepsin G, and myeloperoxidase.
The function of PMNs is primarily associated with the eradication of
bacterial infections and involves a complex interaction with cytokines,
which are produced by epithelial cells, fibroblasts, macrophages, and
lymphocytes. In particular, TNF-, a proinflammatory cytokine, not
only induces the production of chemotactic signals (IL-8), which result
in the recruitment of PMNs to infected sites (14-16), but also is
capable of acting in concert with these signals to enhance PMN function
(17-20). Although the presence of TNF-
has been demonstrated in
both the crevicular fluid and gingival tissues at infected sites of
patients with periodontitis through enzyme-linked immunosorbent assay
or in situ hybridization methodology (21-25), these
procedures are incapable of measuring functional protein. Thus, the
question arises as to whether TNF-
post-secretion is biologically
active.
Here, we report that members of the gingipain family are capable of
degrading TNF-. Additionally, we identify the cleavage points made
by the individual gingipains within the TNF-
molecule. More
importantly, these observations correlate with the loss of the TNF-
cytolytic activity. Significantly, host proteinases, human neutrophil
elastase (HNE), and cathepsin G (cat G) do not degrade TNF-
and thus
do not affect this specific activity. These results suggest that
pathogenic bacterium may use proteolysis as a means of dysregulating
cytokine activity, ultimately contributing to the evasion of host
defenses.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture and Reagents--
The mouse L929 connective tissue
cell line was obtained from the American Type Culture Collection. Cells
were grown at 37 °C in RPMI 1640 medium supplemented with 5% fetal
bovine serum and antibiotics (Sigma). Highly purified TNF- was a
kind gift of Bayer corporation (Berkeley, CA). Three major forms of
gingipain, HRGP, RGP1, and KGP, were purified according to the method
of Pike et al. (26). Cathepsin G and HNE were purified as
described previously (27). Polyclonal antibodies against human
recombinant TNF-
were obtained from Genzyme (Cambridge, MA), and
goat anti-rabbit IgG conjugated to horseradish peroxidase was purchased
from Pierce. H-D-Phe-Pro-Arg-chloromethylketone was from
Bachem Biosciences (Philadelphia, PA).
N
-benzoyl-DL-Arg-pNA,
methoxy-Suc-Ala-Ala-Pro-Val-pNA, and Suc-Ala-Ala-Pro-Phe-pNA were from
Sigma, whereas carboxybenzoyl-Lys-pNA was purchased from NovaBiochem
(La Jolla, CA).
Enzyme Activity Assay--
The amidolytic activity of the
purified gingipains HRGP, RGP2, and KGP was determined with either
N-benzoyl-DL-Arg-pNA or carboxybenzoyl-Lys-pNA. Samples were preincubated in 200 mM
Hepes, 5 mM CaCl2, and 10 mM
cysteine, pH 7.6, for 5 min, 37 °C and then assayed for amidase
activity with 1 mM substrate. The formation of
p-nitroanaline was monitored spectrophotometrically at 405 nm. Amidolytic activities of the host enzymes HNE and cat G were measured spectrophotometrically using the substrates
methoxy-Suc-Ala-Ala-Pro-Val-pNA for HNE and Suc-Ala-Ala-Pro-Phe-pNA for
cat G.
Biological Assay for TNF---
The activity of TNF-
and
fragments of TNF-
was monitored by the method of Mathews and Neale
(28). Briefly, L929 cells were grown in 96-well flat bottom plates
(Falcon Plastics) at 3 × 105 cells/ml and incubated
with a serially diluted test sample of TNF-
or digested reaction
mixtures of TNF-
(equivalent to a final concentration of 0.0375 ng/ml active TNF-
) in the presence of 1 µg/ml actinomycin D (final
concentration) in a humidified atmosphere at 37 °C with 5%
CO2. After 20 h the culture medium was removed, the
plates were washed, the remaining cells were fixed with 5%
formaldehyde, and cell lysis was detected by staining the plates with a
0.2% solution of crystal violet. Stained cells were solubilized with
33% acetic acid. The end point on the microplates was determined
spectrophotometrically by a Vmax microplate
reader (Molecular Devices, Sunnyvale, CA) set for absorption at 562 nm. Cells exposed to culture medium alone were set at 0% lysis, whereas 100% lysis was equivalent to blank wells. Curves were normalized to
zero time point cytolytic activity, expressed as the percentage of
control.
Immunoblot Analysis--
TNF- digest samples were subjected
to 16% SDS-Tricine polyacrylamide gel electrophoresis (29), followed
by electroblotting to nitrocellulose membranes. The primary antibody,
rabbit anti-human recombinant TNF-
serum, was diluted 1:1000 prior
to use. The secondary antibody, goat anti-rabbit IgG conjugated to
horseradish peroxidase, was diluted 1:12,000. Immunoblots were
visualized by chemiluminesence (Amersham Corp.).
Time Course Digest--
Enzymatic digestion of TNF- was
performed at E:S molar ratios of 1:25 or 1:100. Briefly, reaction
mixtures containing 1180 nM TNF-
and either 47.2 or 11.8 nM active enzyme in digest buffer (200 mM
Hepes, 5 mM CaCl2, 10 mM cysteine,
pH 7.6) were allowed to proceed for indicated times, whereupon enzyme
activity was extinguished by the addition of the cysteine proteinase
inhibitor FPRck. Each time point was divided such that 1 µl was set
aside for biological assays, with the remainder being utilized for
immunoblot analysis.
Amino Acid Sequence Determination--
Digestion of TNF- (2 µg) by gingipain HRGP, RGP2, or KGP was performed in digest buffer at
37 °C for 120, 10, or 120 min, respectively, at an E:S molar ratio
of 1:100. Peptides from TNF-
were resolved by 16% SDS-Tricine
polyacrylamide electrophoresis, electrotransferred to PVDF membrane,
and stained with Coomassie Blue. Specific bands were excised and
subjected to automated Edman sequence analysis using the Applied
Biosystems 494 Protein Sequencer (Foster City, CA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To investigate the potential immunomodulatory activities of
cysteine proteinases elaborated from P. gingivalis, we first
examined the ability of homogeneous preparations of three gingipains
(HRGP, RGP2, and KGP) to degrade TNF-. Fig.
1 illustrates cleavage of TNF-
by
gingipains as a function of time, with protein degradation being
monitored by immunoblot analysis, utilizing a polyclonal antibody
raised against human recombinant TNF-
. When reaction digests at an
E:S molar ratio of 1:25 were incubated at 37 °C for up to 8 h,
we observed that all three gingipains were capable of degrading
TNF-
. More specifically, RGP2 was capable of significantly reducing
the level of the 17-kDa TNF-
band within 10 min, whereas both
gingipain HRGP and KGP required additional time, with complete degradation by 240 min. As a comparison study, similar experiments were
performed using homogeneous preparations of the host neutrophil serine
proteinases HNE and cat G. Significantly, no detectable degradation of
TNF-
was observed with either enzyme as determined by immunoblot
analysis (Fig. 2).
|
|
To test whether the proteolytic degradation of TNF- correlated with
decreased biological activity of the cytokine, digest reactions were
also monitored for residual biological activity in a TNF-
-specific
cytolytic assay. Using aliquots from the digest mixtures above, samples
were diluted to within the working range (0.080 pg/ml) of the assay
(Fig. 3). Gingipain RGP2 reduced TNF-
activity by 75% within 10 min (Fig. 3B), whereas both HRGP
and KGP (Fig. 3, A and C, respectively) were
significantly more sluggish, requiring 4 h to eliminate all
TNF-
activity. When parallel activity studies were performed using
the host proteinases HNE and cat G, no significant loss in cytolytic
activity was detected (data not shown), again correlating with the
immunoblot result. Degradation and inactivation by gingipains was also
confirmed in parallel experiments using decreased molar ratios of
enzyme to substrate (1:100), albeit at a slower rate.
|
We next investigated the sites of proteolytic cleavage within the
TNF- molecule by the individual gingipains. The specific digestion
conditions for each proteinase were optimized so as to get several
cleavage products after separation by SDS-polyacrylamide gel
electrophoresis, electrotransfer to PVDF membrane, and Coomassie Blue
staining. Both HRGP and RGP2 were found to cleave TNF-
after Arg2 and Arg6 (Fig.
4). A third point of cleavage, determined
only for RGP2, occurred at residue Arg32. Points of
cleavage by KGP were at Lys63, Lys90, and
Lys98.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Periodontopathic bacterial infection activates the host inflammatory response, leading to the rapid infiltration of PMNs along with an increased flow of crevicular fluid. Significantly, substances from the bacteria initiate and drive the response through the up-regulation of inflammatory mediators such as cytokines, e.g. IL-8, that are synthesized and released from resident fibroblasts, endothelial cells, and epithelial cells, in addition to leukocytes and lymphocytes. These mediators play a major role in amplification and perpetuation of inflammation, as well as in the destruction of host gingival tissues (30).
The gingipains, RGPs and KGP, are believed to be key virulence factors elaborated from P. gingivalis, the Gram-negative bacterium most often associated with adult periodontitis (31, 32). Significantly, these enzymes have been shown to activate several proteolytic cascades associated with the host inflammatory response, including those involved in the complement (6-8), kallikrein/kinin (9-11), and coagulation/fibrinolytic pathways (12, 13). Such activities, together with an ability to inactivate host plasma proteinase inhibitors, can clearly lead to a dysregulation of the inflammatory response.
The proinflammatory cytokine TNF- is synthesized by cells in
response to inflammatory mediators. In addition, it may induce the
production of other cytokines, including IL-1 and IL-8 (14-16). Although up-regulated levels of TNF-
at infected periodontal sites,
as compared with healthy gingival tissue, have been demonstrated through both enzyme-linked immunosorbent assay and in situ
hybridization studies (21-25), the activity of this cytokine was not
tested, and therefore the results are difficult to evaluate. In the
present study we analyzed the ability of three individual gingipains, HRGP, RGP2, and KGP, to digest TNF-
. We chose a pH value of 7.6 for
these experiments, primarily because the pH of the gingival crevicular
fluid at inflamed sites has been shown to be more alkaline (pH
7.4-8.5) than that of clinically healthy sites (pH 6.7-7.2) (33-36).
Significantly, both RGPs reduced the level of the 17-kDa TNF-
band
within 10 min, at an E:S ratio of 1:25. In contrast, KGP needed nearly
4 h for complete digestion of the TNF-
band.
During our studies on gingipain inactivation of TNF-, we identified
the points of cleavage associated with each enzyme. Previous studies,
reviewed in Goh and Porter (37), demonstrated that the first nine
residues of the TNF-
N terminus are not necessary for biological
activity, although a 3.7-fold increase in activity was observed upon
deletion of the first seven residues of the N terminus. Both HRGP and
RGP2 initially cleaved after residues Arg2 and
Arg6. RGP2 additionally caused fragmentation at
Arg32, a residue important for biological activity because
a conserved substitution at this position results in a loss of activity
(37). Although cleavage at Arg32 by HRGP was not confirmed
by this method, the fragmentation pattern was similar to that of RGP2.
For KGP, cleavage occurred after residues Lys63,
Lys98, and Lys90. Taken together these results
demonstrate that the gingipain cysteine proteinases are capable of not
only trimming the N terminus of TNF-
but also of cleaving the
polypeptide at several internal residues buried within the molecule,
thus rendering the proinflammatory cytokine inactive.
It is significant that in these studies protein degradation correlated
with the loss of TNF- cytolytic activity (Fig. 3), which was
significantly reduced within 10 min upon treatment with gingipain RGP2.
Additional incubation did not reduce activity further. As expected from
the protein fragmentation pattern, cytolytic inactivation by either
HRGP or KGP occurred at a slower rate but was still complete within
4 h. Despite the apparent disappearance of the 17-kDa substrate
within the first 60 min of digestion with KGP (Fig. 1C),
these samples possessed essentially 100% biological activity (Fig.
3C), suggesting an artificially low representation of
substrate and/or limitation of immunodetection method. Nevertheless, these results, taken in total, indicate that TNF-
is rapidly degraded and inactivated by the gingipains tested, albeit most rapidly
by RGP2.
In addition to non-host proteinases we also investigated the ability of
the host PMN proteinases HNE and cat G to degrade TNF-, because
these enzymes are also likely to be present at inflammatory sites.
Significantly, they were ineffectual in both degradation (Fig. 2) and
inactivation, suggesting that TNF-
is resistant to proteolytic
digestion by PMN-derived enzymes. We are continuing these studies to
determine whether TNF-
and/or other cytokines are also refractory to
proteinases secreted by host cells, in general, and more specifically
to those classified as metalloproteinases because the latter are
secreted by numerous cell types that would be present at inflammatory
sites.
In conclusion, we have demonstrated that the bacterial cysteine
proteinases elaborated by P. gingivalis are capable of
digesting TNF-, an important inflammatory mediator. This strongly
suggests a local dysregulation of the cytokine network and, ultimately, interruption of the host defense response, including the dysfunction of
PMNs. Moreover, this study supports the belief that proteolysis can be
used as a mechanism of virulence by pathogenic organisms, ultimately
leading to host immune evasion.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant DE-09761 from the NIDR, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Medical College of Georgia, Augusta, Georgia 30912.
To whom correspondence should be addressed. Tel.:
706-542-1711; Fax: 706-542-3719; E-mail: jtravis{at}uga.cc.uga.edu.
1
The abbreviations used are: PMN,
polymorphonuclear leukocytes; TNF-, tumor necrosis factor-
; HRGP,
adhesion domain containing Arg-gingipain 1; RGP2, Arg-gingipain 2; KGP,
Lys-gingipain; HNE, human neutrophil elastase; cat G, cathepsin G; pNA,
p-nitroanilide; Suc, succinyl; PVDF, polyvinylidene
difluoride; IL, interleukin; Tricine,
N-tris(hydroxymethyl)methylglycine.
2 J. Mikolajczyk-Pawlinska, J. Potempa, J. Travis, manuscript in preparation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|