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Introduction
A significant percentage of the human population
suffers from perio dontitis, a chronic inflammatory disease
characterized by the breakdown of periodontal
tissue[1]. The incidence and rate of progression of this disease involve
complex interactions among periodontopathic bacteria as well
as between periodontopathic bacteria and host
cells[2,3]. These interactions lead host cells to release a broad array of
inflammatory cytokines, chemokines, and mediators, some
of which result in the destruction of periodontal supporting
tissue, namely alveolar bone, periodontal ligament, and
cementum around teeth[2,3]. The Gram-negative anaerobic
bacterium Porphyromonas gingivalis is implicated as one of
the important etiological agents of chronic and aggressive
forms of periodontitis[4_7]. This organism expresses a
number of potential virulence factors, including fimbriae,
superoxide dismutase, hemagglutinin, the arginine-X-specific
cysteine proteases, and the lysine-X-specific
proteases[8]. In addition, collagenolytic activity of P gingivalis has been described and is associated with the production of several
proteases[8]. Since collagen is an important component of
the periodontium, collagenase activity plays a critical role in
tissue destruction and progression of
periodontitis[9]. Specific cleavage of type I collagen has been attributed to the
function of the prtC gene product, which is referred to as
collagenase[8]. Takahashi and Kato were the first to report
the cloning and expression of the prtC gene from P gingivalis ATCC 33277[10,11]. While the prtC gene product did not exhibit structural similarity with eukaryotic collagenases, it was
able to degrade soluble and reconstituted fibrillar type I
collagen, heat-denatured type I collagen, and azocoll, but
did not degrade synthetic collagenase substrates and did
not contain a partial zinc-binding region that is consensus in
these enzymes[12]. These features may reflect the unique
nature of this collagenase or perhaps question its status as
a true collagenase[12]. Nonetheless, the inability of a
collagenolytic enzyme to degrade native fibrillar collagen does
not preclude its involvement in the pathogenesis of
periodontal disease[12]. Proteinase produced by some bacteria
could have a direct effect on inducing inflammation besides
its specific enzyme activity[13] and may work in combination
with true bacterial or host
collagenases[14,15]. Recent findings indicate that P gingivalis induces host collagen degradation by affecting expression, activation, and inhibition of
matrix metalloproteinases (MMP) produced by host
cells[8,16]. MMP is thought to be transcriptionally upregulated by
pro-inflammatory mediators, such as interleukin (IL)-1 and
TNF-α, as well as post-translationally activated by proteases from P gingivalis[16]. However, studies on the prtC gene product of P gingivalis mainly concentrate on its collagenolytic
activity. Its ability to induce host cells to produce
inflammatory cytokines has yet not been examined, which is of great
importance in understanding its role in the pathogenesis
and development of periodontitis.
Several studies have demonstrated that P
gingivalis is able to invade and activate different cell types in the
surrounding tissue of teeth, including endothelial, gingival
epithelial cells, as well as periodontal ligament
cells[14,15,17]. Endothelial cells, therefore, can act as primary target cells
during infection with P gingivalis. Endothelial cells are key
players during inflammatory reactions and are able to
produce an array of pro-inflammatory mediators, including
cytokines such as IL-1, IL-6, IL-8, TNF-α, and lipid
mediators like prostaglandins or platelet-activated
factor[17,18]. A previous report has indicated that
P gingivalis can infect human umbilical vein endothelial cells (HUVEC) and trigger
a cascade of events that could lead to endothelial damage,
as well as local and systemic
inflammation[17]. Moreover, host cells sense live
P gingivalis and its components such as lipopolysaccharides (LPS) or fimbriae differently, which
translate into the expression of different inflammatory
cytokine profiles[19]. The objective of the present work is to
investigate the effect of the P gingivalis PrtC protein on the
expression of cytokines in HUVEC-originated ECV304 cells,
which has been shown to be a suitable in
vitro model for the study of endothelial
cells[20,21,22], and subsequently, to
determine the possible association of P
gingivalis PrtC levels with periodontal conditions.
Materials and methods
In vitro study
P gingivalis strains and growth
condition The P gingivalis strain ATCC 33277 was supplied by the
Department of Medical Microbiology and Parasitology,
College of Medicine, Zhejiang University, China. They were
inoculated on trypticase soy agar supplemented with 5
µg/mL haemin, 1 µg/mL vitamin K1, 5%
(v/v) sheep blood, and 1 µg/mL menadione. The plates were incubated in an
anaerobic chamber under atmosphere condition of 80%
N2, 10% CO2, and 10%
H2 at 37 °C for 7 d.
Cloning, sequencing and construction of recombinant
expression vector The P gingivalis prtC was amplified
from type strain ATCC 33277 by PCR with a forward primer
containing the site of BamH I:
5'-GGGGGATCCCTCATGCG-CTCCGTCATC-3', and a reverse primer with the site of
Xho I: 5'-GGGCTCGAGTTATTCTTCTCTTTTGTC-3', according to
published sequence (GenBank Accession No AB006973).
The total volume per reaction was 100 µL, containing 0.25
mmol/L each dNTP, 1 µmol/L each of the primers, 3.0 U
Taq-Pfu polymerase, 20 mmol/L
MgCl2, 100 ng DNA template, and 1×PCR buffer (pH 8.3). The PCR conditions were as
follows: 94 °C for 5 min, 1 cycle; 94 °C for 30 s, 50 °C for 30 s,
72 °C for 90 s, 10 cycles; 94 °C for 30 s, 50 °C for 30 s, 72 °C for
100 s, 20 cycles; and 72 °C for 10 min, 1 cycle. The PCR
products were analyzed by 1.5% agarose gel prestained with
ethidium bromide and visualized under UV light. The
expected size of the target amplification fragment for
prtC was 1005 bp.
The prtC fragment was cloned into the pUCm-T vector
by using the T-A cloning kit according to the manufacturer's
instructions (Biocolor, Shanghai, China). The recombinant
plasmid (pUCm-T-prtC) was first transformed into the
Escherichia coli strain DH5α. After nucleotide sequencing
confirmed the inserted fragments, the E
coli DH5α strains containing
pUCm-T-prtC or the prokaryotic expression
vector pET32a were amplified and then the plasmids were
extracted. pUCm-T-prtC and the pET32a vector were both
digested with BamHI and XhoI. The target fragments of the
prtC gene and pET32a were recovered and then ligated with
a ligation kit (TaKaRa Biomedicals, Otsu, Shiga, Japan). The
recombinant expression vector pET32a-prtC was transformed
into E coli strain BL21 (DE3). Then the plasmids were
extracted and sequenced again.
Expression and identification of the target
recombinant protein The constructed prokaryotic expression
system pET32a-prtC-E coli BL21DE3 was rotationally cultured
in Luria-Bertani (LB) medium at 30 °C under inducement of
0.5 mmol/L isopropyl-1-thio-β-galactoside (IPTG). The
supernatant and precipitate were isolated by centrifugation
after the bacterium was ultrasonically broken. SDS-PAGE
(10%) was used to examine molecular weight, output, and
location of the target recombinant protein (rPrtC). rPrtC was
then collected and purified by affinity chromatography on a
Ni-NTA column (QIAGEN, Hilden, Germany). Following
elution with imidazole, contaminated LPS was removed by
Triton X-114 extraction. Then the protein was chromatographed
on Sephadex G25 columns (Amersham Pharmacia Biotech,
Piscataway, NJ, USA) and eluted in phosphate-buffered
solution. Solution of the recombinant protein 10 µg/mL was
found to be free of endotoxins by the
Limulus amoebocyte lysate assay (Bio Whittaker, Walkersville, MD, USA).
Rabbits were routinely immunized with 1 mg purified rPrtC or 1
mg whole cells of P gingivalis supplemented with complete
Freund's adjuvant, administered subcutaneously 4 times.
Slide agglutination reaction and ELISA were used to detect
the immunoreactivity of rabbit anti-rPrtC antiserum against
P gingivalis ATCC 33277, Prevotella
melaninogenica ATCC 25845, Fusobacterium
nucleatum ATCC 25586, E coli ATCC 25922,
Actinomycetemcomitans actinobacillus Y3,
Veillonella parvula 990116, and 100 µg/mL of their
sonicated supernatants, respectively. After electrophoresis on
10% SDS-PAGE, the rPrtC protein was then transferred to a
polyvinylidene fluoride (PVDF) membrane using the
Trans-Blot SD apparatus (Bio-Rad, Richmond, CA, USA). The blot
was blocked for 1 h in Tris-buffered saline containing 0.1%
Tween-20 (TBST) and 5% (w/v) skim milk and then
incubated with the rabbit antisera (1:1500 dilution) for 1 h. After
washing unbound primary antibodies with TBST 3 times for
10 min each, the blot was treated with horseradish
peroxidase (HRP)-conjugated sheep anti-rabbit immunoglobin G
(1:3000 dilution; Jackson ImmunoResearch, West Grove, PA,
USA) and developed with the ECL system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
ELISA detection of inflammatory cytokine secretion
in ECV304 cells induced by rPrtC HUVEC ECV304 was
maintained at a density of 5×103 cells/well in a 96-well plate
in RPMI-1640 medium (Gibco BRL, Gaithersburg, MD, USA)
supplemented with 10% heat-inactivated fetal bovine serum
(FBS) in a humidified atmosphere of 5%
CO2 in air at 37 °C for 24 h. Then the ECV304 cells were cultured in RPMI-1640
medium supplemented with 2% FBS which contained 1, 5,
and 10 µg/mL rPrtC for 12, 24, or 48 h, respectively. Each
concentration and time interval was repeated 3 times. The
supernatants were collected for the ELISA detection of
IL-1α, IL-8, and TNF-α (R&D Systems, Minneapolis, MN, USA).
Recombinant TNF-α (0.1µg, SibEnzyme, Novosibirsk, Siberia,
Russia) and RPMI-1640 medium containing 2% FBS were
used as positive and negative controls, respectively.
In vivo study
Patients The patients were 49 Chinese chronic
periodontitis (CP) patients [24 males aged 29_66 years (mean
age, 45.7±8.9 years) and 25 females aged 33_67 years (mean
age, 44.3±7.6 years)], and 25 individuals with healthy
periodontium [11 males aged 26_64 years (mean age, 41.8±5.8
years) and 14 females aged 22_63 years (mean age, 41.3±6.8
years)] who were referred to the dental clinic in the Second
Affiliated Hospital, College of Medicine, Zhejiang
University for dental or periodontal treatment or health monitoring.
All the patients were non-smokers without any systemic
disease, and had at least 14 teeth. Those who had received
professional cleaning or had history of antibiotic therapy
during the preceding 3 months were excluded. All of the
patients and the healthy individuals underwent a full mouth
examination. The criteria of diagnosis for chronic
periodontitis were based on the Classification of the Periodontal
Diseases issued by American Academy of Periodontology in
1999[23]. Briefly, the 49 generalized CP patients had >30%
sites showing periodontal probing depth ¡Ý3 mm, clinical
attachment loss >1 mm, and radiographic evidence of
alveolar bone loss. Individuals with periodontal probing depth
less than 3 mm without any clinical attachment loss or
radiographic evidence of bone loss and without inflammation of
gingivae were considered healthy. All the patients received
detailed information concerning the nature of the study and
the procedures involved, and their informed consent was
obtained. The ethical committee of the College of Medicine
at Zhejiang University approved the study protocol.
Collection of samples For each patient, 4 subgingival
plaque samples were taken from the bottom of the 4 deepest
periodontal pockets of the dentition, preferably 1 pocket from
each quadrant, with separate sterile Gracy curettes after
supragingival plaque was gently removed. After initial
periodontal treatment that mainly included oral hygiene
instruction, full mouth supragingival and subgingival
scaling and root planning, subgingival plaque samples from the
same sites were collected again 4 months later. For
individuals with healthy periodontium, samples from the bottom of
gingival sulcular were taken with the same method. Each of
the samples was placed in 200 µL lysis buffer, which
consisted of 1 mmol/L EDTA, 1% Triton X-100, and 10 mmol/L
Tris-HCl (pH 8.0), and stored at -20 °C until use. Clinical
parameters, such as probing depth (PD), attachment loss
(AL), and bleeding on probing (BOP) at 6 sites of each tooth
of the full dentition were recorded at baseline and after initial
periodontal treatment. The 6 sites were the mesial-buccal,
middle-buccal, distal-buccal, mesial-lingual/palatal,
middle-lingual/palatal, and distal-lingual/palatal sites of each tooth.
PCR detection of P gingivalis in subgingival plaque
samples Lysis buffer 100 µL containing subgingival plaque
samples was boiled for 10 min, and 10 µL supernatant was
directly used as a template in the PCR. A multiplex PCR
assay was established to detect the P
gingivalis 16SrDNA and prtC genes. The sequences of the primers specific for
the P gingivalis 16SrDNA gene were: 5'-AGG CAG CTT GCC
ATA CTG CG-3' (sense) and 5'-ACT GTT AGC AAC TAC CGA TGT-3'
(antisense)[24]. For the prtC gene they were:
5'-ACA ATC CAC GAG ACC ATC-3' (sense) and 5'-TTC AGC
CAC ACC GAG ACG-3'
(antisense)[25]. PCR amplification was
performed in a total volume of 100 µL, containing 10 µL of
the template, 10 µL PCR buffer (pH 8.3), and 3U
EX-Taq polymerase (TaKaRa Biomedicals, Japan), 0.25 mmol/L dNTP, 2.5
mmol/L MgCl2, 250 nmol/L primers each for
P gingivalis 16SrDNA and prtC genes. The PCR programs for the
detection of P gingivalis included an initial denaturation step at
94 °C for 5 min, followed by 35 cycles of denaturation at
94 °C for 30 s, primer annealing at 52 °C for 30 s, extension at
72 °C for 1 min, and then a final extension step at 72 °C for 7
min. In each of the PCR assays, 10 ng P
gingivalis ATCC 33277 DNA preparation was co-amplified with the
subgingival plaque samples as the positive control for the detection
of P gingivalis. The expected sizes of the PCR products
amplified from the P gingivalis 16SrDNA and
prtC genes was 404 bp and 584
bp[24,25]. Each reaction product 10 µL
were mixed with 10 µL of 2×loading buffer and fractionated
in a 2% agarose gel containing ethidium bromide (1 µg/mL),
using a 100 bp DNA ladder (Promega, Madison, WI, USA)
as a size marker, and visualized under UV light.
ELISA detection of PrtC levels in subgingival plaque
samples The subgingival plaque samples were sonicated,
and the protein concentration in each of the samples was
measured by UV spectrophotometry. Each sample was
diluted to 20 µg/mL protein with coating buffer in ELISA. By
using 0.1 mL of the diluted samples as the coating antigen,
the self-prepared rabbit anti-rPrtC serum (1:1000 dilution) as
the first antibody, and HRP-labeled sheep anti-rabbit IgG
(1:3000 dilution) as the second antibody, the PrtC levels in
the samples were detected. A model 680 microplate reader
(Bio-Rad, USA) was used to detect the
A490 value. The ELISA result of a sample was considered positive if its
A490 value was over the mean plus 3 times that of standard deviation
(SD) of the sonicated sulcular samples with the same protein
concentration from the 25 individuals with healthy
periodontium who were used as negative controls in the test. Five
wells of 20 µg/mL rPrtC were used as the positive control.
Statistical analysis The clinical and laboratory data were
presented as mean±SD. Student's t-test and ANOVA were
used to determine the significance of the differences
between the sub-groups. P<0.05 was considered
statistically significant.
Results
PCR detection of P gingivalis in subgingival plaque
samples The 25 individuals with healthy periodontium were
all negative for P gingivalis. In the 196 subgingival samples
from CP patients, 95.9% (188/196) and 91.8% (180/196) of the
samples were positive for P gingivalis
16SrDNA and prtC, respectively. None of the samples were positive for
prtC and negative for 16SrDNA (Figure 1).
Nucleotide sequence analysis The target fragments with
the expected size amplified from genomic DNA of
P gingivalis strain ATCC 33277 are shown in Figure 2. Compared with the
published sequences (GenBank Accession No AB006973),
the homology of the nucleotide and putative amino acid
sequences of the amplification fragment were 98.46% and
99.07%, respectively.
Expression of target fusion protein IPTG 0.5 mmol/L
was able to efficiently induce rPrtC expression (Figure 3).
Purified rPrtC showed 1 single band on 10% SDS-PAGE. rPrtC
was mainly presented in ultrasonic precipitation and the
output was approximately 50% of the total bacterial proteins.
Antigenicity and immunoreactivity of rPrtC
The rabbit anti-rPrtC antisera was found positive for
P gingivalis ATCC 33277 by slide agglutination reaction; the
A490 value of the supernatants was 0.89±0.10 as determined by ELISA. The
slide agglutination reactions with the other 5 species were
all negative; the A490 value was 0.01~0.03±0.02~0.03, which
indicated that the antisera were specific. Furthermore,
Western blotting indicated that rPrtC could combine with the
rabbit anti-rPrtC serum and rabbit antiserum against the whole
cell of P gingivalis, and only one distinct band was
shown(Figure 4). It demonstrated that rPrtC could induce rabbit to
produce specific antiserum, showing good antigenicity of
the recombinant protein. The fact rPrtC could combine with
these antiserum also implied its immunoreactivity.
Effects of rPrtC on the secretion of inflammatory
cytokines in ECV304 cells After the ECV304 cells were
treated with 1 µg/mL rPrtC for 24 h or with 5 and 10 µg/mL
rPrtC for 12 h, the secretion of IL-1α, IL-8 and
TNF-α in the supernatants increased significantly
(P>0.05), among which the IL-1α level peaked at 24 h and the IL-8 and
TNF-α levels increased gradually with time. Slightly higher levels of
IL-1α and TNF-α were shown in the ECV304 cells stimulated with
5 or 10 µg/mL rPrtC than with 1 µg/mL rPrtC for the same time
intervals (Figure 5).
PrtC level in subgingival plaque samples before and
after treatment The mean±SD at
A490 of the negative sulcular samples was 0.06±0.06, and the positive reference value was
0.24. According to this, 90.8% (178/196) of the subgingival
plaque samples were positive for PrtC with an
A490 value ranging from 0.26 to 1.23. All these 178 PrtC positive results
were from prtC gene positive samples. Only 2 samples were
PrtC negative, but were prtC gene positive. The correlation
between the levels of P gingivalis PrtC in subgingival
plaques and clinical parameters are shown in Table 1. The
A490 value in the BOP-positive sites was significantly higher
than that in BOP-negative sites (P=0.029). The enhanced
level of PrtC was found in the ¡Ý5 mm AL sites than that in the
¡Ü2 mm AL sites (P=0.016), but with no significant difference
between the ¡Ý5 and >2_<5 mm AL sites or between the
¡Ü2 and >2_<5 mm AL sites (P=0.114, 0.362). A higher
A490 value was detected in the >6 mm PD pockets than in
>4_¡Ü6 mm or in the 3_4 mm sites (P=0.039, 0.023). No statistical difference
could be distinguished between the >4_¡Ü6 mm and the 3_4
mm pockets (P=0.506).
Overall, the periodontal conditions in the periodontitis
patients were improved significantly 4 months after the
initial periodontal treatment (Table 2). Only 56 of the 157 sites
were still positive for BOP (Table 1). The
A490 value decreased after treatment, with 66.8% (131/196) of the samples positive
for PrtC in a range of 0.25_0.88. Although the PrtC level in
the BOP-positive sites was still higher than that in the
BOP-negative sites after treatment (P=0.004), no significant
difference was found between the
A490 values before and after treatment
(P=0.261, 0.286). Higher values were detected in
the ¡Ý5 mm AL sites than those in ¡Ü2 mm AL sites
(P=0.011), but with no statistical difference between the
¡Ý5 and the >2_<5 mm AL sites or between the
¡Ü2 and the >2_<5 mm AL sites (P=0.186, 0.237). A remarkably enhanced level of PrtC was
observed in the >6 mm PD pockets than that in the
>4_¡Ü6 mm or in the 3_4 mm pockets
(P=0.002, 0.000), but the
A490 values between the
>4_¡Ü6 mm and the 3_4 mm PD pockets were almost similar
(P=0.416). After treatment, the PrtC level in
the different AL groups or the ¡Ü6 mm PD groups decreased
markedly compared with that before treatment
(P=0.010_
0.047). Although the A490 values also dropped slightly
compared with that before treatment in the sites with >6 mm PD,
no statistical difference could be found (P=0.246).
Discussion
In the present study, a prokaryotic expression system
was constructed to obtain plentiful purified recombinant PrtC,
which would be helpful in investigating the potential role of
P gingivalis collagenase in periodontal destruction, because
it was difficult to extract a great deal of purified collagenase
directly from P gingivalis. A nucleotide sequence analysis
showed 98.46% and 99.07% homology of the nucleotide and
putative amino acid sequences of our cloned
prtC gene compared with what was registered in GenBank, indicating high
fidelity of the method. A low dosage of 0.5 mmol/L IPTG
could efficiently induce rPrtC expression with a 50% output
of the bacterial total proteins, suggesting the efficiency of
the constructed prokaryotic expression system. The fact
that rPrtC could be recognized by the antibody against the
whole cell of P gingivalis and that it was able to induce
rabbit antisera to produce the specific antibody,
demonstrated good immunoreactivity and antigenicity of the
recom-binant protein. A high frequency of infection of
P gingivalis in the subgingival plaque sample in CP patients (95.9%) was
revealed by PCR. In the 196 samples, 188 and 180 were
positive for P gingivalis 16SrDNA and
prtC, respectively. The data implied that the
prtC gene might be present in most P
gingivalis strains (180/188), which was consistent with
previous reports[25,26]. Furthermore, compared with the PCR
detection of the prtC gene, our established ELISA was highly
sensitive. Among the 180 samples positive for the
prtC gene, 178 were found to be PrtC positive by ELISA. This indicated
that the prtC gene was frequently expressed in this species.
This is the first study to demonstrate that rPrtC is able to
directly promote the production of IL-1α, IL-8 and
TNF-α in endothelial cells. Initial studies indicated that the
prtC gene product could cleave type I collagen, suggesting that this
enzyme might play a role in connective tissue destruction in
periodontitis[4,11]. More recent findings however questioned
the role of the prtC gene product in the collagenolytic
activity of P gingivalis[27,28]. Some researchers have found that
proteinase from some bacteria could have direct
inflammation-causing ability on host cells besides its specific enzyme
activity[13,29]. However, studies of the influence of the
prtC gene product of P gingivalis on host cells are still lacking.
On the other hand, neutrophil-endothelial cell interactions
are the prerequisite for the transendothelial migration of
leukocytes, activation of T cells, and then establishment of
local inflammation[29], so endothelial cells play a critical role
in the development of inflammation. Moreover, endothelial
cells may be important target cells for periodontal pathogens,
including P gingivalis[17]. In this study, we investigated the
effect of recombinant PrtC on the secretion of cytokines in
ECV304 endothelial cells. After co-incubation with 1 µg/mL
rPrtCfor 24 h and with 5 or 10 µg/mL rPrtC for 12 h, the levels
of IL-1α, IL-8, and TNF-α increased significantly
(P<0.05). IL-1α and TNF-α exert multiple effects on inflammatory cells,
including the synthesis of other cytokines and the
modulation of intracellular adhesion molecule-1 upregulation, a critical
step in the development of neutrophil-endothelial cell
interactions[13,19,29]. IL-8 is key mediator of neutrophil migration
to sites of inflammation[13,19]. IL-8 is produced in response to
cytokines such as TNF-α, IL-1, and
LPS[13,19]. So the ability of PrtC to induce the expression of these cytokines in
endothelial cells implies an important role of PrtC in the
inflammation of periodontal tissue. Much attention has been given
to proteinase produced by host cells. MMP, enzymes
secreted by endothelial cells, macrophages, and fibroblasts,
are known to be involved in tissue remodeling,
organo-genesis, angiogenesis, wound repair, and inflammatory
cellular infiltration by degrading extracellular
matrix[16,30]. At the inflammatory site, inflammatory cells are mobilized and MMP
are produced as pro-enzymes and activated by various
stimulatory factors[30]. Recent studies demonstrated that MMP
expression was upregulated by pro-inflammatory mediators
including IL-1 and TNF-α[16]. Taken together, these findings
suggest that although the prtC gene product may not be a
true bacterial collagenase, it could have a strong effect on
inducing host cells to secret inflammatory cytokines and
further enhance the collagenase activity of host cells such
as MMP, which lead to the destruction of periodontal
connective tissue.
Our data further demonstrated that the PrtC level in
clinical samples was correlated with BOP, AL, and PD before and
after treatment (Table 1). ELISA, with the self-prepared
rabbit anti-rPrtC serum, was established to measure the PrtC
level in subgingival samples to understand the association
between PrtC and periodontal inflammation or tissue
destruction. The PrtC level was higher in the BOP-positive
sites or in serious periodontal destruction sites
(¡Ý5 mm AL) than in the BOP-negative or in the
¡Ü2 mm AL sites (P<0.05). The
A490 value of PrtC was higher in the deep pockets (>6 mm
PD) than that in shallow or moderate pockets
(P<0.05). This indicates that the PrtC level is related to periodontal
inflammation and tissue destruction. A previous study in
domestic cats showed that serum antibody response to the
recombinant PrtC of a feline strain of P
gingivalis was associated with the severity of periodontal
disease[12]. The present in
vivo study provided strong evidence that PrtC correlated
with periodontal tissue destruction and inflammation. PrtC
might play a role in the initiation and development of
periodontitis by working in combination with other factors
produced by P gingivalis or host cells, including
P gingivalis LPS or MMP[31,32], since the relationships between PrtC
levels and these clinical parameters seem to be rather
com-plicated. It was notable that the PrtC-positive subgingival
samples were reduced from 90.8% to 66.8%, and the PrtC
level decreased remarkably in different AL sites or in the
¡Ü6 mm PD pockets (P<0.05) after treatment, which might be
explained by the fact that mechanical debridement will
significantly decrease the number of P
gingivalis and other putative periodontal
pathogens[32,33]. However, the
A490 value of PrtC in the BOP-positive samples and in the sites with >6
mm PD changed insignificantly (P>0.05). Previous studies
have indicated that deep pockets and furcations are most
likely inadequately instrumented[34] and microorganisms,
including P gingivalis residing in biofilms left in such
locations, correlated with BOP and greater pocket
depths[35], which may require surgical
intervention[32]. Our results also contributed to the above viewpoints.
In conclusion, periodontal diseases are multifactorial
infections elicited by a complex of bacterial species that
interact with host tissues and cells, which lead to secretion
of various cytokines, including IL-1, IL-6, IL-8, and
TNF-α, as well as prostaglandin E2 and
MMP[2,3,8]. These cytokines and enzymes play critical roles in periodontal tissue
destruc-tion. The results obtained in this study demonstrated that
the prtC gene product could induce host cells to synthesize
and secrete inflammatory cytokines to exert a destructive
effect on periodontal tissue. Further investigation is needed
to reach a conclusive result.
Acknowledgement
We would like to thank Dr Wei-lian SUN for assistance in
the recruitment of patients and the collection of clinical
samples.
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