Department of Microbiology, Oral Health
Science Center, Tokyo Dental College, 1-2-2 Masago, Mihama-ku,
Chiba 261-8502, Japan,1 and
Department
of Oral Biology and Microbiology, State University of New York at
Buffalo, Buffalo, New York 142142
Prolyl-phenylalanine-specific serine protease (dentilisin) is a
major extracellular protease produced by Treponema
denticola. The gene, prtP, coding for the protease
was recently cloned and sequenced (K. Ishihara, T. Miura, H. K. Kuramitsu, and K. Okuda, Infect. Immun. 64:5178-5186, 1996). In order
to determine the role of this protease in the physiology and virulence
of T. denticola, a dentilisin-deficient mutant, K1, was
constructed following electroporation with a
prtP-inactivated DNA fragment. No chymotrypsin-like
protease activity was detected in the dentilisin-deficient mutant. In
addition, the high-molecular-mass oligomeric protein characteristic of
the outer sheath of the organism decreased in the mutant. Furthermore, the hydrophobicity of the mutant was decreased, and coaggregation of
the mutant with Fusobacterium nucleatum was enhanced
compared to that of the wild-type organism. The results obtained with a mouse abscess model system indicated that the virulence of the mutant
was attenuated relative to that of the wild-type organism. These
results suggest that dentilisin activity plays a major role in the
structural organization of the outer sheath of T. denticola. The loss of dentilsin activity and the structural
change in the outer sheath affect the pathogenicity of T. denticola.
 |
INTRODUCTION |
Treponema denticola is a
helically shaped microorganism isolated from the human periodontal
region (29, 30) and dermatitis lesions in cattle
(4). Increased levels of the organism parallel the
destruction of periodontal tissue. In addition, several potential virulence factors, such as an immunosuppressive factor (21, 48), proteolytic activity (32, 35, 43, 52, 53), and attachment factors (8, 19), are expressed by the organism. These observations suggested that this microorganism is potentially a
pathogen involved in periodontitis.
Proteases are considered to be significant pathogenic factors in
periodontal disease. Several proteases or peptidases of T. denticola have been described, and their pathogenic effects have been characterized (32-35, 43, 52, 53). Of these enzymes, a
prolyl phenylalanine-specific protease (dentilisin; also called chymotrypsin-like protease) has a broad substrate specificity, including bioactive peptides (23, 34, 53). In addition, this
enzyme is cytotoxic for human epithelial cells (52). These results suggested that the protease is a major pathogenic factor of
T. denticola. The purified protease consists of three
proteins (23, 37, 53), and activity was lost when the
complex was dissociated (23). Several reports have also
indicated that high-molecular-mass cell surface oligomeric proteins are
expressed in T. denticola and were detected by sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)
(6, 58). A 53-kDa major outer sheath protein (Msp) was also
observed in an oligomeric form (11, 54). Recently, the DNA
sequence of the major surface protease was determined (23).
The protease is approximately 100 kDa under nonreducing conditions.
However, it dissociated to 72-, 43-, and 38-kDa proteins on SDS-PAGE.
The sequences of the 43- and 72-kDa proteins indicated that the open
reading frames of the two proteins are tandemly oriented. The
results of a homology search indicated that the 72-kDa protein is a
protease with a molecular weight of 77,471. In the present study, we
constructed a prtP-deficient mutant in order to determine
the physiological role of the protease in the expression of cell
surface proteins and its potential role in virulence.
| |
MATERIALS AND METHODS |
Microorganisms and plasmids.
The microorganisms and plasmids
used in this study are listed in Table 1.
T. denticola ATCC 35405 was propagated in TYGVS medium
(43), while Porphyromonas gingivalis and
Fusobacterium nucleatum were maintained on Tripticase soy
agar (Becton Dickinson and Company, Cockeysville, Md.) containing 10%
defribrinated horse blood, 5 µg of hemin per ml, and 0.5 µg of
menadione per ml. The bacteria were incubated at 37°C under anaerobic
conditions as described previously (22).
Escherichia coli HB101 was used to construct plasmids for
isolation of the prtP mutant. Strain HB101 was grown on
Luria-Bertani agar plates or in Luria-Bertani broth. Plasmids were
maintained in cultures containing the following antibiotics: pMCL191,
pDLCK3, and pKO3, 30 µg of chloramphenicol per ml; PCR II and pTA2,
100 µg of ampicillin per ml.
Construction of the prtP mutant.
The process for
construction of the prtP mutant is illustrated in Fig.
1. The sequence of mature prtP
was amplified by the PCR with synthetic oligonucleotide primers
(prtP forward primer, 5'-CGGTCTGACAGACGGTAATTATTTGG-3';
prtP reverse primer,
5'-ACGGATCCCCTGTAAACCGTAACTC-3') as described previously
(23). The amplified fragment was inserted into pCR II
(Invitrogen, San Diego, Calif.) according to the supplier's instructions. An EcoRI-BamHI fragment containing
the prtP gene was isolated from the resulting plasmid and
ligated to pMCL191 (41). The plasmid obtained was designated
pDLCK3. An ermF-ermAM cassette (13) was isolated
from plasmid pVA2198 following KpnI-PstI digestion and inserted into pDLCK3. The resulting plasmid, pKO3, was
linearized following EcoRI and BglII digestion
and used in electroporation (28). T. denticola
ATCC 35405 was inoculated into 500 ml of TYGVS medium and incubated for
3 days as described above. Cells were placed on ice for 15 min, washed
with 500 ml of ice-cold distilled water, and centrifuged at 4,000 × g for 10 min. Cells were resuspended in 250 ml of
ice-cold distilled water, centrifuged, and resuspended in 10 ml of
ice-cold distilled water containing 10% glycerol. After
centrifugation, the cells were suspended in 1 ml of 10% glycerol.
Eighty microliters of competent cells (approximately 5 × 1010 cells) was mixed with 10 µg of linearized pKO3.
Competent cells were electroporated as previously described
(28) and mixed with 2 ml of TYGVS medium. After the cells
were incubated for 24 h under anaerobic conditions, 1 ml of the
culture was mixed with 35 ml of TYGVS medium containing 0.8% agarose
(TYGVS plates) and 40 µg of erythromycin per ml. The resulting plates
were incubated for 4 to 8 days under anaerobic conditions. After
incubation, individual colonies were isolated with a capillary pipette
and reinoculated into TYGVS medium containing 40 µg of erythromycin per ml. One mutant, designated K1, was selected for further evaluation.
Growth rate of the mutant.
Cultures of T. denticola ATCC 35405 and mutant K1 were adjusted to an absorbance
of 0.2 at 660 nm in TYGVS medium, and 1.0 ml of each was inoculated
into 100 ml of TYGVS medium and incubated at 37°C under anaerobic
conditions. Growth rates were determined by measuring the absorbance at
660 nm and by use of a Petroff-Hauser bacterial counting chamber.
Southern blot analysis.
Genomic DNAs from T. denticola ATCC 35405 and K1 were isolated and hybridization was
performed as described previously (22). Briefly, chromosomal
DNA from T. denticola was digested with
HindIII, electrophoresed through 1.0% agarose gels,
denatured, and transferred to Hybond-N+ paper by capillary transfer
(49). DNA probes (571-bp KpnI-PstI
frag ment of the prtP gene and
KpnI-BamHI fragment from pVA2198; Fig. 1) were
labeled with digoxigenin-dUTP by use of a DIG DNA labeling system
(Boehringer Mannheim Biochemicals, Indianapolis, Ind.) according to the
manufacturer's protocol. Hybridization was performed at 42°C for
18 h in an aqueous buffer containing 50% formamide, 5 × SSC
(1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1% blocking
buffer (Boehringer), 0.1% sarcosine, and 0.01% SDS. Hybridized
membranes were washed as specified by the supplier, and hybridizing
bands were detected on Hybond-N+ paper by use of a DIG DNA detection
kit (Boehringer).
Assays of dentilisin activity of the mutant.
Proline-phenylalanine-specific protease activity was measured with the
synthetic substrate
N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide (SAAPNA; Sigma Chemical Company, St. Louis,
Mo.). The cells were disrupted by sonication (Branson, Danbury, Conn.) at 100 W for 5 min on ice. Insoluble material was removed by
centrifugation at 8,000 × g for 20 min. The protein
concentration of the sonicate was determined by the DC protein assay
(Bio-Rad Laboratories, Hercules, Calif.). A 5-µl aliquot of sonicate
was mixed with 150 µl of 100 mM Tris-HCl buffer (pH 8.0) containing
1.0 mM SAAPNA. The mixture was incubated at 37°C for 15 min, and the
reaction was stopped by the addition of 50 µl of 20% acetic acid.
The release of p-nitroaniline was determined by measuring
its absorbance at 405 nm. One unit of enzyme was defined as the amount
required to release 1.0 µmol of p-nitroaniline in 1 min at
37°C under these conditions.
Hydrolysis of fibronectin (Sigma) was performed as described before
(23). Samples containing 10 µg of fibronectin were
incubated with 2 µg of the sonicate of T. denticola for
6 h at 37°C. Reaction mixtures were subjected to SDS-PAGE
analysis, and the protein bands were stained with Coomassie brilliant
blue R-250.
Antisera.
Rabbit antiserum against T. denticola
ATCC 35405 whole cells and rabbit antiserum against dentilisin were
prepared as described previously (23). Rabbit antiserum
against T. denticola ATCC 35404 Msp was kindly provided by
T. Umemoto (Asahi University, Gifu, Japan).
SDS-PAGE and immunoblot analyses.
Wild-type cells and cells
of the prtP mutant were examined by SDS-PAGE with or without
1 µM serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF)
by use of the discontinuous system of Laemmli (26). Proteins
(10 µg) were separated on a 10 to 20% gradient resolving gel
(Daiichi-Kagaku, Tokyo, Japan). Cells or sonicates were treated either
at 100°C for 5 min or at 4°C overnight in the presence of
-mercaptoethanol. Protein bands were visualized by staining with
Coomassie brilliant blue R-250.
For zymography, the cells were incubated overnight with SDS sample
buffer at 4°C and the mixtures were separated by SDS-6% PAGE with
gels containing 200 µg of gelatin per ml. After electrophoresis, the
gels were incubated in 100 mM Tris-HCl buffer (pH 8.0) for 1 h and
stained with Coomassie brilliant blue R-250.
For immunoblot analysis, 2 µg of proteins from treponemal cells was
electrophoresed as described above and transferred by the method of
Towbin et al. (51) with a Transblot cell (Bio-Rad). The
blotted membranes were immunostained with rabbit antidentilisin serum
(23), anti-T. denticola ATCC 35405 whole-cell
serum, or anti-T. denticola ATCC 35404 Msp serum. Antibody
bound to protein immobilized on the membranes was detected with
peroxidase-conjugated goat anti-rabbit immunoglobulins (Bio-Rad).
Coaggregation assays.
Coaggregation was determined by a
modification of the method of Cisar et al. (5). Briefly,
cells were washed with phosphate-buffered saline (pH 7.2) and suspended
in coaggregation buffer (1 mM Tris-HCl buffer containing 0.1 mM
CaCl2, 0.1 mM MgCl2, 0.02% NaN3,
and 0.15 M NaCl). All suspensions were adjusted to an optical density of 2.0 at 660 nm with a U2000 spectrophotometer (Hitachi, Tokyo, Japan). Aliquots of 500 µl from each bacterial suspension and coaggregation partner suspension were vortexed for 10 s, allowed to stand at room temperature for 1 h, and visually scored for coaggregation. Tubes containing each cell suspension alone were included as controls. When the bacterial suspensions autoagglutinated, the tubes were mixed again, and the existence of coaggregated cells was
reexamined. Coaggregation was confirmed by phase-contrast microscopy.
Briefly, the mixtures were allowed to stand at room temperature
overnight, and an aliquot was gently vortexed and placed between a
glass slide and coverslip for visual confirmation of coaggregation.
Determination of hydrophobicity.
Evaluation of cell
hydrophobicity was carried out as described by Rosenberg et al.
(46) and Gibbons et al. (15). Bacterial suspensions in PUM buffer (K2HPO4 · H2O, 22.2 g; KH2PO4, 7.3 g; urea, 1.8 g; MgSO4 · 7H2O,
0.2 g) were adjusted to an optical density of approximately 0.5 at
400 nm. Duplicate samples of bacterial suspensions (1.2 ml) in PUM
buffer were placed in 10- by 70-mm glass tubes, and 600 µl of
hexadecane (Sigma) was added. The tubes were vigorously vortexed for
60 s and allowed to stand for 15 min, after which the
A400 of the aqueous phase was measured. The percent hydrophobicity was calculated as follows:
[(A400 before mixing 
A400 after mixing)/A400
before mixing)] × 100. Each isolate was assayed twice, and the values
obtained were averaged.
Evaluation of pathogenicity of the mutant.
Virulence was
assessed with a mouse abscess model described by Kesavalu et al.
(24). Briefly, T. denticola ATCC 35405 and K1
were grown for 72 h under anaerobic conditions as described above
and harvested. Cells were resuspended in phosphate-buffered saline (pH
7.2) and quantitated with a Petroff-Hauser bacterial counting chamber.
Twenty-two BALB/c mice (6 to 8 weeks old) were separated into two
groups and challenged subcutaneously (s.c.) on the posterior
dorsolateral surface with 200 µl (3 × 109 cells) of
ATCC 35405 or K1 cell suspension. Following challenge, the animals were
examined at least once daily for 14 days for lesion formation, and the
size of each lesion was measured with a caliper gauge. At 3 and 5 days
after s.c. challenge, the mice were euthanatized by CO2
asphyxiation, and the contents of each abscess were aspirated with a
syringe after disinfection of the lesion skin with ethyl alcohol. The
viability of the spirochetes was evaluated by dark-field microscopy and
inoculation on TYGVS plates under anaerobic conditions.
Statistical analysis.
Statistical differences in lesion area
were determined by the Mann-Whitney U test.
| |
RESULTS |
Construction of the dentilisin-deficient mutant.
To determine
the role of dentilisin in the physiology of T. denticola, an
isogenic mutant defective in the prtP gene was constructed by allelic exchange mutagenesis (Fig. 1). The 2.1-kbp
ermF-ermAM cassette was cloned into the
KpnI-PstI site of plasmid pDLCK3. The recombinant
plasmid was then linearized with EcoRI and BglII and electroporated into T. denticola ATCC 35405. Since the
plasmid was linearized, erythromycin-resistant transformants could
arise as a result of a double-crossover event between the regions
flanking the erm cassette and the wild-type gene on the
chromosome. This event would result in the replacement of the central
Kpn-PstI fragment of the prtP gene with a
fragment conferring erythromycin resistance.
We obtained 24 Emr colonies following 7 days of incubation.
The efficiency of the recombination event was approximately 1.2 colonies per µg of DNA. We isolated seven of the putative mutants and
designated them K1 to K7. The growth rate of the mutants was the same
as that of the wild type in TYGVS medium. The mutants also exhibited
pronounced autoaggregation activity in TYGVS medium at the stationary
phase. To confirm the predicted recombination event, Southern blot
analysis was carried out (Fig. 2). A
3.3-kbp band was observed for wild-type strain ATCC 35405 when the
KpnI-PstI fragment from the prtP gene
was used as a probe (Fig. 2, lane 1). Since the
KpnI-PstI fragment was replaced with the
ermF-ermAM cassette in the transformants, no positive band
was observed in Emr transformant K1 when the
KpnI-PstI fragment was used as a probe (Fig. 2,
lane 2). When the ermF-ermAM cassette was used as a probe, no band was observed for the wild-type cells (Fig. 2, lane 3). Since
the ermF-ermAM cassette contains a single
HindIII site, two bands (2.0 and 2.5 kbp) were observed
in the Emr mutant (Fig. 2, lane 4) when the
ermF-ermAM cassette was used as a probe. Likewise, the sizes
of the amplified fragments of wild-type T. denticola and
mutant K1 in PCR with the prtP forward and reverse primers
were approximately 2 and 4 kbp, respectively (data not shown). These
data suggested that the predicted recombination event had occurred,
resulting in the interruption of the wild-type protease gene by the
antibiotic resistance gene cassette. Identical results were observed
for the other mutants, and one mutant, K1, was chosen for further
analysis.
View larger version (87K):
[in this window]
[in a new window]
|
FIG. 2.
Southern blot analysis of T. denticola ATCC
35405 and prtP mutant K1. Chromosomal DNAs from T. denticola ATCC 35405 (lanes 1 and 3) and prtP mutant K1
(lanes 2 and 4) were digested with HindIII and
hybridized with a digoxigenin-labeled KpnI-PstI
fragment from the prtP gene (lanes 1 and 2) or the
KpnI-BamHI fragment from pVA2198 (lanes 3 and 4).
Numbers at right are kilobase pairs.
|
|
Proteolytic activity of mutant K1.
The proteolytic activities
of whole cells and sonic extracts from T. denticola ATCC
35405 and K1 were assayed with the synthetic chymotrypsin
substrate SAAPNA. Wild-type T. denticola ATCC 35405 exhibited SAAPNA-hydrolyzing activity (1.0 × 10
4 ± 0.020 × 104 U/1.1 × 109 cells)
whereas K1 displayed little SAAPNA-hydrolyzing activity (0.010 × 104 ± 0.012 × 104 U/1.1 × 109 cells) in whole cells. Zymography with gelatin as a
substrate (Fig. 3) indicated that a
proteolytic band of approximately 100 kDa could be readily detected for
wild-type strain ATCC 35405 but that this band was absent in mutant K1.
The wild-type strain also hydrolyzed fibronectin, while mutant K1 did
not (data not shown). The zymography data indicated that K1 was missing
the predicted proteolytic band of approximately 100 kDa corresponding to the chymotrypsin-like protease activity of T. denticola
(53).
SDS-PAGE and immunoblot analyses of the mutant.
The results of
SDS-PAGE analysis of the proteins expressed by the wild type and mutant
K1 are shown in Fig. 4. In unheated samples, high-molecular-mass oligomeric proteins of 180 to 200 kDa were
observed in the wild type (Fig. 4, lanes 1 and 5), and smaller amounts
of low-molecular-mass proteins were also observed (lane 5).
Pretreatment with PMSF, which prevents proteolysis in SDS-PAGE analysis
(57), increased the intensity of the bands smaller than 100 kDa (Fig. 4, lane 1). On the other hand, the 180- to 200-kDa oligomeric
proteins were less intense in the mutant with or without PMSF
pretreatment (Fig. 4, lanes 2 and 6). The 78-kDa band was observed only
in the mutant and not in the wild type. Boiling the samples resulted in
alterations in the protein profiles for the wild-type and mutant cells.
In the K1 mutant, multiple bands were combined around the position of
Msp. The results of immunoblot analysis with antidentilisin antibody
further indicated that antidentilisin serum reacted with a 72-kDa
protein in boiled samples of the wild-type strain. However, this
protein band was not observed in the prtP mutant extracts
(Fig. 5).
View larger version (112K):
[in this window]
[in a new window]
|
FIG. 4.
SDS-PAGE analysis of sonicates of T. denticola ATCC 35405 and K1. Samples from lanes 1 to 4 were
treated with PMSF. Lanes 1 and 5, T. denticola ATCC 35405 (without boiling); lanes 2 and 6, T. denticola K1 (without
boiling); lanes 3 and 7, T. denticola ATCC 35405 (with
boiling); lanes 4 and 8, T. denticola K1 (with boiling).
After electrophoresis, the gel was stained with Coomassie brilliant
blue R-250. Arrowheads indicate the high-molecular-mass oligomeric
protein and the Msp band.
|
|
View larger version (40K):
[in this window]
[in a new window]
|
FIG. 5.
Immunoblot analysis of T. denticola ATCC
35405 (lane 1; boiled) and K1 (lane 2; boiled) with antidentilisin
serum.
|
|
It was previously reported that a high-molecular-mass oligomeric
protein consisted of polymers of Msp (11, 54). We analyzed the antigenic proteins with antiserum against T. denticola
ATCC 35405 whole cells (Fig. 6). In the
wild-type strain, the oligomeric protein band was observed from about
180 to 230 kDa with or without PMSF treatment (Fig. 6, lanes 1 and 5),
but the reactivity of the antibody with the oligomeric protein from the
mutant was decreased (lanes 2 and 6). Boiled samples of both strains
contained the 53-kDa Msp and a similar band pattern below Msp. However,
the 68-, 70-, 131-, 175-, and 200-kDa bands were observed only in unboiled samples of the mutant (Fig. 6, lanes 3 and 4). We also analyzed the size of Msp in unboiled and boiled samples with antiserum against T. denticola ATCC 35405 Msp. This serum showed
reactivity with both T. denticola ATCC 35404 and T. denticola ATCC 35405 Msp (data not shown). As Fig.
7 indicates, anti-Msp serum reacted with
178- to 224-kDa oligomeric proteins in unboiled samples of wild-type
cells. On the other hand, the corresponding bands appeared faint and a
weak band was observed at 83 kDa in mutant cells. An Msp of 53 kDa was
observed in boiled samples of both the wild type and the mutant. These
results indicated that Msp was expressed in both the wild type and
mutant K1 but that in the mutant the ability of the organization of the
high-molecular-mass oligomeric protein decreased.
View larger version (122K):
[in this window]
[in a new window]
|
FIG. 6.
Immunoblot analysis of T. denticola ATCC
35405 and K1 with anti-T. denticola ATCC 35405 whole-cell
serum. Samples from lanes 1 to 4 were treated with PMSF. Lanes 1 and 5, T. denticola ATCC 35405 (without boiling); lanes 2 and 6, T. denticola K1 (without boiling); lanes 3 and 7, T. denticola ATCC 35405 (with boiling); lanes 4 and 8, T. denticola K1 (with boiling). Arrowheads indicate the
high-molecular-mass oligomeric protein and the Msp band.
|
|
View larger version (90K):
[in this window]
[in a new window]
|
FIG. 7.
Immunoblot analysis of T. denticola ATCC
35405 and K1 with anti-T. denticola ATCC 35404 Msp serum.
Samples from lanes 1 to 4 were treated with PMSF. Lanes 1 and 5, T. denticola ATCC 35405 (without boiling); lanes 2 and 6, T. denticola K1 (without boiling); lanes 3 and 7, T. denticola ATCC 35405 (with boiling); lanes 4 and 8, T. denticola K1 (with boiling). Arrowheads indicate the
high-molecular-mass oligomeric protein and the Msp band.
|
|
Hydrophobicity measurements.
Previous data suggested that the
hydrophobicity of bacterial cells is sometimes correlated with
colonization properties which may be relevant to the oral cavity
(39). To determine the relationship between dentilisin and
the cell surface architecture of T. denticola, the cell
surface hydrophobicities of T. denticola wild-type and mutant K1 cells were compared. The cell surface hydrophobicity was
dramatically decreased in the prtP mutant (23.0% ± 11.9%) compared with the wild type (60.0% ± 3.71%).
Coaggregation activity between T. denticola and other
oral bacteria.
T. denticola was previously reported to
coaggregate with P. gingivalis and F. nucleatum
(17, 25, 44). To determine if the alteration of cell surface
hydrophobicity following inactivation of the prtP gene
influenced such interactions, the coaggregation reactions of the
wild-type and mutant cells were evaluated (Table 2). The mutant exhibited autoaggregation
activity in TYGVS medium but did not exhibit autoaggregation activity
in coaggregation buffer. The coaggregation reaction between T. denticola K1 and F. nucleatum ATCC 25586 was enhanced
relative to that of the wild-type strain. In fact, the interaction
between wild-type strain ATCC 35405 and F. nucleatum ATCC
25586 was relatively weak. The coaggregation score for T. denticola K1 and P. gingivalis was not different from
that of the wild-type strain, but a significant rapid coaggregation reaction was observed. Pretreatment of T. denticola cells
with PMSF just prior to the reaction did not alter either coaggregation reaction.
Virulence of the prtP mutant in the mouse abscess model
system.
To evaluate the effects of the surface structure
alterations displayed by mutant K1 on the virulence of the
microorganism, the wild-type and mutant strains were injected s.c. into
the posterior dorsolateral surface of two groups of mice (Fig.
8). The lesion areas of the group
infected with the prtP mutant were smaller than those of the
group injected with the wild type over a 3- to 14-day period after
infection (days 3, 6, 9, 10, 11, 12, 13, and 14, P < 0.001; days 4, 5, and 8, P < 0.05). T. denticola ATCC 35404 and K1 were detected by microscopy following
aspiration of samples of the abscesses at days 3 and 5. We also
isolated on TYGVS plates viable T. denticola ATCC 35405 and
K1 from the abscesses at days 3 and 5. No gross pathology of the
animals was detected over this time period. However, the animals were
not examined for any internal organ damage. The stability of the
prtP mutation was not assessed, since the double-crossover
recombination event used to construct mutant K1 should result in a
stable mutation in the absence of antibiotic selection pressure.
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 8.
Mean areas of lesions at infection sites after challenge
with T. denticola ATCC 35405 and prtP mutant K1.
Mice were injected with live T. denticola ATCC 35405 (open
bars) or mutant K1 (hatched bars), and lesion areas were determined at
the indicated times following infection.
|
|
| |
DISCUSSION |
Several reports on the proteases of T. denticola have
appeared in the literature (1, 23, 31, 32-35, 38, 43, 45, 53). One of these proteases, dentilisin, is present in the outer sheath of the organism (23). Dentilisin has been proposed to participate in the adhesion of the microorganism to epithelial cells
(27), to interfere with the host immune response
(53) and infiltration of the tissues (18), and to
have cytotoxic effects on human epithelial cells (52).
Therefore, this protease may be involved in the etiology of periodontal
diseases. However, it is not yet clear what role this enzyme plays in
the normal physiology of T. denticola. It has been
suggested that proteases play a role in the formation of the surface
layer of some bacteria. For example, the activity of the protease
Arg-gingipain is required for the maturation and expression of the
fimbriae of P. gingivalis (42, 50). It has been
demonstrated that there is an outer sheath surrounding T. denticola and other spirochete cells (20) and that the
sheath contains high-molecular-mass oligomeric proteins (6, 55,
58). Masuda and Kawata (36) reported that a major protein component was observed in the outer sheath. Weinberg and Holt
(58) reported that oligomeric proteins were observed in the
outer sheath of T. denticola and were not dissociated with sarcosine extraction. Furthermore, Fenno et al. (11)
determined the DNA sequence of the gene expressing Msp, the major
protein of the outer sheath, and stated that Msp in its native form is an oligomeric protein of 150 to 200 kDa. Nevertheless, in the case of
recombinant Msp, an oligomeric form could not be detected. Therefore,
it is possible that the proteases in the outer sheath play a role in
organizing high-molecular-mass oligomeric proteins. As a result, in the
present study, by use of a defective strain of T. denticola
that did not express the dentilisin following site-specific
mutagenesis, it was possible to assess the role of dentilisin in the
pathogenicity of this microorganism and the conversion of Msp into its
normal oligomeric form in the outer sheath.
When the dentilisin-deficient mutant, K1 was compared with the
wild-type strain by means of SDS-PAGE, it was found that a high-molecular-mass oligomeric protein was present in the wild-type strain in unboiled samples but that in the K1 mutant there was a marked
decrease in the size of this complex. This finding suggests that
dentilisin plays a role in the formation of the oligomeric complex.
Dentilisin may serve as a component of the oligomeric complex or may
contribute to the formation of the oligomeric complex via processing of
another protein. On the basis of the results of zymography, which
revealed no protease activity associated with the high-molecular-mass
oligomeric protein complex, it is unlikely that dentilisin is a
component of this complex with Msp. Immunoblotting with a polyclonal
antibody against strain ATCC 35405 whole cells showed that there was
less high-molecular-mass oligomeric protein in the unboiled samples of
the mutant strain than in those of the wild-type strain. However, in
the boiled samples, the size and amount of Msp were the same in both
strains. This result suggests that Msp does not require processing by
dentilisin. The 83-kDa protein was weakly observed in the PMSF-treated
wild-type strain. It is possible that in the dentilisin-deficient
mutant, Msp associated with an 83-kDa complex but not with
higher-molecular-mass complexes. Fenno et al. (12) suggested
that the amino acid sequence of Msp resembled those of bacterial porins
and that the protein exhibited properties consistent with this proposal
(37). It is possible that in the K1 mutant, part of Msp
associated with the 83-kDa complex because of the formation of porins
(high-molecular-weight oligomers) or because of autodegradation. This
suggestion may indicate that dentilisin assists in the formation of
high-molecular-mass oligomeric proteins. Sela et al. (47)
analyzed the lipoproteins of T. denticola and reported that
Msp is also a lipoprotein. Cox et al. (7) proposed a model
in which the N-terminal portions of the mature polypeptides of
Treponema pallidum are inserted into lipid sites of the
cytoplasmic membrane. We propose that dentilisin assists in the
formation of high-molecular-mass oligomeric proteins by a mechanism
which has not been determined.
The ability of bacteria to adhere to cell surfaces is a fundamental
aspect of their pathogenicity. Surface hydrophobicity has been reported
to contribute to bacterial adhesion (15). In the present
study, we demonstrated that there was a distinct decrease in cell
surface hydrophobicity in the dentilisin-deficient mutant. The result
of immunoblotting with an antiserum against T. denticola
ATCC 35405 whole cells indicated that the oligomeric protein band was
observed from 180 to 230 kDa. This change may have been caused by the
loss of dentilisin activity. The change in hydrophobicity was
paralleled by the observed decreased concentration of the
high-molecular-mass oligomeric protein band in unboiled samples in
mutant K1 and the increased concentration of additional bands. These
results suggest that the hydrophobicity of T. denticola is
influenced by the high-molecular-mass proteins of the outer sheath.
Some microorganisms in the oral cavity undergo coaggregation reactions
and may colonize the oral cavity following interaction with
early-colonizing bacteria. Some of these intraoral bacteria possess
cryptic receptors (cryptitopes) (16) whose function is
related to their colonization. T. denticola has been
reported to coaggregate with F. nucleatum and P. gingivalis (17, 25, 44). The strong coaggregation of
P. gingivalis with T. denticola was given a score
of 3, in contrast to the data reported earlier by Grenier
(17). This difference may result from the use of higher cell
numbers in the present study than in the earlier study. Kolenbrander et
al. (25) reported visible coaggregation between F. nucleatum and T. denticola ATCC 35405. The reaction
varied depending upon the strain of F. nucleatum examined.
An increase in aggregation may reflect differences in the expression of
surface receptors between the strains. It is possible that dentilisin digests the surface proteins of the microorganisms and exposes cryptitopes. However, our observations indicated that coaggregation was
strengthened in a dentilisin-deficient strain of T. denticola. Grenier (17), on analyzing the coaggregation
mechanisms of T. denticola and P. gingivalis,
demonstrated that chymotrypsin and trypsin treatments reduced the
degree of coaggregation and suggested that this result was due to the
degradation of receptor protein. In our study, coaggregation activity
was not decreased by PMSF treatment, indicating that dentilisin did not
affect coaggregation activity. Coaggregation activity did not decrease
in the dentilisin-deficient mutant, although the hydrophobicity of the
cells in the mutant decreased. This result indicated that
hydrophobicity did not play a role in coaggregation between T. denticola and F. nucleatum or P. gingivalis.
The decrease in the hydrophobicity of the K1 mutant may reflect the
change in surface structure. This change may increase the exposure of
the receptor protein for its coaggregation reaction with P. gingivalis and F. nucleatum.
It has been suggested that surface structures such as capsules, the
S-layer, and the outer sheath contribute to the pathogenicity of
microorganisms (10, 11, 14, 56). In this regard, Msp, the
major protein of the outer sheath of T. denticola, exhibits fibronectin adhesiveness (11), cytopathogenicity for host
cells (37), and pore formation in artificial membrane
systems (9). On the other hand, dentilisin exhibited a
cytopathic effect for epithelial cells. We demonstrated that when mice
were inoculated s.c., strain ATCC 35405 produced larger lesions than
did dentilisin-deficient mutant K1. No significant differences could be
detected in the structures of the outer sheaths by electron microscopy
(data not shown), while both the cellular protein profile and the cell
surface hydrophobicity of the mutant were altered. The ultrastructure of the outer sheath of mutant K1 was not visibly different from that of
the wild type when both were viewed by transmission electron microscopy. The results from SDS-PAGE analysis revealed that the concentration of the high-molecular-mass oligomeric protein
complex decreased in the mutant but was still detectable. Therefore,
the decreased level of the complex may still have been sufficient to
maintain the structure of the cell surface in mutant K1. In addition,
immunoblot analysis indicated that Msp was not degraded by dentilisin.
The major components of the outer sheath appear to be lipid material
and Msp (58). Therefore, these latter components may be
sufficient to maintain the outer surface of spirochetes, at least as
far as can be detected by such analysis. The decreased virulence
displayed by mutant K1 may directly result from a decrease in
dentilisin activity, from alterations in the expression of the
high-molecular-mass oligomeric complex containing Msp in the outer
sheath of T. denticola, or from a decreased growth rate in
vivo. As the results indicated that the growth rate of the mutant was
the same as that of the wild type, protease activity and Msp are major
factors in the change in pathogenicity. The multiple defects of the
dentilisin-deficient mutant make it difficult to clearly explain the
molecular bases for virulence attenuation. However, the isolation of a
dentilisin-deficient mutant now makes it possible to genetically
analyze the role of proteases in oral spirochete pathogenicity.
We thank Y. Nakano for providing pMCL191. We thank M. Kitamura
for assistance in the statistical analysis. We thank T. Umemoto for
supplying anti-Msp serum.
This study was partially supported by Oral Health Science Center grant
961A02 from Tokyo Dental College and grant 06671833 from the Ministry
of Education, Science, Culture and Sport of Japan.
| 1.
|
Arakawa, S., and H. K. Kuramitsu.
1994.
Cloning and sequence analysis of a chymotrypsinlike protease from Treponema denticola.
Infect. Immun.
62:3424-3433[Abstract/Free Full Text].
|
| 2.
|
Boyer, H. W., and D. Roulland-Dussoix.
1969.
A complementation analysis of the restriction and modification of DNA in Escherichia coli.
J. Mol. Biol.
41:459-472[Medline].
|
| 3.
|
Chan, E. C. S.,
R. Soiboo,
T. Keng,
N. Psarra,
R. Hurley,
S. L. Cheng, and I. Iugovaz.
1993.
Treponema denticola (ex Brumpt 1925) sp. nov. nom. rev. and identification of new spirochete isolates from periodontal pockets.
Int. J. Syst. Bacteriol.
43:196-203[Medline].
|
| 4.
|
Choi, B.-K.,
H. Nattermann,
S. Grund,
W. Haider, and U. B. Gröbel.
1997.
Spirochetes from digital dermatitis lesions in cattle are closely related to treponemes associated with human periodontitis.
Int. J. Syst. Bacteriol.
47:175-181[Medline].
|
| 5.
|
Cisar, J. O.,
P. E. Kolenbrander, and F. C. McIntire.
1979.
Specificity of coaggregation reaction between human oral streptococcus and strains of Actinomyces viscosus and Actinomyces naeslundii.
Infect. Immun.
24:742-752[Abstract/Free Full Text].
|
| 6.
|
Cockayne, A.,
R. Sanger,
A. Ivic,
R. A. Strugnell,
J. H. MacDougall,
R. R. Russell, and C. W. Penn.
1989.
Antigenic and structural analysis of Treponema denticola.
J. Gen. Microbiol.
135:3209-3218[Medline].
|
| 7.
|
Cox, D. L.,
P. Chang,
A. W. McDowall, and J. D. Radolf.
1992.
The outer membrane, not a coat of host proteins, limits antigenicity of virulent Treponema pallidum.
Infect. Immun.
60:1076-1083[Abstract/Free Full Text].
|
| 8.
|
Dawson, J. R., and R. P. Ellen.
1990.
Tip-oriented adherence of Treponema denticola to fibronectin.
Infect. Immun.
58:3924-3928[Abstract/Free Full Text].
|
| 9.
|
Egli, C.,
W. K. Leung,
K.-H. Mäller,
R. E. W. Hancock, and B. C. McBride.
1993.
Pore-forming properties of the major 53-kilodalton surface antigen from the outer sheath of Treponema denticola.
Infect. Immun.
61:1694-1699[Abstract/Free Full Text].
|
| 10.
|
Evenberg, D., and B. Lugtenberg.
1982.
Cell surface of the fish pathogenic bacterium Aeromonas salmonicida. II. Purification and characterization of a major cell envelope protein related to autoagglutination, adhesion, and virulence.
Biochim. Biophys. Acta
684:249-254[Medline].
|
| 11.
|
Fenno, J. C.,
K. H. Muller, and B. C. McBride.
1996.
Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola.
J. Bacteriol.
178:2489-2497[Abstract/Free Full Text].
|
| 12.
|
Fenno, J. C.,
G. W. Wong,
P. M. Hannam,
K. H. Muller,
W. K. Leung, and B. C. McBride.
1997.
Conservation of msp, the gene encoding the major outer membrane protein of oral Treponema spp.
J. Bacteriol.
179:1082-1089[Abstract/Free Full Text].
|
| 13.
|
Fletcher, H. M.,
H. A. Schenkein,
R. M. Morgan,
K. A. Bailey,
C. R. Berry, and F. L. Macrina.
1995.
Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene.
Infect. Immun.
63:1521-1528[Abstract].
|
| 14.
|
Fujimoto, S.,
A. Takade,
K. Amako, and M. J. Blaser.
1991.
Correlation between molecular size of the surface array protein and morphology and antigenicity of the Campylobacter fetus S-layer.
Infect. Immun.
59:2017-2022[Abstract/Free Full Text].
|
| 15.
|
Gibbons, R. J.,
I. Etherden, and S. Skobe.
1983.
Association of fimbriae with the hydrophobicity of Streptococcus sanguis FC-1 and adherence to salivary pellicles.
Infect. Immun.
41:414-417[Abstract/Free Full Text].
|
| 16.
|
Gibbons, R. J.,
D. I. Hay,
W. C. Childs, and G. Davis.
1990.
Role of cryptic receptors (cryptitopes) in bacterial adhesion to oral surfaces.
Arch. Oral Biol.
35:107S-224S.
|
| 17.
|
Grenier, D.
1992.
Demonstration of a bimodal coaggregation reaction between Porphyromonas gingivalis and Treponema denticola.
Oral Microbiol. Immunol.
7:280-284[Medline].
|
| 18.
|
Grenier, D.,
V. J. Uitto, and B. C. McBride.
1990.
Cellular location of a Treponema denticola chymotrypsinlike protease and importance of the protease in migration through the basement membrane.
Infect. Immun.
58:347-351[Abstract/Free Full Text].
|
| 19.
|
Haapasalo, M.,
U. Singh,
B. C. McBride, and V. J. Uitto.
1991.
Sulfhydryl-dependent attachment of Treponema denticola to laminin and other proteins.
Infect. Immun.
59:4230-4237[Abstract/Free Full Text].
|
| 20.
|
Holt, S. C.
1978.
Anatomy and chemistry of spirochetes.
Microbiol. Rev.
42:114-160[Free Full Text].
|
| 21.
|
Ishihara, K.,
I. Takazoe, and K. Okuda.
1992.
Immunomodulating activity of oral Treponema strains on proliferation of mouse lymphocyte.
Bull. Tokyo Dent. Coll.
33:45-50.
|
| 22.
|
Ishihara, K., and H. K. Kuramitsu.
1995.
Cloning and expression of a neutral phosphatase gene from Treponema denticola.
Infect. Immun.
63:1147-1152[Abstract].
|
| 23.
|
Ishihara, K.,
T. Miura,
H. K. Kuramitsu, and K. Okuda.
1996.
Characterization of the Treponema denticola prtP gene encoding a prolyl-phenylalanine-specific protease (dentilisin).
Infect. Immun.
64:5178-5186[Abstract].
|
| 24.
|
Kesavalu, L.,
S. G. Walker,
S. C. Holt,
R. R. Crawley, and J. L. Ebersole.
1997.
Virulence characteristics of oral treponemes in a murine model.
Infect. Immun.
65:5096-5102[Abstract].
|
| 25.
|
Kolenbrander, P. E.,
K. D. Parrish,
R. N. Andersen, and E. P. Greenberg.
1995.
Intergeneric coaggregation of oral Treponema spp. with Fusobacterium spp. and intergeneric coaggregation among Fusobacterium spp.
Infect. Immun.
63:4584-4588[Abstract].
|
| 26.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature (London)
227:680-685[Medline].
|
| 27.
|
Leung, W. K.,
M. Haapasalo,
V.-J. Uitto,
P. M. Hannam, and B. C. McBride.
1996.
The surface proteinase of Treponema denticola may mediate attachment of the bacteria to epithelial cells.
Anaerobe
2:39-46.
|
| 28.
|
Li, H.,
J. Ruby,
N. Charon, and H. K. Kuramitsu.
1996.
Gene inactivation in the oral spirochete Treponema denticola: construction of an flgE mutant.
J. Bacteriol.
178:3664-3667[Abstract/Free Full Text].
|
| 29.
|
Loesche, W. J.
1976.
Periodontal disease and the treponemes, p. 261-275.
In
R. C. Johnson (ed.), The biology of parasitic spirochetes Academic Press, Inc., New York, N.Y.
|
| 30.
|
Loesche, W. J.,
S. A. Syed,
E. Schmidt, and E. C. Morrison.
1985.
Bacterial profiles of subgingival plaques in periodontitis.
J. Periodontol.
56:447-456[Medline].
|
| 31.
|
MacDougall, J. H.,
D. Beighton, and R. R. B. Russell.
1991.
Cloning and expression of protease genes from Treponema denticola in Escherichia coli.
Oral Microbiol. Immunol.
6:270-274[Medline].
|
| 32.
|
Mäkinen, K. K.,
C.-Y. Chen, and P.-L. Mäkinen.
1996.
Proline iminopeptidase from the outer cell envelope of the human oral spirochete Treponema denticola ATCC 35405.
Infect. Immun.
64:702-708[Abstract].
|
| 33.
|
Mäkinen, K. K.,
P.-L. Mäkinen, and S. A. Syed.
1992.
Purification and substrate specificity of an endopeptidase from human oral spirochete Treponema denticola ATCC 35405, active on furylacryloyl-Leu-Gly-Pro-Ala and bradykinin.
J. Biol. Chem.
267:14285-14293[Abstract/Free Full Text].
|
| 34.
|
Mäkinen, P.-L.,
K. K. Mäkinen, and S. Syed.
1995.
Role of the chymotrypsin-like membrane-associated proteinase from Treponema denticola ATCC 35405 in inactivation of bioactive peptides.
Infect. Immun.
63:3567-3575[Abstract].
|
| 35.
|
Mäkinen, P.-L.,
K. K. Mäkinen, and S. A. Syed.
1994.
An endo-acting proline-specific oligopeptidase from Treponema denticola ATCC 35405: evidence of hydrolysis of human bioactive peptides.
Infect. Immun.
62:4938-4947[Abstract/Free Full Text].
|
| 36.
|
Masuda, K., and T. Kawata.
1982.
Isolation, properties, and reassembly of outer sheath carrying a polygonal array from an oral treponeme.
J. Bacteriol.
150:1405-1413[Abstract/Free Full Text].
|
| 37.
|
Mathers, D. A.,
W. K. Leung,
J. C. Fenno,
Y. Hong, and B. C. McBride.
1996.
The major surface protein complex of Treponema denticola depolarizes and induces ion channels in HeLa cell membranes.
Infect. Immun.
64:2904-2910[Abstract].
|
| 38.
|
Mikx, F. H.,
F. Jacobs, and C. Satumalay.
1992.
Cell-bound peptidase activities of Treponema denticola ATCC 33520 in continuous culture.
J. Gen. Microbiol.
138:1837-1842[Medline].
|
| 39.
|
Naito, Y.,
H. Tohda,
K. Okuda, and I. Takazoe.
1993.
Adherence and hydrophobicity of invasive and noninvasive strains of Porphyromonas gingivalis.
Oral Microbiol. Immunol.
8:195-202[Medline].
|
| 40.
|
Nakano, J. Y., and H. K. Kuramitsu.
1992.
Mechanism of Streptococcus mutans glucosyltransferases: hybrid enzyme analysis.
J. Bacteriol.
174:5639-5646[Abstract/Free Full Text].
|
| 41.
| Nakano, Y. Personal communication.
|
| 42.
|
Nakayama, K.,
F. Yoshimura,
T. Kadowaki, and K. Yamamoto.
1996.
Involvement of arginine-specific cysteine protease (Arg-gingipain) in fimbriation of Porphyromonas gingivalis.
J. Bacteriol.
178:2818-2824[Abstract/Free Full Text].
|
| 43.
|
Ohta, K.,
K. K. Mäkinen, and W. J. Loesche.
1986.
Purification and characterization of an enzyme from Treponema denticola capable of hydrolyzing synthetic trypsin substrates.
Infect. Immun.
53:213-220[Abstract/Free Full Text].
|
| 44.
|
Onagawa, M.,
I. Ishihara, and K. Okuda.
1994.
Coaggregation between Porphyromonas gingivalis and Treponema denticola.
Bull. Tokyo Dent. Coll.
35:171-181[Medline].
|
| 45.
|
Rosen, G.,
R. Nator,
S. Kutner, and M. N. Sela.
1994.
Characterization of fibrinolytic activities of Treponema denticola.
Infect. Immun.
62:1749-1754[Abstract/Free Full Text].
|
| 46.
|
Rosenberg, M.,
D. Gutnik, and E. Rosenberg.
1980.
Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity.
FEMS Microbiol. Lett.
9:29-33.
|
| 47.
|
Sela, M. N.,
A. Bolotin,
R. Noar,
A. Weinberg, and G. Rosen.
1997.
Lipoproteins of Treponema denticola: their effect on human polymorphonuclear neutrophils.
J. Periodontal Res.
32:455-466[Medline].
|
| 48.
|
Shenker, B. J.,
M. A. Listgarten, and N. S. Taichman.
1984.
Suppression of human lymphocyte responses by oral spirochetes: a monocyte-dependent phenomenon.
J. Immunol.
132:2039-2045[Abstract].
|
| 49.
|
Southern, E. M.
1975.
Detection of specific sequences among DNA fragments separated by gel electrophoresis.
J. Mol. Biol.
98:503-517[Medline].
|
| 50.
|
Tokuda, M.,
M. Duncan,
M.-I. Cho, and H. K. Kuramitsu.
1996.
Role of Porphyromonas gingivalis protease activity in colonization of oral surfaces.
Infect. Immun.
64:4067-4073[Abstract].
|
| 51.
|
Towbin, H.,
T. Staehelin, and J. Gordon.
1979.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76:4350-4354[Abstract/Free Full Text].
|
| 52.
|
Uitto, V.-J.,
Y.-M. Pan,
W. K. Leung,
H. Larjava,
R. P. Ellen,
B. B. Finlay, and B. C. McBride.
1995.
Cytopathic effects of Treponema denticola chymotrypsin-like proteinase on migrating and stratified epithelial cells.
Infect. Immun.
63:3401-3410[Abstract].
|
| 53.
|
Uitto, V. J.,
D. Grenier,
E. C. Chan, and B. C. McBride.
1988.
Isolation of a chymotrypsinlike enzyme from Treponema denticola.
Infect. Immun.
56:2717-2722[Abstract/Free Full Text].
|
| 54.
|
Umemoto, T.,
I. Namikawa, and S. Asai.
1989.
A major antigen on the outer envelope of a human oral spirochete, Treponema denticola.
Infect. Immun.
57:2470-2474[Abstract/Free Full Text].
|
| 55.
|
Walker, S. G.,
J. L. Ebersole, and S. C. Holt.
1997.
Identification, isolation, and characterization of the 42-kilodalton major outer membrane protein (MompA) from Treponema pectinovorum ATCC 33768.
J. Bacteriol.
179:6441-6447[Abstract/Free Full Text].
|
| 56.
|
Wang, E.,
M. M. Garcia,
M. S. Blake,
Z. Pei, and M. J. Blaser.
1993.
Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus.
J. Bacteriol.
175:4979-4984[Abstract/Free Full Text].
|
| 57.
|
Weidner, M.-F.,
D. Grenier, and D. Mayrand.
1996.
Proteolytic artifact in SDS-PAGE analysis of selected periodontal pathogens.
Oral Microbiol. Immunol.
11:103-108[Medline].
|
| 58.
|
Weinberg, A., and S. C. Holt.
1991.
Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains.
J. Bacteriol.
173:6935-6947[Abstract/Free Full Text].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
