The oral anaerobic bacterium Porphyromonas (Bacteroides) gingivalis, which belongs to the
bacteroides-flavobacterium phylum of the bacterial phylogenetic tree (1) , has been implicated as one of the major causative agents
for advanced adult periodontitis(2, 3, 4) .
The microorganism possesses several potential virulence factors for
periodontopathogenicity(5) . Among these factors the
proteolytic enzymes are of special importance, since some of them have
the abilities to destroy periodontal tissue directly or
indirectly(6, 7) , to activate or degrade host
inflammatory
proteins(8, 9, 10, 11) , and to
disturb host defense mechanisms(12, 13, 14) .
There have been many attempts to isolate a variety of proteinases
produced by P. gingivalis in both cell-free and
cell-associated forms (see (15) for a review). In addition,
several genes encoding proteinases have been cloned from P.
gingivalis(16, 17, 18, 19, 20, 21) .
Although it has been found that the multiple forms of trypsin-like
activity of P. gingivalis are due to the presence of either
Arg-gingipain or Lys-gingipain(22) , the number and properties
of proteinases that are actually associated with virulence of the
organism remain to be clarified.
Protoheme is an absolute
requirement for growth of P.
gingivalis(23, 24, 25) , and it is
probably derived from erythrocytes in the natural niche for the
organism. Therefore, it is particularly important for the organism to
agglutinate and lyse erythrocytes in order to survive in
vivo(26, 27) . The close relationship between
hemagglutinin and cysteine proteinase has been pointed out by several
researchers(19, 28, 29, 30, 31) .
However, there has been little agreement as yet on the identity of the
two molecules. Nishikata and Yoshimura (29) found that one
molecule possesses both the proteinase and hemagglutinin activities. On
the other hand, Pike et al.(30) and Shah et al.(31) reported that the proteinase and hemagglutinin are
separate molecules, although the two molecules are noncovalently bound
to each other. Recently, based on the cloning and sequence analysis of
the gene encoding arginine-specific cysteine proteinase
(argingipain)(19) , we have suggested that the enzyme results
from processing of a 109-kDa preproenzyme comprising four domains, i.e. the signal peptide, the amino-terminal domain, the
proteinase domain, and the carboxyl-terminal hemagglutinin domain. This
finding is consistent with the results of Ciborowski et
al.(28) , who have suggested that the proteinase and
hemagglutinin molecules are formed by processing of the primary product
from the same gene.
Pavloff et al.(21) also have
sequenced the gene encoding arginine-specific cysteine proteinase
(Arg-gingipain-1) from a different P. gingivalis strain.
Comparison of the amino acid sequences deduced from the nucleotide
sequences of argingipain and Arg-gingipain-1 genes have revealed that
they are essentially identical, except that the argingipain gene lacks
a sequence intervening between direct repeats in the carboxyl-terminal
domain. Especially, the proteinase domains of argingipain and
Arg-gingipain-1 genes were completely identical. Therefore, the
arginine-specific cysteine proteinase ``argingipain'' was
renamed ``Arg-gingipain'' in the present study to avoid
redundancies in nomenclature.
We have shown previously that
Arg-gingipain is a major cysteine proteinase of P. gingivalis,
a part of which is secreted extracellularly, and have strongly
suggested that the enzyme is directly involved in the destruction of
periodontal tissue(32) . Furthermore, based on its strong
inhibition of the chemiluminescence (CL) (
)response of
polymorphonuclear leukocytes (PMNs) and its ability to degrade human
immunoglobulins G and A, we have proposed that the enzyme can impair
host defense mechanisms(32) . However, we do not understand to
what extent Arg-gingipain contributes to the virulence of P.
gingivalis. Also, there is little information available on the
physiological significance of the enzyme in the organism. To gain some
insight into these questions, it is necessary to undertake the
molecular genetic approach. For this, we constructed
Arg-gingipain-deficient mutants via disruption of the Arg-gingipain
gene by use of suicide plasmid systems (33) . In the course of
construction, we found that two different Arg-gingipain genes existed
on the chromosome of P. gingivalis ATCC33277. The results
obtained with the Arg-gingipain-deficient mutants provide the evidence
that Arg-gingipain is a major virulence factor of P.
gingivalis.
EXPERIMENTAL PROCEDURES
Bacterial Strains and Plasmids
P. gingivalis ATCC33277, P. gingivalis 381, and Escherichia coli DH5 were used. Plasmids pUC18(34) , pKDCMZ(33) ,
pMJF-3(35) , and R751 (36) were used for construction
of suicide/integration plasmids for P. gingivalis. Plasmid
P.g./pUC118 (19) was used as a source of the Arg-gingipain
gene. Plasmids pKD274, pKD279, pKD280, and pKD290 and P. gingivalis mutants KDP110, KDP111, and KDP112 were obtained in this study.
Media and Conditions for Cell Growth
P.
gingivalis cells were grown anaerobically (10% CO
, 10%
H
, 80% N
) in enriched BHI broth (containing,
per liter, 37 g of brain heart infusion (Difco), 5 g of yeast extract
(Difco), 1 g of cysteine, 5 mg of hemin, and 1 mg of vitamin
K
) and on enriched tryptic soy agar (containing, per liter,
40 g of Trypto-Soya agar (Nissui, Tokyo, Japan), 5 g of brain heart
infusion, 1 g of cysteine, 5 mg of hemin, and 1 mg of vitamin
K
). L broth (containing, per liter, 10 g of tryptone
(Difco), 5 g of yeast extract, and 5 g of sodium chloride) was used for
growing E. coli cells. For selection or maintenance of the
antibiotic-resistant strains, antibiotics were added to the media at
the following concentrations (ampicillin, 50 µg/ml;
chloramphenicol, 25 µg/ml; erythromycin, 10 µg/ml; gentamicin,
100 µg/ml; and tetracycline, 1 µg/ml).
Chemicals and Proteins
Proteinase inhibitors, N
-p-tosyl-L-lysine
chloromethyl ketone (TLCK) and leupeptin were purchased from Sigma and
Peptide Institute Inc. (Osaka, Japan), respectively. Synthetic
substrates, carbobenzoxy-L-phenylalanyl-L-arginine
4-methyl-7-coumarylamide (Z-Phe-Arg-MCA) and t-butyloxycarbonyl-L-phenylalanyl-L-seryl-L-arginine
4-methyl-7-coumarylamide (Boc-Phe-Ser-Arg-MCA) were obtained from
Peptide Institute Inc. Casein, bovine hemoglobin, oyster glycogen, and
zymosan A were obtained from Sigma. DNA restriction enzymes and T4
ligase were purchased from Takara (Kyoto, Japan).
DNA Manipulations
Plasmid DNA was purified from E. coli cells by using the Wizard(TM) DNA purification
system (Promega, Madison, WI). Chromosomal DNA was isolated from P.
gingivalis cells by the guanidine isothiocyanate method (37) with the IsoQuick DNA extraction kit (MicroProbe, Garden
Grove, CA).
Plasmid Construction
Suicide plasmids constructed
in this study are depicted in Fig. 1. A 1.8-kbp SmaI-BamHI fragment of P.g./pUC118 plasmid was
ligated with a HincII-BamHI digest of pUC18. The
resulting plasmid was then digested with EcoRV and ligated
with a BglII linker oligonucleotide to convert the EcoRV site to BglII, giving rise to plasmid pKD274.
Plasmid pKD274 was digested with BglII and BamHI and
self-ligated to yield pKD279. A 0.7-kbp BglII-PstI
fragment of pKD274, which corresponded to the EcoRV-PstI fragment within the Arg-gingipain gene,
was ligated with a BamHI-PstI digest of pKDCMZ
plasmid to yield pKD280. A 0.7-kbp HindIII-BglII
fragment of pKD274 was ligated with a HindIII-BamHI
digest of pMJF-3 plasmid, resulting in plasmid pKD290.
Figure 1:
Plasmid
construction. Ap, ampicillin resistance; Cm,
chloramphenicol resistance; Em, erythromycin resistance; Tc, tetracycline resistance; GUS,
-glucuronidase
gene; Mob, region for plasmid mobilization; ori,
replication origin functioning in E. coli. Black region of Arg-gingipain gene, signal sequence; dark region of Arg-gingipain gene, amino-terminal domain; open region of Arg-gingipain gene, proteinase domain; hatched region of Arg-gingipain gene, carboxyl-terminal domain.
Restriction sites in parentheses no longer exist on the
plasmids.
DNA Probes and Southern Blot Hybridization
A
2.8-kbp SmaI fragment of P.g./pUC118 (probe I) and a 0.7-kbp PstI-EcoRI fragment of pKD279 (probe II) were labeled
with digoxigenin-dUTP (Boehringer GmbH, Mannheim, Germany). Synthetic
oligonucleotides, 5`-GTAGCTTGTGTGAATGGCGATTTCC-3` (probe III),
5`-CGGCACGAAGATCAAGGAAGGTCTG-3` (probe IV),
5`-TTGGACTCGGAGACTTTGTGCAGAC-3` (probe V), and
5`-CCGAATCCAAATCCGAATCCGAATC-3` (probe VI) were obtained from Funakoshi
(Tokyo, Japan) and were labeled with fluorescein-dUTP (Amersham
International plc, Little Chalfont, United Kingdom). Southern blotting
was performed by using a nylon membrane (Hybond(TM)-N; Amersham)
essentially according to Southern(38) . Hybridization with
probes I and II was done by using the Boehringer nonradioactive DNA
labeling and detection kit, and the ECL 3`-oligolabeling and detection
systems (Amersham) were used for Southern analyses with the
oligonucleotide probes.
Mobilization of a Suicide Plasmid (pKD280) from E. coli
to P. gingivalis
The procedure for the mobilization was
described previously(33) . Briefly, the culture of E. coli DH5 harboring pKD280 and R751 plasmids was mixed with an equal
volume of the culture of P. gingivalis ATCC33277 and the
cultures were harvested by centrifugation. The cell pellet was
resuspended in prewarmed enriched BHI broth and spotted on enriched
tryptic soy agar. The plates were aerobically incubated at 37 °C
for 4 h prior to anaerobic incubation at 37 °C for 36 h. Bacterial
cells on the plates were collected with a cotton swab, resuspended in
enriched BHI broth, spread on enriched tryptic soy agar containing
erythromycin and gentamicin, and incubated anaerobically at 37 °C
for 7 days. Erythromycin-resistant (Em
) transconjugants
were obtained at the frequency of 2.5
10
/recipient cell.
Electrotransformation of P. gingivalis with pKD290
Plasmid DNA
P. gingivalis cells were anaerobically
grown to 6
10
/ml at 37 °C in enriched BHI
broth. The cells were then harvested by centrifugation, washed with the
electroporation solution (300 mM sucrose), and resuspended in
0.1 volume of the same solution. Fifteen microliters of pKD290 plasmid
DNA solution (270 µg of DNA/ml in TE buffer) were added to 0.4 ml
of the cell suspension. The whole volume of the DNA-containing cell
suspension was poured into a cuvette for electroporation (Pulser(TM)
cuvette with 0.2-cm electrode gap; Bio-Rad). Electroporation was
performed on the condition (voltage, 2.0 kV; time constant, 5 ms) with
an electroporation apparatus (Gene Pulser(TM); Bio-Rad). These
procedures were carried out at 4 °C. The cell suspension was
immediately mixed with 10 ml of prewarmed enriched BHI broth and
incubated anaerobically at 37 °C for 15 h. Cells of the culture
were spread on enriched tryptic soy agar containing tetracycline and
incubated anaerobically at 37 °C for 7 days. Tetracycline-resistant
(Tc
) transformants were obtained at the frequency of 3.7
10
/recipient cell.
Preparation of Culture Supernatants and Cell
Extracts
Twenty-four-hour cultures were harvested by
centrifugation at 10,000
g for 20 min at 4 °C.
Ammonium sulfate was added to the culture supernatant to a final
concentration of 75% saturation. The precipitated proteins were
collected by centrifugation at 10,000
g for 20 min and
suspended in 10 mM sodium phosphate buffer (pH 7.0) containing
0.05% Brij 35. After overnight dialysis against the same buffer at 4
°C, insoluble materials were removed by centrifugation at 25,000
g for 30 min. The resulting supernatant was used as a
culture supernatant in this study. On the other hand, bacterial cells
were washed with phosphate-buffered saline (PBS) and resuspended in 10
mM sodium phosphate buffer (pH 7.0) containing 0.05% Brij 35.
Cell extracts were prepared by ultrasonication followed by
centrifugation at 25,000
g for 30 min.
Gel Electrophoresis and Immunoblot Analysis
Sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed
according to the method of Laemmli(39) . The proteinase
inhibitor leupeptin was added to a solubilizing buffer to avoid
proteolysis by endogenous proteinases. For immunoblotting, proteins on
SDS gels were electrophoretically transferred to nitrocellulose
membranes according to the method of Towbin et
al.(40) . The blotted membranes were immunostained with
anti-Arg-gingipain IgG, essentially according to the procedure
described previously(41) .
Enzymatic Assays
Arginine-specific cysteine
proteinase activity on the two synthetic substrates, Z-Phe-Arg-MCA and
Boc-Phe-Ser-Arg-MCA, was determined by the method described previously (32) . Briefly, the reaction mixture (1 ml) contained various
amounts of cell extracts or culture supernatants, 10 µM
synthetic substrates, and 5 mM cysteine in 20 mM sodium phosphate buffer (pH 7.5). After incubation at 40 °C
for 10 min, the reaction was terminated by adding 1 ml of 10 mM iodoacetic acid (pH 5.0), and the released
7-amino-4-methylcoumarin was measured at 460 nm (excitation at 380 nm).
The casein- and hemoglobin-hydrolyzing activities were determined by
measuring acid-soluble products by the method described earlier (42) with slight modification. The reaction mixture (0.5 ml)
containing various amounts of culture supernatants, 2.5 mg of casein or
bovine hemoglobin, and 1 mM dithiothreitol in 20 mM sodium phosphate buffer (pH 7.5) was incubated at 40 °C for 10
min. The reaction was stopped by adding 0.5 ml of 5% trichloroacetic
acid and keeping on ice for 10 min. After centrifugation, 300 µl of
the resulting supernatant were added to 2 ml of 0.2 M borate
buffer (pH 8.9) and then mixed with 1 ml of fluorescamine (0.1 mg/ml in
acetone). The fluorescence was measured at 475 nm (excitation at 390
nm) on a fluorescence spectrophotometer (model F-3010; Hitachi, Tokyo,
Japan).
Measurement of Luminol-dependent CL Response
CL
response of PMNs was measured according to the method described
previously(32) . In summary, sterilized oyster glycogen (0.2%
in saline) was intraperitoneally injected into guinea pigs. At 14 h
after the injection, the cells in the peritoneal exudate were collected
and washed twice with Hanks' balanced salt solution (Nissui), and
suspended in the same medium. The cell suspension (1
10
cells/ml) was preincubated at 37 °C for 30 min with the
indicated concentrations of bacterial culture supernatants. Then, PMNs
were washed with PBS and resuspended in PBS at a final cell
concentration of 2
10
cells/ml. Zymosan A (20 mg/ml
in PBS) was boiled for 10 min and washed with PBS prior to being
opsonized to reduce clumping. The zymosan suspension was incubated with
an equal volume of serum from guinea pig at 37 °C for 30 min. The
particles were washed twice with PBS and suspended in the original
volume of PBS. The cuvette that contained the reaction mixture
consisting of 0.1 ml of freshly diluted luminol solution (0.2
mM), 0.1 ml of the PMNs suspension (2
10
cells/ml), and 0.1 ml of the opsonized zymosan (20 mg/ml) was
maintained at 37 °C in a luminescence analyzer (model LB9505AT;
Berthold, Wildbad, Germany). The intensity of light emitted in the
cuvette was measured for 30 min. The CL response is expressed by the
peak intensity of CL.
Hemagglutination Assay
Overnight cultures of P. gingivalis strains were centrifuged, washed twice with PBS,
and resuspended in PBS at an optical density at 660 nm of 0.44. The
bacterial suspensions were then diluted in a two-fold series with PBS.
A 100-µl aliquot each of the dilutions was mixed with an equal
volume of sheep erythrocyte suspension (2.5% in PBS) and incubated in a
round-bottom microtiter plate at room temperature for 3 h. The
hemagglutination titer was determined as the last dilution exhibiting
full agglutination.
RESULTS
Southern Hybridization Analyses with DNA Probes for the
P. gingivalis Arg-gingipain Gene
The Arg-gingipain-encoding gene has been cloned from P.
gingivalis 381, and its nucleotide sequence has been determined (19) . Since it was difficult to use this strain for
construction of Arg-gingipain-deficient mutants because of its low
efficiency in mobilization and electrotransformation, (
)we
chose P. gingivalis ATCC33277 from which we had isolated a
superoxide dismutase-deficient mutant by mobilization of a suicide
plasmid(33) . To examine whether the Arg-gingipain gene is
located on the chromosome of P. gingivalis ATCC33277, Southern
hybridization of its chromosomal DNA was performed with a 2.8-kbp SmaI fragment of P.g./pUC118 corresponding to the SmaI-PvuII region of Arg-gingipain gene (probe I) as
a DNA probe (Fig. 2). Interestingly, probe I DNA hybridized to
four and five separate HindIII fragments of the chromosomes of P. gingivalis ATCC33277 and 381, respectively. Since probe I
DNA has no HindIII site, the result indicates that P.
gingivalis chromosome may possess several regions which share
homology with the probe DNA. Then, we analyzed the chromosome of
ATCC33277 with other Arg-gingipain gene-associated DNA probes. A
0.7-kbp PstI-EcoRI fragment of pKD279, which
corresponded to the SmaI-EcoRV region encoding the
amino-terminal region of the proteinase domain (probe II) and an
oligonucleotide probe encoding a putative catalytic site for the
proteinase (probe III)(19) , hybridized to two separate HindIII fragments, suggesting that P. gingivalis ATCC33277 may possess two different chromosomal loci encoding
Arg-gingipain (Fig. 2). One of the gene loci (12.5-kbp HindIII fragment) and the other (7.8-kbp HindIII
fragment) were tentatively designated rgpA and rgpB,
respectively. The rgpA locus was also hybridized with two
oligonucleotide probes for the carboxyl-terminal domain of
Arg-gingipain (probe IV) and for the region downstream from the
Arg-gingipain gene (probe V), whereas the rgpB locus was
hybridized with neither of them (Fig. 2). The result suggests
that the rgpA locus may encode the carboxyl-terminal domain of
Arg-gingipain in addition to the proteinase domain, while the rgpB locus may not. Recently, Pavloff et al.(21) have
sequenced the Arg-gingipain-1 gene (rgp1) of P. gingivalis H66. Comparison between the amino acid sequences of Arg-gingipain
gene of 381 and Arg-gingipain-1 gene of H66 revealed that their
proteinase domains were identical but their carboxyl-terminal domains
were different (Fig. 2). The Arg-gingipain-1 gene contains three
direct repeats of the nucleotide sequence that encodes 17 amino acids
starting from YTYTVYRD in its carboxyl-terminal region. A DNA region
intervening between the first repeating sequence and the third one is
completely deleted from the carboxyl-terminal region of the
Arg-gingipain gene. To determine whether the rgpA locus of
ATCC33277 contains this intervening sequence, the oligonucleotide probe
for the intervening sequence (probe VI) was used. The rgpA locus was hybridized with this probe, and the restriction maps of
the rgpA locus of ATCC33277 and the rgp1 locus of H66
were so far identical, suggesting that the rgpA of ATCC33277
is probably equivalent to the rgp1 of H66. From these Southern
analyses, we also found that at least two separate chromosomal regions
other than the rgpA locus might encode the carboxyl-terminal
domain of Arg-gingipain-1.
Figure 2:
Southern blot analyses of P.
gingivalis ATCC33277 chromosomal DNA with the Arg-gingipain and
Arg-gingipain-1 gene probes. Panel A, restriction maps of
Arg-gingipain gene of P. gingivalis 381 and Arg-gingipain-1
gene of P. gingivalis H66 and location of the DNA regions
complementary to their DNA probes. The restriction maps of
Arg-gingipain and Arg-gingipain-1 genes are according to Okamoto et
al.(19) and Pavloff et al.(21) ,
respectively. Openboxes above restriction maps show
Arg-gingipain DNA probes I and II. Triangles indicate the DNA
regions complementary to the oligonucleotide probes III, IV, V, and VI.
The dark, dotted, open, and hatched regions of Arg-gingipain gene represent the signal sequence, the
amino-terminal domain, the proteinase domain, and the carboxyl-terminal
domain, respectively. Black regions of Arg-gingipain and
Arg-gingipain-1 genes represent a direct repeat encoding 17 amino acids
starting from YTYTVYRD. Restriction sites: B, BamHI; E, EcoRV; Ps, PstI; Pv, PvuII; S, SmaI. Panel B, Southern
blots. The chromosomal DNA of P. gingivalis was digested with
several restriction enzymes. The digested DNA was subjected to agarose
gel electrophoresis and Southern blot hybridization with the indicated
DNA probes. a, chromosomal DNA of ATCC33277 (lanes1 and 3) and 381 (lanes2 and 4) was digested with HindIII (lanes1 and 2) and PvuII (lanes3 and 4). b, chromosomal DNA of ATCC33277 was digested with HindIII (lane1), PvuII plus PstI (lane2), PvuII plus EcoRV (lane3), PvuII (lane4), and StuI (lane5). c-f, chromosomal DNA of ATCC33277 was digested with HindIII (lane1), StuI (lane2), PvuII (lane3), PvuII plus PstI (lane4), and PvuII plus EcoRV (lane5). The
blots (c and d) were subjected to rehybridization
with probe VI (e) and probe V (f), respectively,
after removal of probes III and IV.
Construction of Arg-gingipain-deficient Mutants
To determine the importance of Arg-gingipain for
pathogenecity of P. gingivalis, we constructed
Arg-gingipain-deficient mutants via gene disruption by use of a suicide
plasmid containing an internal DNA fragment of the Arg-gingipain gene.
Thus, Em
transconjugants were obtained after mobilization
of pKD280 containing the 0.7-kbp EcoRV-PstI fragment
of the Arg-gingipain gene into P. gingivalis ATCC33277.
Southern hybridization analysis of the chromosomes of the Em
transconjugants showed that two classes of transconjugants were
obtained with respect to the location of the integrated plasmid. In one
class (a representative strain, KDP110) the rgpA locus is
disrupted, while in the other class (a representative strain, KDP111)
disruption occurred at the rgpB locus ( Fig. 3and Fig. 4). Thus, integration of pKD280 plasmid DNA into the
chromosome at the rgpA locus resulted in the disappearance of
the 12.5-kbp hybridizing HindIII fragment and the appearance
of the 7.9-kbp HindIII fragment. This new hybridizing fragment
overlaps with the 7.8-kbp HindIII fragment from the rgpB locus on the blot. Integration of pKD280 at the rgpB locus eliminated the 7.8-kbp HindIII fragment and the
5.4-kbp HindIII fragment appeared. PvuII digestions
of the rgp mutants clarified that the 7.5-kbp PvuII
fragment DNA of ATCC33277 contained two different PvuII
fragments hybridizing to probe II, confirming the result of the HindIII digestion.
Figure 3:
Southern blot analysis of chromosomal DNA
of the rgp mutants. The chromosomal DNA of ATCC33277 (lanes4 and 8), KDP110 (lanes3 and 7), KDP111 (lanes2 and 6), and KDP112 (lanes1 and 5) was
digested with HindIII (lanes 1-4) and PvuII (lanes 5-8). These digested DNA were
subjected to agarose gel electrophoresis and Southern blot
hybridization with probe II.
Figure 4:
Chromosomal structures at the rgpA and rgpB loci of the rgp mutants. a, rgpA loci of ATCC33277 and an rgpA mutant, KDP110. b, rgpB loci of ATCC33277 and an rgpB mutant, KDP111. c, rgpA loci of KDP111 and an rgpA rgpB mutant, KDP112. Closedboxes represent the DNA regions complementary to probe II. Restriction
sites: E, EcoRV; H. HindIII; Ps, PstI; Pv, PvuII.
Next, we constructed an rgpA rgpB double mutant from KDP111 (rgpB) by electrotransformation
with the suicide/integration plasmid (pKD290), which consisted of the
pMJF-3 plasmid DNA and the SmaI-EcoRV fragment DNA of
the Arg-gingipain gene. Southern blot hybridization with the
Arg-gingipain gene probe revealed that pKD290 plasmid DNA was
integrated to the chromosome of a transformant (KDP112) at the rgpA locus ( Fig. 3and Fig. 4). Thus, integration of
pKD290 plasmid DNA at the rgpA locus resulted in the
disappearance of a 12.5-kbp HindIII fragment and the
appearance of 6.1- and 16.8-kbp HindIII fragments.
Characterization of the rgpA, rgpB, and rgpA rgpB Mutants
Western Blot Analysis with Anti-Arg-gingipain
IgG
Cell extracts of the wild type strain ATCC33277 showed a
discrete protein band with an apparent molecular mass of 44 kDa and
smearing protein bands with molecular masses of 60-90 kDa (Fig. 5a). The 44-kDa protein band almost disappeared
in the rgpA mutant extract (KDP110). In contrast, the 44-kDa
protein remained in the rgpB mutant (KDP111), but most of the
upper smearing bands disappeared with several new protein bands
appearing. These new protein bands may be explained by the truncated
products from the disrupted rgpB gene. The rgpA rgpB double mutant (KDP112) showed only protein bands similar to the
new protein bands of KDP111, suggesting that rgpA and rgpB are mainly responsible for the production of the protein band of
44 kDa and the protein bands of 60-90 kDa, respectively, and that
the rgpA rgpB double mutant loses most of the proteins
immunoreacting with anti-Arg-gingipain IgG. The culture supernatant of
the wild type strain showed immunoreactive protein bands with molecular
masses of 76, 70, and 44 kDa (Fig. 5b). Despite the
presence of smearing bands of 60-90 kDa in its cell extracts,
clear proteins immunoreactive with anti-Arg-gingipain IgG were barely
detectable in the culture supernatant of KDP110 under the condition
used. However, when a large excess of the fraction was applied to
SDS-gels, faint but clear immunoreactive bands with apparent molecular
masses of 76 and 70 kDa, but not 44 kDa, were detected (data not
shown). Therefore, it may be that the disruption of rgpA gene
affects extracellular translocation of these rgpB-derived
proteins. The culture supernatant of the rgpB mutant (KDP111)
revealed the 44-kDa protein alone. No immunoreactive bands were
detected in the culture supernatant of the double mutant (KDP112), even
when a large excess of this fraction was used. These results strongly
suggest that proteins of 44 and 60-90 kDa are products from the rgpA and rgpB genes, respectively.
Figure 5:
Immunoblot analysis of culture
supernatants and cell extracts of the rgp mutants with
anti-Arg-gingipain antibody. Cell extracts (10 µg of protein) (a) and culture supernatants (30 µg of protein) (b) were subjected to SDS-polyacrylamide gel electrophoresis
on 7-12% gradient gels. Protein bands on the gels were
transferred to nitrocellulose membranes and immunoreacted with
anti-Arg-gingipain antibody. Lanes 1, ATCC33277; lanes
2, KDP110; lanes 3, KDP111; lane 4,
KDP112.
Proteolytic Activity
Arg-gingipain can cleave the
two synthetic substrates Boc-Phe-Ser-Arg-MCA and
Z-Phe-Arg-MCA(32) . The hydrolytic activity on these substrates
was markedly decreased in both the culture supernatants and cell
extracts of KDP110 (rgpA) and KDP111 (rgpB).
Moreover, KDP112 (rgpA rgpB) showed complete loss of the
hydrolytic activity in both its culture supernatant and cell extract (Table 1). Residual activities observed in KDP110 and KDP111 were
inhibited by leupeptin, TLCK, and EDTA (data not shown). The residual
activities of the single mutants apparently did not balance. The marked
and disproportioned decrease in the proteolytic activities of
Arg-gingipain in the single mutants suggests the possibility that the
disruption of one rgp gene has an inhibitory effect on
expression from the other gene. When the protein substrates casein and
hemoglobin were used in assay, only 5-10% of the hydrolytic
activity of the culture supernatant of ATCC33277 was observed in that
of KDP112 (rgpA rgpB) (Table 1). These results indicate
that Arg-gingipain is a major extracellular proteinase of P.
gingivalis.
Effect on CL Response of PMNs
The culture
supernatant of P. gingivalis possesses a potent virulence
factor, which disrupts the bactericidal function of PMNs(14) .
The virulence factor was successfully purified and turned out to be
arginine-specific cysteine proteinase (Arg-gingipain)(32) . To
determine whether the virulence factor in the culture supernatant is
attributable to Arg-gingipain, we examined the rgp mutants for
the effect of their supernatants on CL response of PMNs stimulated by
serum-activated zymosan. The supernatant of KDP112 (rgpA rgpB)
almost completely lost the inhibitory effect on CL response of PMNs,
whereas KDP110 (rgpA) and KDP111 (rgpB) partially
lost the effect (Fig. 6). The result suggests the possibility
that the effect of the culture supernatant of P. gingivalis on
CL response of PMNs is mainly due to Arg-gingipain.
Figure 6:
Effect of the culture supernatants of the rgp mutants on the CL response of PMNs. Guinea pig PMNs (1
10
cells/ml) were preincubated with indicated
amounts of culture supernatant at 37 °C for 30 min. Then, PMNs were
washed and resuspended in PBS at a final concentration of 2
10
cells/ml. The CL response of the PMNs was measured after
stimulating by opsonized zymosan. The values are means of three
determinations.
, ATCC33277;
, KDP110;
, KDP111;
, KDP112.
Hemagglutination
We found in the previous study (19) that the carboxyl-terminal domain of Arg-gingipain
contained the sequence identical to the amino-terminal sequence of a
hemagglutinin fraction suggested by Pike et al.(30) .
To determine whether the rgp genes are related with
hemagglutination of P. gingivalis, we examined the rgp mutants for hemagglutination. KDP110 (rgpA) and KDP111 (rgpB) showed only a little decrease of hemagglutination,
whereas the rgpA rgpB double mutant (KDP112) showed a greater
decrease of hemagglutination (Fig. 7). These results indicate
that the rgp genes are directly or indirectly involved in the
hemagglutination activities of P. gingivalis.
Figure 7:
Hemagglutinating activity of the rgp mutants. The hemagglutination titers of P. gingivalis ATCC33277, KDP110, KDP111, and KDP112 are 32, 16, 16, and 1,
respectively.
DISCUSSION
We constructed rgpA and rgpB single mutants
of Arg-gingipain by integration of the Em
suicide plasmid
pKDCMZ containing an internal DNA fragment of the gene. To construct
the rgpA rgpB double mutant, we needed to use a second suicide
plasmid carrying a different antibiotic resistance gene and having
little or no homology with pKDCMZ. The shuttle vector pMJF-3 contains a
Tc
gene, which functions in Bacteroides spp. (35) and P. gingivalis.
Although pMJF-3
can replicate in some Bacteroides spp., it cannot in P.
gingivalis.
Furthermore, pMJF-3 DNA has only a little
homology with pKDCMZ DNA. Therefore, this vector was used to construct
the second suicide plasmid pKD290 for construction of the double
knockout mutant. We used electroporation to introduce pKD290 into P. gingivalis cells, although P. gingivalis strains
including ATCC33277 were thought to possess DNA restriction
systems(43) . All the Tc
transformants analyzed
contained the suicide plasmid DNA in the homologous chromosomal region.
This result indicates that electroporation can be applied to construct
gene-disrupted mutants in P. gingivalis.
Several lines of
evidence show that P. gingivalis ATCC33277 possesses two
separate genes responsible for arginine-specific cysteine proteinase
activity in its chromosome. First, the DNA probes for the proteinase
domain of Arg-gingipain hybridized to two different restriction
fragments of the chromosomal DNA. Second, homologous recombination took
place between each of the two genetic loci (rgpA and rgpB) on the chromosome and the Arg-gingipain DNA on the
suicide plasmids. Third, the rgpA and the rgpB single
mutants showed the marked reduction of the hydrolytic activity on the
two synthetic substrates for arginine-specific proteinase but still
retain some of the activity; however, the rgpA rgpB double
mutant showed no hydrolytic activity on the substrates. Fourth,
residual proteinase activity seen in the culture supernatants and the
cell extracts of the rgpA and rgpB single mutants was
sensitive to leupeptin, TLCK, and EDTA, all of which are potent
inhibitors for Arg-gingipain. Fifth, the culture supernatants and cell
extracts of the rgpA and rgpB single mutants still
contained proteins that immunoreacted with anti-Arg-gingipain antibody.
However, the amounts of immunoreacting protein made by the mutants were
much less than that of the wild type strain, and the profiles of the
immunoblots were also different from that of the wild type strain.
These immunoreacting substances almost disappeared in the rgpA rgpB mutant.
Gene duplication has been observed in other prokaryotic
systems (44, 45, 46) . Among them, the
cholera toxin operon (ctxAB) in Vibrio cholerae is a
good example with respect to the direct correlation between copy number
and virulence(46) . The proteinase domain region of
Arg-gingipain gene was probably duplicated at some time in the
evolution of P. gingivalis, and a strain possessing two or
more Arg-gingipain-related genes may selectively survive in conflict
with its host and other microorganisms.
Although the rgpA and rgpB genes are very similar to each other in their
DNA regions for the proteinase domains, their DNA regions for the
carboxyl-terminal domains are not identical, as revealed by the
Southern analyses. Judging from the immunoblot analyses, the rgpA and rgpB gene products may be different from each other
in the processing step. The difference in this step could come from the
difference of carboxyl-terminal domains of these gene products. Further
study including cloning and sequencing of rgpA and rgpB genes is needed to determine the similarity of the two genes and
their products. This work is now in progress.
Several proteinases
with specificities similar to Arg-gingipain have been purified from
various P. gingivalis strains(20, 28, 30, 47, 48, 49, 50, 51, 52) .
In this study, we found that all of the enzymatic activity for
arginine-specific cysteine proteinase in P. gingivalis ATCC33277 might be derived from the rgpA and rgpB, as suggested by the total loss of the activity in the rgpA rgpB mutant. Therefore, some of the proteinases
previously described may be homologs of the rgpA or rgpB gene product and others may not be virtually expressed in the P. gingivalis cells, although we cannot rule out the
possibility that a protease gene(s) could be located downstream of rgpA or rgpB and inactivated by polar effect of
plasmid integration. Usually, complementation of a mutation by a
plasmid carrying a wild type gene can be used in many microorganisms to
prove the cause and effect relationship. However, it is difficult to
apply complementation to P. gingivalis because no stable
plasmid has been found in this organism.
Hydrolytic activities on
casein and hemoglobin were significantly reduced in the culture
supernatant of the rgpA rgpB mutant, indicating that
Arg-gingipain is particularly important among extracellular proteinases
secreted from P. gingivalis for general protein degradation.
In addition, Arg-gingipain has the specific ability to disturb the
function of PMNs. The present study indicates that the inhibitory
effect of the culture supernatant of P. gingivalis on CL
response of PMNs is mainly attributable to the presence of
Arg-gingipain in the supernatant. Thus, it is feasible to consider that
Arg-gingipain plays an important role in periodontopathogenicity of P. gingivalis.
A role for Arg-gingipain in P.
gingivalis hemagglutination is indicated by the marked decrease in
this activity in the rgpA rgpB double mutant, although we
should reserve the possibility that hemagglutinin genes could be
located downstream of both of the rgpA and rgpB genes
and simultaneously inactivated in the double mutant by integration of
the suicide plasmids. Pike et al.(30) found that the
high molecular mass Arg-gingipain has hemagglutinin activity that is
inhibited by arginine, whereas the low molecular mass Arg-gingipain is
devoid of the activity. We also found that the extracellular
Arg-gingipain (44 kDa) purified from the culture supernatant has no
hemagglutinin activity. (
)These results suggest that the
carboxyl-terminal domains of Arg-gingipain and Arg-gingipain-1
(starting from SGQAEIVL) could be responsible for the hemagglutinin
activity; however, the possibility that the proteinase domain is also
necessary for the activity cannot be excluded since the rgpB gene seems not to possess the same carboxyl-terminal domain. In
this connection, we found in this study that there were at least two
other chromosomal regions which may share homology with the
carboxyl-terminal domain, suggesting that these regions might encode
other hemagglutinins.