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J Biol Chem, Vol. 274, Issue 25, 17955-17960, June 18, 1999
From the Departments of Porphyromonas gingivalis produces
arginine-specific cysteine proteinase (Arg-gingipain, RGP) and
lysine-specific cysteine proteinase (Lys-gingipain, KGP) in the
extracellular and cell-associated forms. Two separate genes
(rgpA and rgpB) and a single gene
(kgp) have been found to encode RGP and KGP, respectively.
We constructed rgpA rgpB kgp triple mutants by homologous
recombination with cloned rgp and kgp DNA
interrupted by drug resistance gene markers. The triple mutants showed
no RGP or KGP activity in either cell extracts or culture supernatants.
The culture supernatants of the triple mutants grown in a rich medium
had no proteolytic activity toward bovine serum albumin or gelatin
derived from human type I collagen. Moreover, the mutants did not grow
in a defined medium containing bovine serum albumin as the sole
carbon/energy source. These results indicate that the proteolytic
activity of P. gingivalis toward bovine serum albumin and
gelatin derived from human type I collagen appears to be attributable
to RGP and KGP. The hemagglutinin gene hagA of P. gingivalis possesses the adhesin domain regions responsible for
hemagglutination and hemoglobin binding that are also located in the
C-terminal regions of rgpA and kgp. A
rgpA kgp hagA triple mutant constructed in this study
exhibited no hemagglutination using sheep erythrocytes or hemoglobin
binding activity, as determined by a solid-phase binding assay with
horseradish peroxidase-conjugated human hemoglobin, indicating that the
adhesin domains seem to be particularly important for P. gingivalis cells to agglutinate erythrocytes and bind hemoglobin,
leading to heme acquisition.
Porphyromonas gingivalis is a Gram-negative anaerobic
bacterium that is implicated as an important etiological agent of adult periodontal disease (1). P. gingivalis is asaccharolytic and highly proteolytic. Proteinases with trypsin-like activity, which are
major extracellular and cell-associated proteinases of P. gingivalis, are now found to consist of arginine-specific cysteine proteinase (Arg-gingipain,
RGP)1 and lysine-specific
cysteine proteinase (Lys-gingipain, KGP) (2). Molecular genetic
analyses have revealed that RGP is encoded by the two genes
rgpA (rgp-1, prpR1, and prtR) and
rgpB (rgp-2, prR2, and prtRII) (3-6),
and KGP is encoded by the single gene kgp (prtP
and prtK) (7-11). In addition to rgp and
kgp, several proteinase-encoding genes have been cloned and
characterized (12-14). Because of asaccharolysis, P. gingivalis is totally dependent on amino acids and peptides for
its growth. However, it has not yet been determined what proteinase(s)
is actually responsible for the degradation of environmental proteins
and the generation of amino acids and peptides as carbon/energy sources.
Nucleotide sequencing revealed that rgpA consists of three
DNA regions: (i) an N-terminal propeptide, (ii) a proteinase domain, and (iii) a C-terminal adhesin domain region (15). rgpB
shares a high similarity in the N-terminal propeptide and proteinase domain with rgpA, and, importantly, the proteinase domains
of the two genes are almost identical (4). Most of the C-terminal adhesin domain region is absent in rgpB (4). On the other
hand, kgp has the same gene structure (an N-terminal
propeptide, a proteinase domain, and a C-terminal adhesin region) as
rgpA (7). Although the proteinase domains of kgp
and rgpA are divergent, their C-terminal adhesin domain
regions are very similar to each other (7). In addition to
rgpA and kgp, part of the C-terminal adhesin
domain region is also encoded by hagA and tla of
P. gingivalis (16, 17). The C-terminal adhesin domain region
of rgpA consists of four domains (HGP44, HGP15, HGP17, and
HGP27) (15). One of the domain proteins, HGP15, was found to have the
ability to bind hemoglobin by surface plasmon resonance detection using
a recombinant HGP15 protein, and we proposed to designate this protein
"hemoglobin receptor (HbR) domain protein" (18). The three other
non-HbR domains (HGP44, HGP17, and HGP27) have a 49-amino acid-long
sequence in common (15). At least two of the non-HbR domain proteins (HGP44 and HGP17) seem to be involved in hemagglutination of P. gingivalis, as suggested by the finding that monoclonal antibodies inhibiting hemagglutination recognize a particular amino acid sequence
within the domain proteins (19-22).
Construction and analysis of a rgpA rgpB double mutant and a
kgp mutant revealed that rgpA and rgpB
are responsible for hemagglutination, the disruption of the
bactericidal function of leukocytes, and the maturation of several
P. gingivalis surface proteins such as fimbrilin (3, 23,
24), whereas kgp contributes to heme accumulation on the
cell surface, resulting in colonial black pigmentation on blood agar
plates (11). Although rgp and kgp seem to play
different roles in cell metabolism, functional complementation between
rgp and kgp may occur, judging from the
structural similarity. To further elucidate the roles of these genes,
we constructed rgpA rgpB kgp and rgpA kgp hagA
triple mutants and examined them for proteolysis, hemoglobin binding,
and hemagglutination.
Media and Conditions for Cell Growth--
P.
gingivalis cells were grown anaerobically (10% CO2,
10% H2, and 80% N2) in enriched brain heart
infusion (BHI) broth (3) and on enriched tryptic soy agar (3). For
blood agar plates, defibrinated laked sheep blood was added to enriched
tryptic soy agar at 5%. As a defined minimal medium, we used
Construction of Plasmids and Bacterial Strains--
A
promoterless cat DNA block (end-filled HindIII
fragment; 0.75 kilobase pairs) of pCM7 (Amersham Pharmacia Biotech) was
inserted into the end-filled EcoRI site within the
kgp gene of pNKD (11), resulting in pKD362, which contained
two tandem inserts of the cat block at the same orientation
as kgp. A PstI fragment of pKD362 containing the
kgp::cat operon fusion was introduced
into P. gingivalis ATCC33277 and KDP112
(rgpA1::Tcr
rgpB1::Emr) by electroporation to produce
the Cm-resistant (Cmr) transformants KDP129
(kgp-2::Cmr) and KDP128
(rgpA1::Tcr
rgpB1::Emr
kgp-2::Cmr), respectively. An ermF
ermAM DNA block (end-filled EcoRI-BamHI fragment) of pVA2198 (26) was inserted into the EcoRV site
within the rgpA gene of P. g./pUC118 plasmid
(27), resulting in pKD373. An EcoRI-BamHI
fragment of pKD373 containing
rgpA2::Emr was used for
electrotransformation of ATCC33277 and KDP129 to yield KDP131
(rgpA2::Emr) and KDP134
(rgpA2::Emr
kgp-2::Cmr), respectively. An
EcoRI-SphI fragment of pKD314 (4) was ligated to
the EcoRI-SphI fragment of pKD296 (4) to give
rise to pKD317 containing the whole rgpB gene. A unique
SmaI site within rgpB of pKD317 was converted to
a BglII site using a BglII linker DNA to yield
pKD376. A tetQ DNA block (2.7-kilobase pair
BamHI-BglII fragment) of pKD375 that was derived
from pMJF-3 (28) was inserted into the BglII site of pKD376,
resulting in pKD377. A PstI fragment of pKD377 containing
rgpB2::Tcr was introduced into
ATCC33277, KDP129, and KDP134 to produce the Tcr
transformants KDP132 (rgpB2::Tcr),
KDP135 (rgpB2::Tcr
kgp-2::Cmr), and KDP136
(rgpA2::Emr
rgpB2::Tcr
kgp-2::Cmr), respectively. A DNA region (936 base pairs) in the vicinity of the 5' end of the hagA gene
was polymerase chain reaction-amplified from the chromosomal DNA of
P. gingivalis ATCC33277 with two primers (5'-CGCTGCAGAAAGGTATTCGAACATC-3' and 5'-TCGGATCCGAGGGTTTCTTCCAGTA-3') and inserted into pMOSBlue plasmid by using a T-vector
system (pMOSBlue T-vector kit; Amersham Pharmacia Biotech).
A PstI-BamHI fragment of the resulting plasmid
that contained the internal region of the hagA gene was then
inserted into the PstI-BamHI region of pMJF-3,
giving rise to pKD363. ATCC33277 and KDP134 were transformed to
Tcr by electroporation with pKD363 circular plasmid DNA to
yield KDP130 (hagA1::Tcr) and KDP137
(rgpA2::Emr
kgp-2::Cmr
hagA1::Tcr), respectively. Proper DNA
replacement and integration in KDP136 and KDP137 were confirmed by
Southern hybridization.
Enzymatic Assays--
Lys-X and Arg-X
specific cysteine proteinase activities were determined by use of the
synthetic substrates
N-p-Tosyl-Gly-Pro-Lys-p-nitroanilide and
N- Hemagglutination Assay--
Forty-eight-h cultures of P. gingivalis strains in enriched BHI broth were centrifuged, washed
with phosphate-buffered saline (PBS), and resuspended in PBS. The
bacterial suspensions were then diluted in a twofold series with PBS. A
100-µl aliquot of each of the dilutions was mixed with an equal
volume of sheep erythrocyte suspension (2.5% in PBS) and incubated in
a round-bottomed microtiter plate at room temperature for 3 h.
Solid-phase Binding Assays--
Forty-eight-h cultures of
P. gingivalis strains in enriched BHI broth were diluted in
a twofold series with PBS, and a 10-µl aliquot of each of the
dilutions was applied to nitrocellulose membranes and allowed to dry.
The membranes were immersed in PBS containing 1% skim milk for 1 h at room temperature to block nonspecific protein binding. For
hemoglobin binding activity, the membranes were then probed with
horseradish peroxidase (HRP)-conjugated hemoglobin in PBS containing
0.5% BSA for 1 h at room temperature. HRP-conjugated hemoglobin
was made according to the method of Kishore et al. (29).
After three 10-min washes with PBS, peroxidase activity was detected
(29). For antibody binding, rabbit anti-HbR antiserum (18) and mouse
monoclonal antibody (mAb) 61BG1.3 for the detection of the non-HbR
domain proteins (30) were used as the primary antibody, and
HRP-conjugated anti-rabbit and anti-mouse IgGs were used as the
secondary antibody, respectively.
Gel Electrophoresis and Immunoblot
Analysis--
SDS-polyacrylamide gel electrophoresis was performed
essentially according to the method of Laemmli (31). Before being
solubilized in a sample buffer, P. gingivalis cells were
treated with 10% trichloroacetic acid to inactivate endogenous
proteinases. For immunoblotting, proteins on SDS gels were
electrophoretically transferred to nitrocellulose membranes using a
semi-dry blotting system (Amersham Pharmacia Biotech). The blotted
membranes were immunostained with anti-HbR antiserum or mAb 61BG1.3,
and signals were detected using an ECL detection system (Amersham
Pharmacia Biotech).
Chemicals and
Proteins--
N-p-Tosyl-Gly-Pro-Lys-p-nitroanilide,
N- Other Methods--
Electrotransformation and Southern blotting
were done as described previously (3).
Construction of the rgpA rgpB kgp and rgpA kgp hagA Triple
Mutants--
We used the promoterless Cm acetyltransferase-encoding
gene for the construction of a kgp insertional mutation
because we had used the Emr gene (ermF) and the
Tcr gene (tetQ) for the construction of
rgpA and rgpB mutations. The
kgp-2::Cmr mutant (KDP129) and the
rgpA1::Tcr
rgpB1::Emr
kgp-2::Cmr mutant (KDP128) were obtained by
the selection of Cmr transformants after the introduction
of the kgp-2::Cmr DNA fragment to the
wild type parent (ATCC33277) and the
rgpA1::Tcr
rgpB1::Emr mutant (KDP112), respectively, by
electroporation. Southern analysis indicated the replacement of
kgp with kgp-2::Cmr in
KDP129 and KDP128 (Fig. 1). KDP129 showed
no KGP activity, and KDP128 showed neither KGP nor RGP activity (Table
I). In addition, KDP129 exhibited reduced
colonial pigmentation on blood agar plates (Fig.
2), which was one of the characteristic
features of a kgp mutant (11). Colonies of KDP128 showed
less color on the blood agar plates than those of KDP129 (Fig. 2).
KDP128 has integration-type mutations at the rgpA and
rgpB loci. Because of the potential problem of instability
in integration-type mutations, another rgpA rgpB kgp triple
mutant (KDP136) was constructed from KDP129 by sequential replacement
with linear DNA fragments containing rgpA2::Emr and
rgpB2::Tcr mutations. The
rgpA2::Emr
kgp-2::Cmr
hagA1::Tcr mutant (KDP137) was obtained by
the introduction of pKD363 circular plasmid DNA containing the internal
region of hagA into KDP134 (rgpA2::Emr
kgp-2::Cmr). Determination of the
proteolytic activities of the various mutants supported the fact that
RGP is encoded by two separate genes, rgpA and
rgpB, whereas KGP is encoded by a single gene, kgp (Table I).
Cell Growth in Enriched BHI Broth--
KDP112 (rgpA
rgpB) and KDP128 (rgpA rgpB kgp) grew faster than
ATCC33277 (wild type) and KDP129 (kgp) in enriched BHI broth (Fig. 3). Moreover, ATCC33277 and KDP129
showed a decrease in absorbance after 100 h of incubation,
indicating cell lysis. Although the absorbance was also decreased in
KDP128 and KDP112, the absorbance decreases of KDP128 and KDP112 were
low and intermediate, respectively, compared with those of ATCC33277
and KDP129. These results indicate that the cell lysis seen after
prolonged incubation appeared to be caused mainly by RGP and KGP.
Degradation of Gelatin and BSA by Culture Supernatants of the rgp-
and kgp-related Mutants--
The rgp- and
kgp-related mutants were grown in enriched BHI broth.
Supernatants of the cultures of a 3-day incubation were mixed with
gelatin derived from human type I collagen or BSA. ATCC33277, KDP129,
and KDP112 showed a complete degradation of gelatin, whereas KDP128
showed no degradation (Fig.
4a). KDP128 also showed no
degradation of BSA (Fig. 4b). These results indicate that
the extracellular proteolytic activity of P. gingivalis is totally attributable to RGP and KGP.
Cell Growth in Lack of Hemoglobin Binding Ability in the rgpA rgpB kgp and rgpA
kgp hagA Triple Mutants--
P. gingivalis has the ability
to bind hemoglobin (32-34). We found that the HbR protein of P. gingivalis was intragenically encoded by the rgpA,
kgp, and hagA genes (18). In addition, another
gene (tla) that was found to encode the HbR domain protein in the C-terminal region has recently been cloned (17). To determine which gene(s) is actually responsible for the production of the HbR
protein, immunoblot analyses with anti-HbR antiserum were performed
using cell lysates and intact cells of various mutants (Fig.
6, a and b). The
wild type parent (ATCC33277), the rgpA rgpB mutants (KDP112
and KDP133), the kgp mutant (KDP129), and the rgpA
kgp mutant (KDP134) exhibited the 19-kDa HbR protein in the
lysates of cells grown in blood agar plates for 7 days, whereas the
rgpA rgpB kgp mutants (KDP128 and KDP136) and the rgpA
kgp hagA mutant (KDP137) produced no HbR protein in cell lysates.
The intact cells of ATCC33277, KDP133, KDP129, and KDP134 reacted to
the anti-HbR antiserum, whereas those of KDP136 and KDP137 showed no
reaction with the antiserum. These results suggest that all three
of the genes (rgpA, kgp, and hagA) contribute to the HbR expression of P. gingivalis. Then we determined the
hemoglobin binding ability of the mutants (Fig.
7). The rgpA rgpB kgp mutants (KDP128 and KDP136) and the rgpA kgp hagA mutant (KDP137)
showed no hemoglobin binding ability, whereas the cells of other
strains (ATCC33277, KDP112, KDP129, KDP133, and KDP134) had the ability to bind hemoglobin, although the binding ability varied among the
different strains. In addition, the fimA mutant KDP98 that is deficient in fimbriation (35) exhibited hemoglobin binding activity.
These results indicate that hemoglobin binding activity appeared to be
correlated to HbR expression. Because the HbR protein had hemoglobin
binding activity in a cell-free system (18), it is plausible to
consider that the hemoglobin binding ability of P. gingivalis is attributable to the HbR protein.
No Hemagglutination of the rgpA rgpB kgp and rgpA kgp hagA Triple
Mutants--
P. gingivalis has the ability to agglutinate
erythrocytes, which is one of the significant features of this
organism. Pike et al. (36) reported that the RGP/adhesin and
KGP/adhesin complexes have hemagglutinating activity. A monoclonal
antibody (mAb 61BG1.3) that inhibits the hemagglutination of P. gingivalis was found to recognize a peptide within the adhesin
domain (HGP44 of rgpA) encoded by rgpA, kgp, and
hagA (20, 21). To determine whether the rgp- and
kgp-related mutants produce mAb 61BG1.3-reactive proteins,
immunoblot analyses were performed using cell lysates and intact cells
(Fig. 8, a and b).
The wild type strain (ATCC33277), the rgpA rgpB mutant
(KDP133), the kgp mutant (KDP129), and the rgpA
kgp mutant (KDP134) produced immunoreactive proteins on the cell
surfaces and in the cell lysates, whereas the rgpA rgpB kgp mutant (KDP136) and the rgpA kgp hagA mutant (KDP137)
produced no reactive proteins on their cell surfaces. Interestingly,
the rgpA kgp hagA mutant showed no reactive proteins in the
cell lysate, whereas the rgpA rgpB kgp mutant produced
immunoreactive proteins with molecular masses of more than 100 kDa that
were probably derived from hagA. The rgpA rgpB
kgp and rgpA kgp hagA mutants showed no
hemagglutinating activity using sheep erythrocytes (Fig. 9). These results indicate that
hemagglutination of P. gingivalis is caused by the
rgpA-, kgp-, and hagA-encoding adhesin domains and that the expression of these adhesin domains on the cell surface is
particularly important for hemagglutination.
P. gingivalis cannot utilize carbohydrates as
carbon/energy sources (37). Therefore, the microorganism has developed
utilization of environmental amino acids and peptides by production of
extracellular proteinases. In the gingiva, macromolecules such as serum
albumin, immunoglobulins, hemoglobin, and various proteins of host
tissues and secretions are target molecules for degradation to amino
acids and peptides by the extracellular proteinases secreted from the organism. Although a number of extracellular and cell-associated proteinases have been found in P. gingivalis, it is still
unclear which proteinase(s) is actually responsible for the production of utilizable amino acids and peptides. In this study, we found that
the culture supernatants of the rgpA rgpB kgp triple mutants had no proteolytic activity to gelatin or BSA, indicating that the
extracellular proteinase activity of P. gingivalis may be totally attributable to the three genes. The inability of the rgpA rgpB kgp mutants to grow in the In our previous study (18), we found that the HbR domain protein that
was intragenically encoded by rgpA, kgp, and hagA had the ability to bind hemoglobin. Immunoblot analysis using anti-HbR
antiserum revealed that the rgpA kgp double mutant produced the 19-kDa HbR protein, whereas the rgpA kgp hagA triple
mutant produced no HbR protein. The result indicates that
hagA is responsible for HbR production as well as
rgpA and kgp. Aduse-Opoku et al. (17)
recently reported that the HbR domain region was also located within
the tla gene cloned from the P. gingivalis W50
chromosome. However, they mentioned in the study that Northern analyses
of mRNA had thus far failed to reveal the presence of a
tla transcript in cells grown under any growth condition,
indicating that there might be very little HbR production from the
tla gene. A restriction map around the tla gene
in ATCC33277 is different from that of W50 (17). An oligonucleotide
probe recognizing the HbR region hybridized to three different
restriction fragments of ATCC33277 chromosomal DNA, which were probably
derived from the rgpA, kgp, and hagA
loci.2 These results suggest
another possibility: that the ATCC33277 chromosome may not possess the
HbR domain region in the tla gene. Further investigation
including the cloning and nucleotide sequencing of tla from
ATCC33277 will be necessary for clarification of this issue. In the
previous study (18), we also found that the rgpA rgpB mutant
produced as much HbR as the wild type parent; however, the N terminus
of the HbR from the rgpA rgpB mutant was Arg1155
(the residue number of the kgp primary gene product
according to Okamoto et al.; Ref. 7) instead of
Ala1156, indicating that cleavage at the N terminus might
be done by KGP in the mutant. In this study, we found that the
rgpA rgpB kgp triple mutant produced no 19-kDa HbR protein.
Because the triple mutant produced mAb 61BG1.3-reactive proteins with
high molecular masses, the hagA gene appears to be expressed
in the triple mutant. Therefore, it is most likely that the processing and maturation of the HbR domain protein of hagA depend on
the presence of both RGP and KGP activities. The finding that the hemoglobin binding activities of the various mutants were consistent with the presence or absence of HbR in the mutants indicates that the
hemoglobin binding ability of P. gingivalis is caused mainly by HbR; however, Kuboniwa et al. (38) recently reported that the KGP proteinase domain itself has the ability to bind hemoglobin.
Hemagglutination is a distinctive characteristic of P. gingivalis that discriminates the microorganism from other
asaccharolytic black-pigmented anaerobic organisms. This feature has
been recognized to have taxonomic value, together with other important
features such as the RGP and KGP activities, in distinguishing P. gingivalis from other Porphyromonas spp. Because
P. gingivalis requires heme for growth, hemagglutination
serves as the first step in heme acquisition from erythrocytes. We have
previously found that the rgpA rgpB double (RGP-null) mutant
showed decreased ability to agglutinate erythrocytes (3). Pike et
al. (36) also reported that the high molecular mass RGP has
hemagglutinin activity. These results suggest that the rgp
genes are involved in hemagglutination. The hemagglutinin gene
hagA of P. gingivalis that confers
hemagglutination on Escherichia coli cells was found to
possess the DNA region homologous to those of the C-terminal adhesin
domains of rgpA and kgp (16). Moreover, mAb
61BG1.3, which reacts with an epitope within the adhesin domains,
inhibits the hemagglutination of P. gingivalis (20, 21). No
hemagglutination of the rgpA kgp hagA triple mutant observed
in this study suggests that all three genes are responsible for
hemagglutination. In addition to the adhesin domain proteins, RGP
proteinase derived from rgpB is thought to be involved in
the hemagglutinating activity because the rgpA rgpB kgp
triple mutants showed less than 1.6% of the activity of the wild type
parent, whereas the rgpA kgp double mutant showed 6.3% of
the activity of the wild type parent. There are at least two possible
explanations for the involvement of RGP in hemagglutination. One is
that because maturation of the adhesin domains requires RGP activity, a
complete defect of RGP would decrease hemagglutination if maturation of
the adhesin domains from hagA is required for the
agglutination. The other is that RGP-mediated modification of putative
erythrocyte surface molecule(s) for binding to P. gingivalis
cells would be necessary for hemagglutination.
Several other candidates such as fimbriae, HagB, and HagC have been
proposed as a hemagglutinin of P. gingivalis (39-41).
However, neither fimbriae nor anti-fimbria antibody inhibits
hemagglutination (42). Purified fimbriae have also been shown to
exhibit no hemagglutinating activity (43). In addition, we found that
the rgpA kgp hagA mutant having no hemagglutinating activity
expressed the fimA gene, resulting in
fimbriation.3 Taken together,
it is unlikely that fimbriae are responsible for hemagglutination of
P. gingivalis, even if synthetic peptides derived from the
amino acid sequence of fimbrilin possess hemagglutinating activity
(44). The expression of hagB and hagC depends on
the phase of bacterial growth and on the levels of hemin (45).
Therefore, we cannot rule out the possibility that these genes
contribute to the hemagglutination of P. gingivalis under
conditions that differ from those used in this study. Although several
problems concerning hemagglutination of P. gingivalis remain
to be solved, it can be said that the non-HbR adhesin domain proteins
encoded by rgpA, kgp, and hagA are the
most important agglutinins for hemagglutination.
We thank Dr. R. Gmur for kindly giving us
monoclonal antibody 61BG1.3. General assistance by K. Sakai is
acknowledged with appreciation.
*
This work was supported by Grants-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports and Culture of
Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Microbiology, Faculty of Dentistry, Kyushu University, 3-1-1, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan. Tel.: 81-92-642-6332;
Fax: 81-92-642-6263, E-mail: knak{at}dent.kyushu-u.ac.jp.
2
Y. Shi, D. B. Ratnayake, and K. Nakayama,
unpublished observations.
3
Y. Shi, D. B. Ratnayake, A. Umeda, and K. Nakayama, unpublished observations.
The abbreviations used are:
RGP, Arg-gingipain;
KGP, Lys-gingipain;
HbR, hemoglobin receptor;
BHI, brain heart
infusion;
Genetic Analyses of Proteolysis, Hemoglobin Binding, and
Hemagglutination of Porphyromonas gingivalis
CONSTRUCTION OF MUTANTS WITH A COMBINATION OF rgpA, rgpB,
kgp, AND hagA*
,,¶
Microbiology and
§ Pharmacology, Faculty of Dentistry, Kyushu University,
Fukuoka 812-8582, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ketoglutarate/bovine serum albumin (-KG/BSA) medium for the
growth of P. gingivalis (25). To make trypsin-pretreated
-KG/BSA medium, trypsin was added to -KG/BSA medium at a
concentration of 50 µg/ml and incubated at 37 °C for 4 h. For
selection and maintenance of the antibiotic-resistant strains,
antibiotics were added to the medium at the following concentrations:
ampicillin, 50 µg/ml; chloramphenicol (Cm), 20 µg/ml; erythromycin
(Em), 10 µg/ml; and tetracycline (Tc), 0.7 µg/ml.
-benzoyl-DL-Arg-p-nitroanilide,
respectively. In brief, various volumes of the cell lysates and
supernatants of the culture were added to a reaction mixture (1 ml)
containing 0.25 mM
N-p-Tosyl-Gly-Pro-Lys-p-nitroanilide, 5 mM L-cysteine, and 20 mM
phosphate buffer (pH 7.5) for KGP and a reaction mixture (1 ml)
containing 0.5 mM
N--benzoyl-DL-Arg-p-nitroanilide, 10 mM L-cysteine, 10 mM
CaCl2, and 100 mM Tris-HCl (pH 8.0) for RGP.
The reaction mixtures were incubated at 40 °C for KGP and at
30 °C for RGP. After the samples were added, absorbance was continuously measured at 405 nm on a spectrophotometer. Proteinase activities in cell extracts and culture supernatants were determined by
the increase in absorbance per minute per milligram of protein and the
increase in absorbance per minute per milliliter, respectively.
-benzoyl-DL-Arg-p-nitroanilide,
human hemoglobin, -KG, BSA (type IV), and trypsin were purchased
from Sigma. Gelatin derived from human type I collagen was obtained
from Seikagaku Co. (Japan). HRP-conjugated anti-rabbit and anti-mouse
IgGs were purchased from Santa Cruz Biotechnology.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (47K):
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Fig. 1.
Southern analysis of the kgp
mutants. Chromosomal DNA of ATCC33277 (wild type)
(lanes 1 and 5), KDP129
(kgp-2::Cmr) (lanes 2 and
6), KDP112 (rgpA rgpB) (lanes 3 and
7), and KDP128 (rgpA rgpB
kgp-2::Cmr) (lanes 4 and
8) was digested with PstI (lanes 1-4)
and BamHI (lanes 5-8). The digested DNA was
subjected to agarose gel electrophoresis and Southern blot
hybridization with a 1.2-kilobase pair HindIII fragment
inside the N-terminal propeptide and catalytic domain-encoding region
of kgp as a DNA probe.
RGP and KGP activities of various P. gingivalis mutants
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Fig. 2.
Colonial pigmentation. P. gingivalis ATCC33277 (wild type), KDP112 (rgpA rgpB),
KDP128 (rgpA rgpB kgp), and KDP129 (kgp) were
anaerobically grown on blood agar plates at 37 °C for 7 days.
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Fig. 3.
Growth of the rgp-
and kgp-related mutants in enriched
BHI broth. An overnight culture was diluted 20-fold with enriched
BHI broth and incubated anaerobically at 37 °C. Growth was monitored
by measuring the optical density at 540 nm. 
, ATCC33277; 
,
KDP129; 
, KDP112; 
, KDP128.
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Fig. 4.
Digestion of gelatin and BSA by culture
supernatants of the rgp- and
kgp-related mutants. P. gingivalis
cells were grown in enriched BHI broth for 3 days, and the culture
supernatant was collected by centrifugation. Two µl of the culture
supernatant were mixed with 2.5 µl of a protein solution (1 mg/ml)
and 7.5 µl of a reaction buffer (80 mM Tris-HCl (pH 7.5),
0.32 M NaCl, 8 mM CaCl2, and 1.6 mM dithiothreitol) and incubated at 37 °C for 2 h.
The reaction was terminated by adding 4 µl of Laemmli sample buffer
and heating at 100 °C for 5 min. Samples were subjected to
SDS-polyacrylamide gel electrophoresis. The protein bands on the gel
were visualized by Coomassie Brilliant Blue R-250 staining. Molecular
mass markers are as follows: phosphorylase b, 94 kDa; bovine serum
albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase b, 30 kDa;
trypsin inhibitor, 20.1 kDa; and -lactoalbumin, 14.4 kDa.
a, gelatin derived from human type I collagen. b,
gelatin derived from BSA. Lanes 1, no supernatants;
lanes 2, KDP128; lanes 3, KDP112; lanes
4, KDP129; lanes 5, ATCC33277.
-KG/BSA Defined Medium--
The -KG/BSA
defined medium contains BSA as the sole carbon/energy source, and this
medium supports the growth of wild type P. gingivalis cells
(25). To determine whether P. gingivalis cells require RGP
and KGP activities to grow in this medium, rgp- and
kgp-related mutants were incubated in the medium. ATCC33277, KDP112, and KDP129 grew in this medium, whereas KDP128 did not grow
(Fig. 5). KDP128 grew in the
trypsin-predigested -KG/BSA medium as well as ATCC33277. These
results strongly indicate that RGP and KGP contribute to protein
degradation, leading to the production of peptides utilizable as
carbon/energy sources. KDP136 (rgpA rgpB kgp) and KDP133
(rgpA rgpB) showed the same results as KDP128 and KDP112,
respectively, in cell growth in enriched BHI broth, degradation of
gelatin and BSA by culture supernatants, and cell growth in the
-KG/BSA defined medium.
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Fig. 5.
Growth of the rgp-
and kgp-related mutants in
-KG/BSA defined medium with or without trypsin
predigestion. An overnight culture of P. gingivalis in
enriched BHI broth was diluted 10-fold with -KG/BSA medium (,
, , and ) or trypsin-pretreated -KG/BSA medium (
and
) and incubated anaerobically at 37 °C. Growth was monitored by
measuring the optical density at 540 nm. and , ATCC33277; ,
KDP112; , KDP129; and , KDP128.
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Fig. 6.
Expression of the HbR protein in the cell
lysates and on the cell surfaces of the rgp-
and kgp-related mutants.
a, an immunoblot using anti-HbR antiserum. P. gingivalis cells were grown on blood agar plates for 7 days,
harvested, and lysed with Laemmli sample buffer. Samples were subjected
to SDS-polyacrylamide gel electrophoresis. Protein bands on the gel
were transferred to a nitrocellulose membrane and immunoreacted with
anti-HbR antiserum. Lane 1, ATCC33277; lane 2,
KDP112; lane 3, KDP133; lane 4, KDP129;
lane 5, KDP134; lane 6, KDP128; lane
7, KDP136; lane 8, KDP137; lane 9, KDP98.
b, solid-phase binding assay with anti-HbR serum. P. gingivalis cells grown in enriched BHI broth for 48 h were
washed with PBS and resuspended in the original volume of PBS. Ten µl
of the suspension were applied to a nitrocellulose membrane and allowed
to dry. The membrane was then subjected to the solid-phase binding
assay using anti-HbR antiserum. Blots are as follows: 1,
ATCC33277; 2, KDP136; 3, KDP137; 4,
KDP129; 5, KDP133; and 6, KDP134.
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Fig. 7.
Solid-phase hemoglobin binding assay.
P. gingivalis cells grown in enriched BHI broth for 48 h were washed with PBS, resuspended in the original volume of PBS, and
diluted in a 2-fold series with PBS. A 10-µl aliquot of each of the
dilutions was applied to a nitrocellulose membrane and allowed to dry.
The membrane was then subjected to the solid-phase binding assay using
HRP-conjugated hemoglobin. Columns are as follows: 1,
ATCC33277; 2, KDP112; 3, KDP133; 4,
KDP129; 5, KDP134; 6, KDP128; 7,
KDP136; 8, KDP137; and 9, KDP98.
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Fig. 8.
Immunoblot and solid-phase analysis using mAb
61BG1.3. a, an immunoblot using mAb 61BG1.3. P. gingivalis cells were grown in enriched BHI broth for 48 h,
harvested, and lysed with Laemmli sample buffer. Protein bands on the
gel were transferred to a nitrocellulose membrane and immunoreacted
with mAb 61BG1.3. Lane 1, ATCC33277; lane 2,
KDP137; lane 3, KDP136; lane 4, KDP134;
lane 5, KDP133; lane 6, KDP129. b,
solid-phase binding assay with mAb 61BG1.3. Procedures were the same as
those described in the legend to Fig. 6b, except that mAb
61BG1.3 was used. Blots are as follows: 1, ATCC33277;
2, KDP136; 3, KDP137; 4, KDP129;
5, KDP133; and 6, KDP134.
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Fig. 9.
Hemagglutinating activity of the
rgp- and kgp-related mutants.
P. gingivalis cells were grown in enriched BHI broth, washed
with PBS, and resuspended in PBS at an optical density at 540 nm of
0.4. The suspension and its dilutions in a 2-fold series were applied
to the wells of a microtiter plate from left to right and mixed with
sheep erythrocyte suspension. 1, ATCC33277; 2,
KDP129; 3, KDP133; 4, KDP134; 5,
KDP136; 6, KDP137.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-KG/BSA defined
medium supported this idea. Several proteinases other than RGP and KGP
have been cloned and characterized (12-14). The results obtained here,
however, suggest that these proteinases may not be located on the
surface or secreted outside or may not be expressed under the culture conditions used in this study. We also found that the autolysis of
P. gingivalis cells observed in prolonged cultures might be due mainly to extracellular and cell-associated RGP and KGP.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
-KG, -ketoglutarate;
BSA, bovine serum albumin;
Cm, chloramphenicol;
Cmr, chloramphenicol-resistant;
Em, erythromycin;
Emr, erythromycin-resistant;
Tc, tetracycline;
Tcr, tetracycline-resistant;
PBS, phosphate-buffered saline;
HRP, horseradish peroxidase;
mAb, monoclonal
antibody.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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D. Grenier, S. Imbeault, P. Plamondon, G. Grenier, K. Nakayama, and D. Mayrand Role of Gingipains in Growth of Porphyromonas gingivalis in the Presence of Human Serum Albumin Infect. Immun., August 1, 2001; 69(8): 5166 - 5172. [Abstract] [Full Text] [PDF]
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S. G. Dashper, L. Brownfield, N. Slakeski, P. S. Zilm, A. H. Rogers, and E. C. Reynolds Sodium Ion-Driven Serine/Threonine Transport in Porphyromonas gingivalis J. Bacteriol., July 15, 2001; 183(14): 4142 - 4148. [Abstract] [Full Text] [PDF]
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K. Nisapakultorn, K. F. Ross, and M. C. Herzberg Calprotectin Expression In Vitro by Oral Epithelial Cells Confers Resistance to Infection by Porphyromonas gingivalis Infect. Immun., July 1, 2001; 69(7): 4242 - 4247. [Abstract] [Full Text] [PDF]
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W. O. Chung, Y. Park, R. J. Lamont, R. McNab, B. Barbieri, and D. R. Demuth Signaling System in Porphyromonas gingivalis Based on a LuxS Protein J. Bacteriol., July 1, 2001; 183(13): 3903 - 3909. [Abstract] [Full Text] [PDF]
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 A. Lourbakos, Y. Yuan, A. L. Jenkins, J. Travis, P. Andrade-Gordon, R. Santulli, J. Potempa, and R. N. Pike Activation of protease-activated receptors by gingipains from Porphyromonas gingivalis leads to platelet aggregation: a new trait in microbial pathogenicity Blood, June 15, 2001; 97(12): 3790 - 3797. [Abstract] [Full Text] [PDF]
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M. Kuboniwa, A. Amano, S. Shizukuishi, I. Nakagawa, and S. Hamada Specific Antibodies to Porphyromonas gingivalis Lys-Gingipain by DNA Vaccination Inhibit Bacterial Binding to Hemoglobin and Protect Mice from Infection Infect. Immun., May 1, 2001; 69(5): 2972 - 2979. [Abstract] [Full Text] [PDF]
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D. P. Henderson, E. E. Wyckoff, C. E. Rashidi, H. Verlei, and A. L. Oldham Characterization of the Plesiomonas shigelloides Genes Encoding the Heme Iron Utilization System J. Bacteriol., May 1, 2001; 183(9): 2715 - 2723. [Abstract] [Full Text]
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H. Abaibou, Z. Chen, G. J. Olango, Y. Liu, J. Edwards, and H. M. Fletcher vimA Gene Downstream of recA Is Involved in Virulence Modulation in Porphyromonas gingivalis W83 Infect. Immun., January 1, 2001; 69(1): 325 - 335. [Abstract] [Full Text] [PDF]
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 M.A. Curtis, J. Aduse-Opoku, and M. Rangarajan Cysteine Proteases of Porphyromonas Gingivalis Critical Reviews in Oral Biology & Medicine, January 1, 2001; 12(3): 192 - 216. [Abstract] [Full Text] [PDF]
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J. Aduse-Opoku, N. N. Davies, A. Gallagher, A. Hashim, H. E. A. Evans, M. Rangarajan, J. M. Slaney, and M. A. Curtis Generation of Lys-gingipain protease activity in Porphyromonas gingivalis W50 is independent of Arg-gingipain protease activities Microbiology, August 1, 2000; 146(8): 1933 - 1940. [Abstract] [Full Text]
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Y. Shi, W. Kong, and K. Nakayama Human Lactoferrin Binds and Removes the Hemoglobin Receptor Protein of the Periodontopathogen Porphyromonas gingivalis J. Biol. Chem., September 22, 2000; 275(39): 30002 - 30008. [Abstract] [Full Text] [PDF]
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