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Infection and Immunity, January 2006, p. 734-739, Vol. 74, No. 1
0019-9567/06/$08.00+0 doi:10.1128/IAI.74.1.734-739.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Departments of Oral Frontier Biology,1 Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, Suita-Osaka 565-0871, Japan2
Received 14 July 2005/ Returned for modification 29 August 2005/ Accepted 9 October 2005
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The following P.
gingivalis strains were used in this study: ATCC 33277
(type I fimbriae), HG1691 (type Ib), OMZ314 (type II), 6/26 (type III),
HG564 (type IV), HNA99 (type V), a fimA-deficient mutant of
ATCC33277 [
fimA (I)]
(26), a
fimA-deficient mutant of OMZ314 [
fimA (II)]
(17), and a
gingipain-deficient mutant of ATCC33277
[
rgpAB
kgp (I)]
(23). Their
vesicles were prepared as described previously
(9). Bacterial
hemagglutination activities were assayed as described previously
(11). The phenotypic
characterizations of P. gingivalis strains were performed with
an API 20A system (BioMérieux Industry, Marcy
l'Etoile, France) as described previously
(24,
28). HeLa
cells were cultured in Dulbecco's modified Eagle medium supplemented
with 10% fetal bovine serum, gentamicin, and 1% fungizone (all from
Invitrogen Co., Carlsbad, CA) at 37°C, and cell viability was
assayed as described previously
(16). Covalent coupling
of vesicles or bovine serum albumin (BSA) (as a control) to fluorescent
MS (Molecular Probes, Eugene, OR) (diameter, 1.0 µm) was
performed as described previously
(16). The
amounts of bound proteins and lipopolysaccharide (LPS) as well as
proteolytic activities of gingipains were measured as described
previously (10,
25). Western blotting of
fimbriae was performed as described previously
(17). Fimbriae on
vesicle-conjugated MS (vcMS) were stained with polyclonal rabbit
anti-fimbriae antibodies (diluted 1:1,000)
(17), followed by Alexa
Fluor 488 goat anti-rabbit antibody (Molecular Probes). Confocal
microscopic analyses of adhesion to and invasion of HeLa
cells by vcMS were performed using a laser scanning confocal
microscope (model LSM510; Carl Zeiss, Thornwood, NY) as described
previously (16). The
cells were incubated with vcMS (200 vcMS/cell) in
Dulbecco's modified Eagle medium containing 10% fetal bovine serum and
then fixed with 4% paraformaldehyde in phosphate-buffered saline, after
which F-actin was stained with Oregon Green 488-conjugated phalloidin
(Molecular Probes). The efficiency of the adhesion and/or invasion was
expressed as the average area measured in images collected in 10
independent experiments. To analyze the distribution of the invaded
vcMS, optical sections were obtained along the z axis at
0.15-µm intervals (60 sections, 9-µm
thickness), and images of the x-z and
y-z planes were reconstructed using LSM510 software.
Cellular apoptosis was identified with a terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling
(TUNEL) assay using a DeadEnd fluorometric TUNEL system
(Promega, Madison, WI) according to the manufacturer's instructions.
All data are expressed as the means ± standard
deviations and were analyzed with an unpaired Student's t
test.
The MS were equivalently conjugated to vesicles
with different types of fimbriae (proteins, 17.13 ± 1.34 to
21.71 ± 1.58 µg/109 vcMS; LPS, 0.24
± 0.011 to 0.30 ± 0.18 µg/109 vcMS).
Adequate coupling of fimbriae to vcMS was confirmed with Western
blotting (Fig.
1A), and the presence of type II fimbriae (Fig. 1B) as well as other types
of fimbriae (data not shown) on the surface of vcMS was also confirmed with a confocal microscope. The interaction of epithelial cells with vcMS was examined as shown in Fig. 1C, while quantitative measurement of vcMS following invasion was performed as described above (Fig. 1D). Using this assay, the adhesion to and/or invasion of epithelial cells by the different types of vcMS was quantitatively evaluated. Type II fimbria vcMS significantly adhered to and invaded the cells at 6 h
after incubation compared to the results seen with other types (Fig.
2A and B). Further, following prolonged
incubation for 12 and 24 h, vcMS of types I and III invaded
at levels similar to those seen with type II vcMS, with the vcMS of
types Ib, IV, and V eventually showing invasion at 56% to 80% of the
type II vcMS level
(Fig. 2B).
Although gingipains and their adhesion domains were
suggested to be involved in the adhesion and invasion
(5,
6), the
fimA-deficient mutants showed negligible adhesion and
invasion, which was similar to the result seen with the
gingipain-deficient mutant
[
rgpAB
kgp (I)]
that lacked fimbriae on the surface
(12; Koji Nakayama,
personal communication). In addition, no positive correlation of
gingipain activities to invasive efficiency was found (Fig.
3). P. gingivalis reportedly suppressed cellular apoptosis and
maintained the viability of infected cells
(18,
29,
31), while its gingipains
were shown to induce cellular apoptosis and death
(5,
6,
22). In the present
study, approximately 30% of the cells had detached from the plates and
lost their viability after 6 h (Fig.
4A). However, the number of floating cells did not increase with prolonged
incubation, suggesting that vcMS suppressed further apoptotic changes.
As shown in Fig. 4B and C, apoptotic changes of the attached cells were most frequently observed
after 6 h, though the ratio was lower than 8% of all cells.
Apoptosis was more frequently observed in association with vcMS
invasion, especially with type II vcMS invasion, compared to noninvaded
cell results. Further, the number of apoptotic cells gradually
decreased as incubation time increased, even among cells invaded by
type II-vcMS. The gingipain-deficient mutant caused very little cell
death and apoptosis, suggesting that gingipains play a major role in
cellular death and apoptosis.
![]() View larger version (26K): [in a new window] |
FIG. 1. Intracellular
localization of type II fimbria vcMS. (A) Western blotting of
fimbriae on vcMS. Lanes: 1, strain ATCC 33277 (I); 2, HG1691 (Ib); 3,
OMZ314 (II); 4, 6/26 (III); 5, fimA (I); 6,
fimA (II); 7, HG564 (IV); 8, HNA99 (V). (B)
Presence of type II fimbriae on vcMS. Fimbriae on the surfaces of vcMS
were visualized with antifimbria antibodies by use of a confocal
microscope. Bar, 1 µm. (C) Epithelial cells were
incubated with type II fimbria vcMS for 6 h. Images (red,
type II vcMS; green, actin) were collected and analyzed with a confocal
microscope. (D) The number of the cells invaded by type II
vcMS increased with incubation time. To analyze the distribution of the
vcMS-invaded cells, 60 optical sections were obtained along the
z axis at 0.15-µm intervals, and images of the
x-z and y-z planes were
reconstructed with LSM510 software. Magnification, x63. Bar, 20
µm.
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FIG. 2. Quantitative
analysis of adhesion to and/or invasion of epithelial cells by vcMS.
(A) Confocal microscopic images of invaded vcMS (red) at
6 h after incubation. BSA-conjugated MS was used as a
control. Magnification, x63. Bar, 20 µm. (B)
Time course of adhesion to and invasion of epithelial cells by vcMS.
The fimbria type of the strains used for vesicle preparation is shown
in parentheses. , adhered and invaded vcMS; , invaded
vcMS; , BSA-coated MS as a control. *, significant
difference (P < 0.01) compared to the results seen
with others at the same time point. Phenotypes of these strains
including the mutants were found to be identical as follows:
hemagglutination (+), proteolytic activity (+),
catalase (), indole production (), urease
(), glucose (), D-mannitol (),
lactose (), sucrose (), maltose (), salicin
(), D-xylose (), L-arabinose
(), ß-glucosidase (), glycerin (),
D-cellobiose (), D-mannose (),
D-melezitose (), D-raffinose
(), D-sorbitol (), L-rhamnose
(), and D-trehalose
().
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FIG. 3. Correlation
between vcMS invasive efficiency and gingipain activities. The
correlation between gingipain activities (Arg-specific gingipain; Rgp-
and Lys-specific gingipain; Kgp) and the area invaded by vcMS at
6 h was analyzed. No positive associations were
observed.
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FIG. 4. Cell
death-apoptosis induced by vcMS. (A) The ratios of epithelial
cells attached to the culture plates during the experimental period are
shown. The fimbria type of the strains used for vesicle preparation is
shown in parentheses. Floating cell viability was found to be nearly
lost. *, significant difference (P < 0.01)
compared to vcMS of other fimbria types at the same time point.
(B) Cellular apoptosis was detected by a TUNEL assay and
visualized with green fluorescence at 6 h after starting
incubation. Magnification, x63. Bar, 20 µm.
(C) Time course of cellular apoptosis associated with
invasion and noninvasion of vcMS. Ten fields were analyzed in each of
the experiments, which were repeated 10 times. Data represent the
ratios (%) of apoptotic cells to total adhered cells, vcMS-invaded
cells, and noninvaded cells. *, significant difference
(P < 0.01) compared to control (BSA) results at the
same time point.
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