Protective immunization against experimental Bacteroids (Porphyromonas) gingivalis infection

Article (PDF Available)inInfection and Immunity 58(10):3394-400 · November 1990with8 Reads
Source: PubMed
Abstract
The effects of immunization in modulating the pathogenesis of Bacteroides (Porphyromonas) gingivalis infection in a murine model system were examined. BALB/c mice were immunized by intraperitoneal injection with B. gingivalis ATCC 53977 (one injection per week for 3 weeks), or with a lithium diiodosalicylate (LIS) extract (one injection per week for 3 weeks), or with lipopolysaccharide (LPS; one intravenous or intraperitoneal injection) from this same strain. Two weeks after the final immunization, the mice were challenged by subcutaneous injection of B. gingivalis ATCC 53977. Mice immunized with bacteria had no secondary lesions and no septicemia, whereas mice immunized with LIS extract had few secondary lesions and no septicemia. Mice immunized with LPS and nonimmunized mice demonstrated secondary abdominal lesions and septicemia after challenge. Bacterial cells and LIS extract, but not LPS, induced serum antibody and antigen reactive lymphocytes, as measured by enzyme-linked immunosorbent assay, immunoblot, Western immunoblot transfer, and in vitro lymphoproliferative responses. The present study suggests that immunization with a LIS extract or whole cells may induce a protective response against experimental B. gingivalis infection.
Vol.
58,
No.
10
Protective
Immunization
against
Experimental
Bacteroides
(Porphyromonas)
gingivalis
Infection
PRISCILLA
B.
CHEN,'*
LYNDA
BETH
DAVERN,1
ROBERT
SCHIFFERLE,"2
AND
JOSEPH
J.
ZAMBON"
2
Departments
of
Oral
Biology'
and
Periodontology,2
School
of
Dental
Medicine,
State
University
of
New
York
at
Buffalo,
Buffalo,
New
York
14214
Received
27
February
1990/Accepted
13
July
1990
The
effects
of
immunization
in
modulating
the
pathogenesis
of
Bacteroides
(Porphyromonas)
gingivalis
infection
in
a
murine
model
system
were
examined.
BALB/c
mice
were
immunized
by
intraperitoneal
injection
with
B.
gingivalis
ATCC
53977
(one
injection
per
week
for
3
weeks),
or
with
a
lithium
diiodosalicylate
(LIS)
extract
(one
injection
per
week
for
3
weeks),
or
with
lipopolysaccharide
(LPS;
one
intravenous
or
intraperi-
toneal
injection)
from
this
same
strain.
Two
weeks
after
the
final
immunization,
the
mice
were
challenged
by
subcutaneous
injection
of
B.
gingivalis
ATCC
53977.
Mice
immunized
with
bacteria
had
no
secondary
lesions
and
no
septicemia,
whereas
mice
immunized
with
LIS
extract
had
few
secondary
lesions
and
no
septicemia.
Mice
immunized
with
LPS
and
nonimmunized
mice
demonstrated
secondary
abdominal
lesions
and
septicemia
after
challenge.
Bacterial
cells
and
LIS
extract,
but
not
LPS,
induced
serum
antibody
and
antigen
reactive
lymphocytes,
as
measured
by
enzyme-linked
immunosorbent
assay,
immunoblot,
Western
immunoblot
transfer,
and
in
vitro
lymphoproliferative
responses.
The
present
study
suggests
that
immunization
with
a
LIS
extract
or
whole
cells
may
induce
a
protective
response
against
experimental
B.
gingivalis
infection.
Bacteroides
(Porphyromonas)
gingivalis
is
a
gram-nega-
tive,
anaerobic
rod
that
has
been
associated
with
adult
periodontitis
and
abscesses
of
oral
origin.
This
species
demonstrates
significant
heterogeneity
as
regards
virulence
in
animal
models.
Studies
in
our
laboratory
(25)
and
other
laboratories
(8,
11,
30,
32)
have
demonstrated
that
B.
gingi-
valis
strains
can
be
classified
as
either
invasive
or
noninva-
sive
depending
on
whether
the
strain
migrates
to
a
site
distant
from
the
injection
site
(invasive)
or
remains
localized
to
the
injection
site
(noninvasive).
Immunization
can
alter
the
course
of
experimental
B.
gingivalis
infections
in
a
mouse
model.
Previously,
we
have
shown
that
immunization
with
invasive
B.
gingivalis
ATCC
53977
(A7A1-28)
resulted
in
localization
of
a
subsequent
challenge
infection
with
the
homologous
strain
(4).
However,
mice
immunized
with
a
noninvasive
strain
of
B.
gingivalis
or
with
Bacteroides
intermedius
or
with
Ringer
solution
(4)
developed
spreading
infections
after
challenge
with
strain
ATCC
53977.
Similarly,
Okuda
and
colleagues
(26)
found
that
there
were
slight
reductions
in
the
number
of
B.
gingivalis
381
recovered
from
hamsters
immunized
with
B.
gingivalis
381
whole
cells
(a
noninvasive
strain)
or
its
hemagglutinin
when
compared
with
sham-immunized
controls.
The
present
study
sought
to
determine
if
bacterial
extracts
such
as
a
membrane
extract
or
lipopolysaccharide
(LPS)
could
confer
protective
immunity
against
an
experimental
invasive
B.
gingivalis
infection.
MATERIALS
AND
METHODS
Bacteria.
B.
gingivalis
ATCC
53977
(A7A1-28)
was
iso-
lated
in
1985
at
the
State
University
of
New
York
at
Buffalo
from
a
deep
periodontal
lesion
in
a
37-year-old
male
patient
with
non-insulin-dependent
diabetes
mellitus.
In
mice,
this
B.
gingivalis
isolate
is
invasive
and
produces
ulcerated
lesions
distant
from
the
injection
site
and
septicemia
and,
often,
death
(25).
The
pathogenic
potential
of
this
strain
is
similar
to
that
reported
for
B.
gingivalis
W50
and
W83
(30,
*
Corresponding
author.
32).
B.
gingivalis
ATCC
53977
was
transferred
from
tryptic
soy
agar
(Difco
Laboratories,
Detroit,
Mich.)
supplemented
with
5%
sheep
blood
(Crane
Laboratories,
Inc.,
Syracuse,
N.Y.),
5
jig
of
hemin
(Sigma
Chemical
Co.,
St.
Louis,
Mo.)
per
ml,
1.0
jig
of
menadione
(Sigma)
per
ml,
and
1%
yeast
extract
(BBL
Microbiology
Systems,
Cockeysville,
Md.),
henceforth
called
enriched
tryptic
soy
agar
(ETSA),
to
trypticase
soy
broth
containing
hemin
(5
,g/ml)
and
mena-
dione
(0.5
,ug/ml).
The
microorganism
was
cultured
in
an
anaerobic
chamber
containing
an
atmosphere
of
85%
N2-
10%
H2-5%
CO2
(Forma
Scientific,
Marietta,
Ohio)
at
37°C
to
late
logarithmic-early
stationary
phase.
Bacterial
purity
was
confirmed
by
phase-contrast
microscopy,
light
micro-
scopic
examination
of
Gram
stains,
and
monitoring
of
colony
morphology
after
anaerobic
culture.
Cells
were
harvested
by
centrifugation,
washed
three
times
in
phosphate-buffered
saline
(pH
7.2)
and
stored
at
-70°C
until
required.
Antigen
preparation
and
characterization.
Two
different
preparations
were
prepared
from
whole
cells
of
B.
gingivalis
ATCC
53977
for
use
in this
study.
(i)
LIS
extract.
Bacterial
cells
were
extracted
with
lithium
diiodosalicylate
(LIS;
Eastman
Kodak
Co.,
Rochester,
N.Y.)
by
stirring
2
x
1010
bacteria
per
ml
in
0.3
M
LIS
(16)
dissolved
in
Tris
buffer
(0.05
M,
pH
7.2)
for
1
h
at
room
temperature
and
then
overnight
at
4°C.
The
extracted
cells
were
removed
by
centrifugation
at
12,000
x
g.
The
super-
natant
was
dialyzed
against
distilled
water
until
the
optical
density
at
323
nm
(OD323)
was
<0.05
and
was
then
concen-
trated
by
ultrafiltration
and
stored
in
aliquots
at
-70°C
until
required.
Protein
content
was
determined
by
the
method
of
Lowry
et
al.
(14)
by
using
bovine
serum
albumin
as
a
standard.
The
LIS
extract
(1
mg
of
protein
per
ml)
was
hydrolyzed
at
100°C
for
6
h
with
2
N
HCl
for
hexosamine
analysis
and
with
6
N
HCl
for
24
h
for
amino
acid
analysis
with
a
Beckman
6300
amino
acid
analyzer.
Neutral
sugar
content
of
the
LIS
extract
was
determined
by
the
method
of
Dubois
et
al.
(5)
by
using
glucose
as
a
standard.
(ii)
LPS.
LPS
was
prepared
by
extracting
bacterial
cells
with
hot
phenol
by
using
a
modification
of
the
method
of
3394
INFECTION
AND
IMMUNITY,
OCt.
1990,
p.
3394-3400
0019-9567/90/103394-07$02.00/0
Copyright
X
1990,
American
Society
for
Microbiology
EXPERIMENTAL
B.
GINGIVALIS
INFECTION
3395
Millar
et
al.
(19).
Briefly,
bacterial
cells
(10
to
15%,
wt/vol)
were
suspended
in
sodium
phosphate
buffer
(0.05
M,
pH
7.4)
containing
NaCl
(0.15
M)
and
disodium
EDTA
(1
mM)
and
broken
by
means
of
glass
beads
with
a
Braun
homogenizer
(five
1-min
bursts)
at
4°C.
The
glass
beads
were
removed
by
centrifugation
at
200
x
g.
The
bacterial
cell
suspension
was
centrifuged
at
12,000
x
g,
and
the
resulting
supernatant
was
ultracentrifuged
at
80,000
x
g.
The
pellet
formed
by
ultra-
centrifugation
was
lyophilized.
The
pellet
was
then
sus-
pended
in
distilled
water
(10
mg/ml)
and
mixed
with
phenol
(1:1)
preheated
to
68°C.
The
phenol-water
mixture
was
reheated
to
68°C
with
stirring.
The
aqueous
phase
was
removed
and
the
phenol
phase
was
reextracted
at
68°C
with
an
equal
volume
of
water.
The
two
aqueous
phases
were
combined,
dialyzed
against
distilled
water
at
4°C,
and
then
lyophilized.
Protein
content
was
determined
by
the
method
of
Lowry
et
al.
(14)
by
using
bovine
serum
albumin
as
a
standard.
The
two
B.
gingivalis
antigens
were
compared
by
sodium
dodecyl
sulfate-polyacrylamide
gel
electrophoresis
by
the
method
of
Laemmli
(13)
with
a
Hoeffer
SE600
Mighty
Tall
slab
gel
unit.
The
LIS
extract
and
LPS
were
electrophoresed
on
a
sodium
dodecyl
sulfate-12%
polyacrylamide
resolving
gel
and
a
3%
polyacrylamide
stacking
gel
and
stained
with
silver
(9)
or
with
Coomassie
brilliant
blue
R
(Sigma).
After
sodium
dodecyl
sulfate-polyacrylamide
gel
electrophoresis,
the
antigens
were
also
compared
by
Western
immunoblot
analysis
with
a
dry
transfer
system
(Polyblot
transfer
appa-
ratus,
Hayward,
Calif.)
for
transfer
to
nitrocellulose.
The
nitrocellulose
was
blocked
with
bovine
serum
albumin
over-
night
and
then
incubated
with
normal
mouse
serum
(1/500)
or
immunized
mouse
serum
(1/500)
for
3
h
at
room
temperature.
The
second
antibody
was
goat
anti-mouse
immunoglobulin
G
(IgG)
or
IgM
conjugated
to
horseradish
peroxidase
(1/
3000;
Bio-Rad
Laboratories,
Richmond,
Calif.),
and
the
substrate
was
4-chloro-1-naphthol
(Bio-Rad).
Infection
model.
Female
BALB/c
mice,
6
to
8
weeks
of
age
(West
Seneca
Labs,
West
Seneca,
N.Y.),
were
immunized
by
intraperitoneal
injection
of
0.1
ml
of
B.
gingivalis
cells
(109
per
mouse),
LIS
extract
or
LPS
(100
,ug/ml
of
pyrogen-
free
saline).
The
LIS
extract
and
B.
gingivalis
cells
were
administered
once
a
week
for
3
weeks,
whereas
LPS
was
administered
once.
Serum
samples
from
each
mouse
were
obtained
before
and
after
immunization.
All
mice
were
challenged
2
weeks
after
the
final
immuni-
zation
by
subcutaneous
injection
of
0.1
ml
of
B.
gingivalis
ATCC
53977
bacteria
(5
x
1010
per
ml)
into
each
of
two
sites
about
1
cm
lateral
from
the
midline
on
the
dorsal
surface.
The
mice
were
then
examined
daily
for
the
following:
(i)
the
size
and
presence
of
lesions
at
the
injection
site;
(ii)
pres-
ence,
location,
and
appearance
of
secondary
lesions;
and
(iii)
health
status.
Five
mice
from
each
group
were
sacrificed
on
days
1,
2,
5,
and
15
after
challenge,
and
samples,
including
blood
from
the
subclavian
artery,
abscess
fluid,
and
serous
exudate
from
any
secondary
lesion,
were
collected
for
microbiological
examination.
Portions
of
each
sample
were
streaked
onto
ETSA
and
anaerobically
cultured
at
37°C
for
7
days.
The
ETSA
plates
were
then
examined
for
growth
of
B.
gingivalis,
and
the
number
of
colonies
was
recorded,
as
was
the
colony
morphology
and
pigmentation.
Bacterial
cells
from
the
colonies
were
gram
stained
and
examined
by
light
microscopy.
Immediately
prior
to
challenge,
spleens
were
obtained
from
five
normal
and
five
immunized
mice
from
each
group
and
tested
as
previously
described
(3).
Briefly,
single-cell
suspensions
were
prepared
from
each
spleen
and
adjusted
to
TABLE
1.
Amino
acid
composition
of
LIS
extract
Amino
acid
No.
of
residues/
1,000
residuesa
Aspartic
acid
.................
......................
124.8
Threonine
.......................................
62.1
Serine
.......................................
49.3
Glutamic
acid
.......................................
138.8
Proline
.......................................
42.7
Glycine
.......................................
113.9
Alanine
.......................................
97.1
Half-cystine
.......................................
7.3
Valine
.......................................
71.4
Methionine
.......................................
23.2
Isoleucine
.......................................
56.8
Leucine
.......................................
71.8
Tyrosine
.......................................
34.8
Phenylalanine
.......................................
35.5
Histidine
.......................................
14.3
Lysine
.......................................
40.1
Arginine
.......................................
16.2
a
Results
are
from
an
amino
acid
analyzer
after
hydrolysis
in
6
N
HCI
at
105°C
for
28
h.
2.5
x
106
cells
per
ml
of
RPMI
1640
(GIBCO,
Grand
Island,
N.Y.)
supplemented
with
antibiotics
and
10%
heat-inacti-
vated
fetal
calf
serum
(GIBCO).
Cells
were
dispensed
into
individual
wells
(200
.lI
per
well)
of
a
96-well
microtiter
plate,
and
the
appropriate
antigen
or
mitogen
was
added
to
triplicate
wells
in
20-,ul
volumes.
The
cell
cultures
were
incubated
at
37°C
in
5%
C02-95%
air
for
72
h.
Six
hours
prior
to
harvest,
[3H]thymidine
(specific
activity,
2.0
Ci/mM,
1
jxCi
per
culture)
was
added
to
each
well.
Cells
were
collected
by
using
a
cell
harvester,
and
the
degree
of
radiolabel