sparfloxacin against. Pseudomonas aeruginosa pneumoniae in neutropenic mice. Antimicrob. Agents. Chemother. 36: 1352-1357. 15 ) Porras,. C.S.,. Svanborg,.
Microbiol. Immunol., 42 (10), 697 - 702, 1998
Attachment of Nontypable Haemophilus to Human Pharyngeal Epithelial Cells by a Ganglioside Receptor Kenji Kawakami*, Kamruddin Ahmed, Yoshiaki Utsunomiya, Kazunori Oishi, and Tsuyoshi Nagatake
influenzae Mediated
Naoto Rikitomi, Akihiro Hori,
Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki Japan
University, Nagasaki, Nagasaki 852-8102,
Received June 3, 1998. Accepted July 6, 1998 Abstract:
Nontypable
infections
and
receptor nificant
Haemophilus
has
the
to which decrease
attachment
NTHI
not
hand,
decrease
the
centration glioside of
D1b
NTHI
the
receptors
Key
words:
in
attachment, in
and
D2
treatment of
ng/ml.
Treatment
Other cells
on
human
a dose-dependent of
NTHI
Haemophilus
isolates
of
with
Ml,
of NTHI
as
manner.
This
Ganglioside,
D3,
inhibited a decrease
monoclonal
indicates
epithelial
epithelial
important
colominic
T lb the
acid
resulted
of the
acid.
The acid,
Only
treatment
the
attachment.
decreased
glycoconjugate,
with On
M1
significantly after
also
cell in a sig-
receptor.
asialoganglioside
in attachment
sialic
epithelial
N-acetyl-neuraminic
attachment
antibody
that
cells
decreased and
respiratory
the
component
of fucose, and
of human
characterized
significantly
M3,
D2
showed
study
pharyngeal
an
acid
M2,
GD2
pathogens
We
1,000 ƒÊg/ml
12.5 ƒÊg/ml
ganglioside
anti-human
influenzae,
acid
with
of
major
cells.
acetyl-salicylic
Only
the
of pharyngeal
sialic
pretreated
gangliosides
NTHI.
of
epithelial
pretreatment
suggesting NTHI
is one
pharyngeal
at a concentration
with
attachment
of 12.5 D2.
(NTHI)
to
N-acetyl-galactosamine,
D1a,
other
attach
Neuraminidase
decreased
N-acetyl-glucosamine,
the
influenzae to
bind.
in NTHI
was
gangliosides
ability
did at
treatment the
not
a con-
with
gan-
attachment
GD2,
is one
of
cells.
Attachment,
Receptor
The attachment of bacteria to pharyngeal epithelial cells is prerequisite for the establishment of respiratory infection. The host epithelial cell expresses the receptor on its surface, to which the bacteria attach by its adhesin (8). Haemophilus influenzae (H. influenzae) is one of the major pathogens of respiratory infections and meningitis. The adherence of this organism to the epithelium is a critical step in the process of colonization and infection caused by nontypable H. influenzae (NTHI) (13). To understand the pathogenesis, several important studies have been done to reveal the attachment mechanism of this bacteria with respiratory epithelial cells. These studies clearly indicated that the receptor of H. influenzae resides in the sequence of gangliosides (6, 9, 10, 22). Although several studies have shown that H. influenzae causing respiratory infections are mostly nontypable (1, 11, 12, 22), till now, only one study was done to evaluate ganglioside interactions with NTHI (6). That study
showed the receptor as a minor ganglioside. Gangliosides are important constituents of the surface membrane of most cells of mammalian tissues. It is generally assumed that the gangliosides occur on the outer side of the cellular plasma membrane (23). It is recognized that gangliosides are important receptor sites for many bacteria and toxins. Interestingly, another study identifled proteoglycan as a receptor for NTHI (13). Therefore, to solve confusion, this study was done for identify the receptor of NTHI for pharyngeal epithelial cells.
Address correspondence to Dr. Kenji Kawakami, Depart-
Abbreviations: GD2, diganglioside 2; IgG3, Immunoglobulin G3; NANA, N-acetyl-neuraminicacid; NTHI, nontypable Haemo-
*
ment of Internal ki University,
Medicine, 1-12-4
Institute
Sakamoto,
of Tropical Nagasaki,
Medicine, Nagasaki
Materials and Methods Bacteria. NTHI isolated from the sputum of patients with respiratory infection were used in this study. Serotypes of H. influenzae strains were determined by type-specific antisera (Difco Laboratories, Detroit, Mich., U.S.A.). H-95-135 was the isolate mainly used in this
Nagasa-
852-8523,
philus influenzae; PB, phosphate buffer; PBS, phosphate-buffered saline; TLC, thin-layer chromatography.
Japan.
697
698
K. KAWAKAMI
experiment.
Other
H-95-120. sion
All
(BHI)
eysville,
broth Md.,
Dickinson tries,
and
Osaka,
on
ing
5%
in
a 5%
Co.),
for
BHI
agar
(Becton
Dickinson
with
were
to
0.75%
washings
hr
were pH
centrifuging
at
in
The 80 •~
were
for
10
min
of
bacteria
cells
at a concentration
with
neuraminidase
gens,
Sigma
water
bath,
room
of
Chemical and
Bacteria with
and
mmol
phos-
washed
three
at
tem-
room
Pure
D2,
min
M1,
D3
and
were
T1b and
a water
cells
treated
with
Corporation,
30
min.
1D3,
bohydrate, quantitative vitalex,
used
1 •~
108
CFU/ml
(purified
from
a
Effects At
10
min.
PB
at
of
the
chain
of
and
of
the
than
glioside
(2)
on
NAD,
BHI
and
cells
were
after
neuraminidase,
31.7•} treated
respectively.
μg/ml. The gangliosides D1a
30
a 30%
at
D1b
104 cells/ml
IgG3
However
mouse
and C
antibody
of
IgG3
resulted
of
M1
also
tested
with
the
lowest
250
T
b
Gangliosides at con(Table
did
not
M2,
Only
500
were
attachment
gangliosides
dose
a
1).
decrease
M3,
ganglioside in
effective
and
mixture T1b.
attachment
gan-
decrease
respectively
and
M1
decreased
the
64-75% 125,
and
125 ƒÊg/ml,
asialoganglioside
manner
in
decreased
and
caused
except
gangliosides
D1b,
sugars
acid)
adherence
of
the
D1a,
12.5
We
nificantly
no
N-acetyl-glucosamine,
concentrations
gangliosides
attachment.
isotype
which
significantly
of
Attachment
1,000 ƒÊg/ml,
acid,
of
Ml,
and
on and
decrease
constituents
Sigma
500
D2,
D3
D2
sig-
dose-dependent being
12.5
ng/ml.
on
Adherence
lipopolysaccharide immunotype-1
with
was agar
were
epithelial
0.001)
acetyl-salicylic
adherence
D1a,
Effects of
and
of
of
mixture,
acid,
37
treatment NTHI
Data
pharyngeal
control
colominic
more
at
Fisher
After
done
if P values
(P•ƒ
Carbohydrates
NANA,
for
C
Different
mix-
incubated
monoclonal
to
4.3%
the
5 units/ml
N-acetyl-galactosamine,
control.
bacteria.
culturing
of
brain;
5 •~
of
and
of
a concentration
37
of
antibody
aeruginosa
viability
and
(fucose,
of
M3,
concentrations
Tokyo)
was
Attachment
NTHI
22.1 •}
0.001)
were
N-acetyl-
M2,
bovine
in
on of
to
(P•ƒ 20
at
in
colominic
suspended
bacteria
(ANOVA).
significant
50 two
average.
analysis
variance
Neuraminidase
decreased
with
N-acetyl-neuraminic
M1,
an
a
on
sample,
attached as
at
on Products
each
Statistical
attachment
10.8%
PB
ganglioside fucose,
gangliosides
a mouse
as of
hemin
min
with g for
acid,
the O-side
Pseudomonas was
10
80 •~
of
monoclonal
against
Viability
for
Industries),
different
GD2
(14)
of
The
treated
at
at a concentration
(Seikagaku
isotype
of
For
statistically
10 min
0.05.
centrations
anti-human
from
than
Effects
bath.
Epithelial
for
considered
for
bacteria
counting
of
min. three
collected
Southern
presented
analysis
30 of
perfrin-
times
N-acetyl-galactosamine,
Co.),
in
C
concentrations
asialoganglioside
Chemical
37
centrifuging
Chemical
were
Clostridium
for
was
g
attached
counted.
made
cells/ml,
Results
Epithelial
cells/ml
three
acetyl-salicylic
(NANA),
D1b,
at
a concentration
glucosamine, acid
104
cells.
from
washed
different
(Wako
V Co.)
then by
at
epithelial
5 •~
(type
temperature
treated
and
The
pharyngeal
a total
were
at
male.
perature.
ture
were
cells
Treatment
(Shandon
England).
7.2,
C for
80 •~
cells
cytospin
analysis.
one-way
by
done
pH
5 X 104
at 37
at
Finally,
were
Statistical
kept
centrifuging
attachment
with
less
swab
1/15
cells
g
adult
a cotton
collected
7.2.
at 80
epithelial
healthy
with
separated
were
bac-
Bacteria
and of
were by
the
was
(PBS),
CFU/ml,
bacteria
cells
the
108
and
by
specimens
isolate.
Pharyngeal
slide
assay
concentration ratios
Astmoor,
and
a magnification in each
a normal
30
a
of
modification.
at equal
in PBS
successive
CX
operated
at
fimbriation cells.
for 100
with
saline
1 •~
at
temperature.
glass
used
acetate
Tokyo)
scrapped
swab (PB),
mixed
Ltd.,
was
a JEM
scanned
from was
the
by
14
from
grid
uranyl
Ltd.,
the
collected
oropharynx
times
for
of
Unattached
at a concentration
under
randomly
epithelial
buffer
grown
copper
examined
determine
Pharyngeal
from
contain-
attachment
15)
in phosphate-buffered
cells
viability
control.
An (3,
concentration
were
day,
the
assay.
epithelial
cul-
Bacteria
saline
(JEOL
were
phate
was
Co.)
observation.
microscope
cells
NTHI
and
a
next
with
before
suspended
Mo.,
The
compared
described
room
staining
were
Louis,
and
A carbon-coated
Bacteria
The
use.
NAD,
in normal
Specimens
cells
and
microscopic
36,000
Indusadenine
St.
overnight.
was Attachment
as
incubator.
suspended
electron
of
hemin
Cock-
Chemical
Co.,
teria
(Becton
(ƒÀ-nicotinamide
C until
negative
kV.
NAD Chemical
at -40
CFU/ml.
sec.
and
incubator
infu-
Co.,
Isovitalex
Pure
kept
CO,
109
(Wako
and
heart
and
5%
and
were
of
Dickinson
hemin
Isovitalex,
H-94-152
in brain
containing
Japan)
Electron agar
H-93-151,
stocked
(Becton
Sigma
U.S.A.),
were were
U.S.A.)
dinucleotide,
tured
isolates
isolates
ET AL
determined
containing
incubated
each
in
carby
5% a 5%
of
Anti-Human
GD2
Antibody
the
NTHI The
adherence
of
a dose-dependent
Iso-
mouse
CO2
3).
anti-human There
NTHI
manner
was
GD2 no
decrease
was
decreased
after
cells
significantly were
monoclonal in
attachment
treated
antibody
in with (Table
when
cells
RECEPTOR
OF NONTYPABLE
H. INFLUENZAE
699
Table 1. Attachment of H. influenzae to pharyngeal epithelial cells after treatment with different gangliosides
were
treated
with
monoclonal
a control
antibody
of
the
same
isotype
mouse
Electron
(1D3).
stain Effect
of
Strains We three treated to
the
gests glioside
Ganglioside
D2
on
the
Adherence
of
found
a significant isolates
of
with
1.25 ƒÊg/ml without
universality D2
for
under
Viability
control the
stains
NTHI.
decrease
in
when
these
NTHI of
the
D2 (Table
receptor
adherence
strains
ganglioside
ganglioside of
the
as 2).
sequence
Observation
were an
nonfimbriated
electron
as
revealed
by
negative
microscope.
Other
of NTHI
other
Microscopic
All
of
were
pre-
compared This in
After this ty
suggan-
study, compared
CFU/ml.
of
Bacteria
treatment there to
with was the
no control.
different significant The
carbohydrates difference viable
count
used of was
in
viabili1 •~
108
K. KAWAKAMI
700
Table
2. Attachment
epithelial
cells
of three after
strains
treatment
of H.
with
influenzae
to
1.25 ƒÊg/ml
Our
pharyngeal
ganglioside
ET AL
D2
findings
clude
with
that
The
the
GD2
from
in
in
the
originated tor
for
and
the
of
the
to con-
of
were
GD2.
purified
antibody
GD2. does
of
was
Therefore,
We
sequence
us
sequence
monoclonal
species.
in the
permit
assay
ganglioside
different lies
in
human
function
NTHI
NTHI
inhibition
against
from
of
lies
the
brain
mouse
possible
strains
receptor
used
bovine
raised
other
not
it is
vary
when
the
recep-
assume GalNAc1ƒÀ1-4
(Neu-
Acƒ¿2-8NeuAcƒ¿2-3). Several
Table 3. Attachment of H. influenzae to pharyngeal epithelial cells after treatment with mouse-anti-human gangliosides D2 and 1D3
our
features
findings.
receptor
of
could
M.
detect
(8)
may
disturb
and
tured
human
and
unidentified
with
acid
for
H.
have
influenzae
pathogens.
cells
(3).
glioside
in
Ml,
of of
that
the
identify
the
ment,
D2
was
and
D3
gangliosides decrease
binding
decreased
sequence further
share
and
all
D2,
where
bition
was cells
attachment
of
these
anti-human significantly
GD2 a
signifi-
with the and
gan-
occurring
of
sequences 4).
A
observed
for in
antibody dose-dependent
also
by
cells
manner.
of the
is due
to
the
which
receptors;
essentially by
aber-
glycosphingolipid
Another
of
bacteria,
reason
may
be
interaction
NTHI
to
epithelial
is
primarily
to
(1).
This
Apicella
et of
indicates as
that
in,
The
attachment and
NTHI found
well
as
al
(4)
reported
the
H.
influenzae
all
are
other
H.
influenzae
study
H.
in
all the
fresh
clinical
nonfimbriated.
influenzae
are
impor-
infection.
cells which
in infection
respiratory
to epithelial
facwere
influenzae
respiratory
for
do (16).
adherence
were
factors
H.
this
that,
nonfimbriated
of bacteria there
in
during
responsible
of
nonfimbrial used
NTHI
epithelium
proteins
that
nonfimbriated
80%
respiratory
as
The We
tant
process
identified
20).
were
fimbriated
surface
been 13,
attachment
Interestingly,
attachment
have (5,
isolates,
decreased
idenone
identifying
adhesin-receptor
attachment
High-molecular-weight
sputum
epithe-
the
of
and
characterized of
(7).
sets
cells
experiments,
of are
surface
fimbriae.
facilitate
nonfimbriated.
inhiof
cell
Gram-negative
with
ganglioside
attachment
treatment
In
tors
treatment
types cancers
of
receptor
Therefore, in
their
nasopha-
the
researchers in
by
proteins
cultured
organization
several
during
M2,
significant
after
used
and
are
mediated not
attach-
gangliosides
different
of
supported
identify
(13).
role
cells.
even the
by cells
different
at the
there
used
was
The
of
discrepancies
animal
synthesis
that
To
inhibit
rant
of
to
a receptor
these
and
expressed
role
D1b
human
gan-
suggest-
to
with
effect
Furthermore,
in
the
gangliosides.
except
a dose-dependent
with
these
(Table not
gangliosides
observed.
express
all
respectively,
common
D 1b was
possibly
(NeuAcƒ¿2-
elucidate
required
treated
which
pharyngeal
as
as
NTHI
(6).
membrane
attempt
shown between
is further
outer
et al also
for
types
significantly, in
reasons of
of
with
D1a
Noel
different
separately to
Another
by
was
oligosaccharide
specific
the cul-
ganglioside
GM1
NTHI
sialylated
proteoglycans
the
attention
tested
NTHI
of
which
using
it
this
near
bac-
and
interaction
however,
mobility
to
(17).
tified
epithe-
of
steps
TLC
the
it
of
bacteria
cells,
to
while
detect
commonly
washing
epithelial
binding
mucin
is
two-dimensional
essential
(3),
binding
between
NTHI;
the
receptor
treatment
12.5 ƒÊg/ml,
resides
D 1a
We
T1b,
and
in attachment
with
lial
NTHI
minimum
NTHI
M3,
125
receptor
our
gangliosides
of
receptor
on
NTHI
and
on
The
GalNAcƒÀ1-4
after
recep-
respirato-
receptor
19).
NTHI.
treated
With
the
the
aeruginosa,
received
for
D 1 a, D 1b
ganglioside.
attachment
be
attachment we
at concentrations
ing
to
as
other
catarrhalis)
gangliosides
mixture,
gliosides each
as
receptor
decrease
for
Pseudomonas
(9,
is proposed
candidate
only
also
bacteria (M.
Therefore,
cant
of
not
but
proposed
binding
cells
proven b,
case
catarrhalis
epithelial
the
the was
for
Moraxella
3)
been type
In
GalNAci31-4Gal lial
in
ryngeal
Gangliosides
is
gangliosides
NTHI
ry
acid
assay
the
method
not
receptor
many
laryngeal
sialic
sialic
require
By
present
could
to
of
M2
(TLC)
interaction
(21).
the
in TLC
critical
identification
ganglioside
the
TLC
ganglioside
tor
in
that,
the
were
the
where
lactosylceramide,
weak
the
by
receptor
It is explained to
that
methodology
chromatography
(18). teria
our
revealed
catarrhalis,
the
thin-layer
Discussion
of
It was
are
is a complex responsible
RECEPTOR
Table
4. Sequences
of different
OF NONTYPABLE
H. INFLUENZAE
gangliosides
in that process (6). In conclusion, we provided here evidence that the receptor for NTHI lies in the sequence of GD2 on human pharyngeal epithelial cells; however, much still needs to be learned about the factors which determine the successful attachment of H. influenzae in the respiratory tract.
350. 9 )
Krivan,
H.C.,
Roberts,
pathogenic
Acad.
Kubiet,
M.,
We thank Akitoyo Ichinoseand Kiwao Watanabefor their excellenttechnicalassistance. Part of the projectwas supported by Grant-in-Aidfor Encouragementof YoungScientists,1996, Ministryof Education,Science,Sportsand Cultureand Grantfor ScientificResearch (InternationalCooperationon the Preventionand Treatmentof AcuteRespiratoryInfections)fromthe Ministryof Healthand Welfare,Japan.
U.S.A.
and
11 )
Ramphal,
3)
4)
5)
6)
7) 8)
to in
1988.
the
some
Many
carbohydrate
glycolipid.
Proc.
6157-6161. R.
1995.
Adhesion
from
blood
and
lactoferin.
mucins
Murphy,
F.T.,
and
Haemophilus antigens,
and
Infect. 12 )
Dis.
Musher,
of
and
nontypable
sputum
to
Infect.
13 )
14 )
the
immune
human
human
Immun.
63:
Kubitschek,
Pneumonia
Love,
bacterial
Immun.
62:
T.,
K.,
and
and
of
febrile
surface
infection.
Rev.
David,
to
J.,
and
Baughn,
tracheobronchitis
Intern. M.
nontypable
adhesion
due
Med.
99:
444-450.
1994.
High-molec-
Haemophilus
cellular
influenzae
proteoglycans.
Infect.
4028-4033.
Sonoda,
F.,
Matsumoto,
Iwagaki,
K.
lipopolysaccharide
A.,
1992.
against
neutropenic
Kobayashi,
Effects
specific
sparfloxacin in
to
Crennan,
acute Ann.
D.C.,
proteins
mediate
Oishi,
Nontypable aspects,
response
R.K., and
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G.J.,
1987.
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1-15.
Haemophilus
Noel,
A.M.
a review
9:
1983.
Apicella,
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M.D.,
E.R.
1) Ahmed, K., Ichinose, A., Dai, T.C., Takahashi, A., Utsunomiya,Y., Kawakami,K., Nagatake,T., and Matsu
2)
85:
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