Microbiol. Immunol. Vol. 36 (2), 113-121, 1992
Immunoelectron Microscopy of Chlamydia psittaci with Monoclonal Antibodies Shuji ANDO,* Ikuo TAKASHIMA,and Nobuo
HASHIMOTO
Department of VeterinaryPublic Health, Faculty of VeterinaryMedicine, Hokkaido University, Sapporo, Hokkaido 060, Japan
(Accepted for publication, November 8, 1991)
Abstract An immunoelectron microscopic study was performed to determine the distribution of antigenic components on particles of Chlamydiapsittaci and infected. cells using a number of monoclonal antibodies (MAbs). Of three anti-lipopolysaccharide (LPS) antibodies (4D5, A2 and 4G5), two antibodies (4D5 and A2) reacted with the surface of reticulate bodies (RBs) but not with that of elementary bodies (EBs). The other antibody (4G5) reacted with both EBs and RBs. Examination of infected cells in thin sections revealed that 4D5 and A2 combined with the membranes of both EBs and RBs. These results indicate that each LPS epitope localized at a different position in the chlamydial membrane. Most MAbs directed to protein antigens reacted on the surface of both EBs and RBs though 3E9 specific for the 90 kDa and 50 kDa protein components combined with RBs only.
Genus Chlamydia is an obligate intracellular parasite that shows a unique developmental cycle in which two distinct forms, an elementary body (EB) and a reticulate body (RB), exist (14). The outer membrane of EB is suggested to play an important role in the attachment to host cells and in the inhibition of phagosomelysosome fusion (4, 10). The surface distribution of antigenic components on the outer membrane of C. trachomatis was examined with respect to the neutralizing mechanism due to antibody and to the structural characteristics of the outer membrane (5, 8, 16, 17, 19, 23). Immunofluorescent assay, peroxidase- and ferritinlabeled immunoelectron microscopy have been used for this purpose (8, 17, 19). In recent years, the immunogold-labeling method for electron microscopy has been applied to antigenic analysis of the surface of C. trachomatisas well as to that of other micro-organisms (2, 9, 12). Kuo and Chi (12) and Collett et al (9) examined the surface distribution of epitopes in C. trachomatisusing several MAbs, and Birkelund et al (2) examined lipopolysaccharide (LPS) distribution on the C. trachomatis EB surface. These studies were performed mainly on LPS, major outer membrane protein (MOMP) and other cysteine-rich proteins, and it was suggested that the surface-exposed
MOMP
might
be responsible
for the neutralization
of C. trachomatis
infection. We previously reported the production and characterization of a number of MAbs against two C.psittaci strains (18, 22). Using these MAbs, epitope distribution in C. psittaci particles was examined by immunoelectron microscopy (IEM). 113
S. ANDO
114
MATERIALS
Organisms. was
Two
derived
from
Chlamydial tion,
organisms
and al
(15)
60
min
of
10 mm
.
at
HEPES at
30,000•~g
for
Percoll-HBS particles
30
at
min
staining
preparations To
(v/v)
Ltd.,
Monoclonal
reactivity
of by
1D4,
21, all
2F4 had
study. primary
3B5
common and
antibody.
(IgG-Gold-5, ZYMED
Immunoelectron was
carried
suspension copper grids (BSA),
out
were 1%
both
with
normal
mouse
at
applied
to
the
with
8 ƒÊl and
20
tests two
(18,
25
kDa
Izawa-1
fluid
(negative
Labs. by
the
et
al
in
The
containing (PBSAG)
used were
5 nm
For
10 min
in
purified
was bovine to
this as
diameter
Chlamydia of
membrane
(w/v)
in used
(IgG-Gold-15,
microliters
suspension
for
(2F4,
Some
P-1041,
of
0.1 %
MAbs (18).
diameter markers.
Eight
Crossstrain-
components.
particles
collodion
5 min.
1. and
control)
15 nm labeling
(9).
characterized Table
Some
and
colloidal or as
in
protein
Immunolabeling
Collett
NaN3
microand
components
strains,
U.S.A.) were used
been
21).
protein
two
PBS
trypsin a
(TAAB
subspecies-
the
IgG-gold
mm
had
subgenus-,
carbon-coated
of
the
IFU/ml.
(w/v)
provided
shown
and
for
2•~106 %
resin
are
kDa
observation. of
for
use. observation,
paraformaldehyde
MAbs
40
Inc., Calif., U.S.A.)
determined (3) and
in
instructions
MAbs
genus-,
ascitic
temperature
glycine
the
with
anti-mouse
method
room
incubated (w/v)
the
3%
g
ordinary
microscopy
centrifuged
K4M
the
antibody
reactions
of
All of
as
the
were
chla-
prepared.
reacted
microscopic by
were grids
with
of 0.1
at (v/v)
of
the
was G-250
a titer
by
30,000 •~
by
with
Lowicryl to
were
defined
F2)
E.Y. Labs. Inc., Calif.,
Labs.
in
according IEM
cells
mixture
immunoreagents.
and
Goat
a
embedding
properties
was
reacted
MAbs
with
immunofluorescent
3E9,
example,
The
band
C until microscopic
at
then
30%
electron
suspended
for
again
at
stained
at -80
organisms
HBS),
of
The
transmission
g
sedimented
ml
again
of the suspensions brilliant blue
were
EDTA.
for
The
MAbs
with
fixed
and
22).
a
Newhall
composed
7.4;
30
4 C.
HBS,
were stored immunoelectron
cells
England) sections
in
under
infected
and
at
10,000 •~
(7).
post-infec-
centrifuged in
min
at
was
was
centrifuged
suspended
infected
After
indirect
3F9,
MAbs
500•~g,
30
hr
buffer
pH
resuspended
and
48
by
of
debris
supernatant
protein content with Coomassie
(w/v)
antibodies
(18,
specific
were
5 ml
saline, cell
Izawa-1 pigeon
described
in
Host
for
HBS
examined
the
thin
previously
30,000 •~g in
as
suspended
was
protein content) sections for
Berkshire,
manufacturer,
pellet
Total assay
glutaraldehyde.
Equip.
The
were
and
purity. binding
0.02% at
the
twice
monolayers
tube
and
organisms
method
containing
min,
at
centrifuged
buffered
sec).
Strain a feral
harvested
and
were
study. from
gradient
(HEPES w,
this
derived
cells,
density
20
at
washed
post-inoculation,
centrifuge 1%
centrifuged
the
cell
hr
C.
229
in
harvested
NaCl
10
4
(200 ƒÊg/ml prepare ultrathin
229
PBS
at
HeLa
pellets
(75
for
Purified
to check protein-dye
in
min
4 C.
(TEM) by the
48
30
was
negative
At
500•~g
and
mydial
mm
settings
at
was
were
cell
145
low
used
P-1041
Percoll
cells
resultant
and
twice
were
and in
by
METHODS
psittaci
grown
infected
The
centrifugation
C.
AND
(13),
purified
Briefly,
4 C.
sonicated
of
were
partially
et
HeLa
strains
a budgerigar
ET AL
on
chlamydial
aspirated serum block
200-mesh and albumin non-specific
the
IMMUNOELECTRON Table
1.
MICROSCOPY Properties
of monoclonal
OF
C. PSITTACI
115
antibodies
binding. The grids were placed on a droplet (30 pl) of primary antibody solution in PBSA (PBSAG without glycine) for 30 min at 37 C, washed 10 times with PBSA and then put on a droplet (30 pl) of IgG-Gold-5 in PBSA for 30 min at 37 C. After careful washings with PBSA, the specimens were incubated with 0.05% (w/v) BSA in distilled water (pH 7.0) for 5 min, and then dried on a filter paper. Immunolabeling of ultrathin sections was performed as follows. The thin sections mounted on a grid were floated on a droplet (40 pl) of 1% (w/v) BSA in PBS for 2 hr at room temperature to block non-specific binding, and then placed on a droplet (40 pl) of primary antibody solution in PBS containing 1% (w/v) BSA for 2 hr at room temperature. After several washings with PBS, the grid was transferred onto a droplet (40 pl) of IgG-Gold-15 in 50 mm Tris, pH 7.4, 150 mm-NaC1 containing 1% BSA and 0.05% NaN3 and incubated for 2 hr at room temperature. After washings with PBS, the sample was fixed with 2.5% (v/v) glutaraldehyde in phosphate buffer (pH 7.4) for 10 min. The sample was washed with distilled water 5 times, air-dried, and then stained with uranyl acetate and lead nitrate solutions. The optimum concentrations of antibodies and IgG-gold were tested by box titration. Each MAb was serially diluted (1: 10, 1: 50, 1: 100, 1: 200, 1: 400, 1: 800 and 1: 1,600) and used in the IEM. IgG-gold and normal mouse ascitic fluid were diluted at 1: 20 and 1: 50 dilutions, respectively. RESULTS
Electron Microscopic Observations of Chlamydial Particles in Negative Stained and ThinSectioned Preparations Partially
purified
organisms
are
shown
in Fig.
la.
EEs
and
RBs
were
distin-
116
S. ANDO
Fig.
1.
Electron
staining HeLa
Fig.
of 229
2.
cells
Electron
antibodies. RB ascitic
micrographs
(b)
(bar,
(bars,
0.5 ƒÊm). as
a negative
organisms
P-1041
harvested
strain
(bar,
at
1 ƒÊm).
48 (b)
hr
post-infection.
Thin
section
(a)
Negative
of
P-1041-infected
after
reaction
1 ƒÊm).
micrographs Genus-specific
fluid
of
Percoll-purified
ET AL
of
Percoll-purified
anti-LPS Izawa-1 control
MAb strain (bars,
EB
chlamydial 4G5 (c)
was and
0.5 ƒÊm).
reacted RB
(d)
EBs with were
and
RBs
Izawa-1 reacted
strain with
EB
a normal
with (a)
and
mouse
IMMUNOELECTRON Table
Fig.
3. with (bars,
2.
Electron IgG-gold.
Reactivities
of monoclonal
micrographs
of chlamydial
Anti-MOMP
0.5 ƒÊm).
MICROSCOPY
MAb
3E9
MAb was
applied
OF
antibodies
EBs
4E11
was to
and reacted
Izawa-1
C. PSITTACI
in immunoelectron
RBs
after
with strain
reaction
Izawa-1 EB
(c)
microscopy
with strain
and
117
RB
MAbs EB
(a)
(d)
(bars,
visualized and
RB
(b)
0.5 ƒÊm).
guished by their size. Thin sections of infected HeLa 229 cells were prepared at 48 hr after infection. In this preparation, the EB/RB ratio in the inclusions was approximately 4: 1 (Fig. 1b). Immunocytochemistry for Pured To define
the specific
C. psittaci
reactions
between
organisms
and
antibodies,
the distribu-
118
S. ANDO
ET AL
Fig. 4. Electron micrograph of ultrathin section of P-1041-infected cells after reaction with a genus-specific anti-LPS MAb 4D5. EBs and RBs were labeled with IgG-gold (arrows) (bar, 0.5 ,um).
tion of the gold particles on isolated chlamydial particles was compared with that on the background on the chlamydial particles treated with normal mouse ascitic fluids (negative control), because gold particles conjugated with immunoglobulin tended to aggregate more often than protein-A gold (20). Eight examples of characteristic reaction patterns on the surface of EBs and RBs are shown in Figs. 2 and 3. The results of all antibodies used in this study are summarized in Table 2. The labeling distribution of genus-specific anti-LPS antibodies (4G5, 4D5 and A2) were divided into two patterns. MAb 4G5 bound over a large part of the surface of RBs but the labelings on EBs were partially localized on the surface in both strains (Fig. 2, a and b, Table 2). In contrast, 4D5 and A2 only reacted with the RB surface, but not with the EB surface. No labeling was seen on the organisms treated with normal mouse ascitic fluid (Fig. 2, c and d). All protein-directing MAbs (4F8 and others) except 3E9 apparently reacted with the surface of both EB and RB particles (Table 2, Fig. 3, a and b). In contrast, a subgenus-specific anti-Izawa-1 MAb, 3E9, showed a different reaction pattern from other protein-indirecting MAbs; 3E9 apparently combined only with the surface of RBs, but not with the surface of EBs (Fig. 3, c and d). Immunoreactionof EBs and RBs in Thin Sections Epitopes corresponding to two genus-specific anti-LPS MAbs (4D5 and A2) were not observed on the surface of purified EB particles (Table 2). Therefore, to examine the presence of LPS epitopes in in situ chlamydial particles, IEM was performed on thin sections of infected cells (Table 2, Fig. 4). The MAbs, 4D5 and A2, evidently reacted with EBs and RBs in the inclusions. Gold particles were located on the membranes of EB and RB particles and also within the particles (Fig. 4). Binding of gold particles was rarely seen in the cyto-
IMMUNOELECTRON
MICROSCOPY
OF
C. PSITTACI
119
plasm surrounding the chlamydial inclusions and no reaction was seen on the thin section treated with normal mouse ascitic fluid (data not shown). These results may indicate that the binding of gold particles to chlamydial particles is specific. DISCUSSION
In this study, the localization of chlamydial antigenic components was analyzed with immunoelectron microscopy. The LPS epitopes of C. psittaci recognized with three anti-LPS MAbs were divided into two groups depending on the labeling patterns. MAbs 4D5 and A2 reacted with RBs but not with EBs when MAbs were applied to intact organisms. In contrast, 4G5 combined with the surface of RB particles and partially with the EB surface. Observations of the infected cells in thin sections demonstrated that LPS epitopes reactive with 4D5 and A2 were present in EB particles. Specificity of the reaction on thin section was supported by the gold distribution limited in chlamydial particles and no labeling in the negative control. Therefore, these results strongly suggest that in situ EBs possess LPS perhaps in the membrane, but the epitopes reactive with 4D5 and A2 are not exposed on the EB surface. Birkelund et al (2) showed that anti-LPS MAbs bound strongly on the surface of formalin-fixed EB and that LPS readily dissociated from the surface of unfixed EBs. In contrast, Collett et al (9) did not detect the LPS antigen on the surfaces of C. trachomatisEB particles using anti-LPS MAbs and suggested that the LPS moiety was not exposed on the EB surface. This confusion, as to the localization of the LPS moiety on the EB surface, might be due to different MAbs used. Chlamydial LPS has at least three antigenic domains (6). Therefore, the MAbs used in this study might recognize different domains of the LPS moiety because the reaction patterns were divided into two groups. The epitopes corresponding to 4G5 were exposed on a limited part of the EB surface, but the epitopes bound with 4D5 and A2 were rarely detected on the EB surface. In contrast, LPS epitopes corresponding to these three MAbs (A2, 4D5 and 4G5) were detected over a wide area of the RB surface. Therefore, there are different modes for integration of the LPS moiety in the RB and EB membranes. One domain might be exposed on the EB surface, while the other might be concealed in the EB membrane. Karimi et al (11) reported the accumulation of chlamydial LPS antigen in the plasma membranes of infected cells, and suggested that LPS antigen was transported from inclusions to the host plasma membranes. However, gold particle in the cytoplasm and plasma membrane was never encountered in the thin sections in the present study. One of 11 MAbs directed to chlamydial protein, 3E9, showed a reaction pattern similar to two anti-LPS MAbs (4D5 and A2). Epitopes of 3E9 were detected on the RB surface but not on the EB surface. We examined the kinetics of the antigen appearance in the infected cells and revealed that antigens corresponding to 3E9 and LPS antigens were detected throughout the growth cycle of Izawa-1 and P-1041 strains (1). Therefore, these antigens may be essential for the chlamydial growth.
120
S. ANDO
ET AL
The protein antigens recognized by most of the anti-protein MAbs except 3E9 were exposed on the surfaces of both RB and EB particles. The neutralization of C. trachomatis was reported to be attributed to the surface-exposed epitopes on MOMP (23). However, it has not been clear how neutralization of C. psittaci infection takes place. Therefore, it would be of special interest to examine whether MAbs against surface-exposed epitopes of EB have neutralizing ability. REFERENCES
1) Ando, S., Suwa, T., Takashima, I., and Hashimoto, N. 1991. Kinetic studies on the appearance of antigens of Chlamydiapsittaci during its developmental cycle. J. Vet. Med. Sci. 53: 691-697. 2) Birkelund, S., Lundemose, A.G., and Christiansen, G. 1989. Immunoelectron microscopy of lipopolysaccharide in Chlamydiatrachomatis. Infect. Immun. 57: 3250-3253. 3) Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytic. Biochem. 72: 248-254. 4) Byrne, G.I., and Moulder, J.W. 1978. Parasite-specified phagocytosis of Chlamydiapsittaci and Chlamydia trachomatisby L and HeLa cells. Infect. Immun. 19: 598-606. 5) Caldwell, H.D., and Perry, L. J. 1982. Neutralization of Chlamydia trachomatis infectivity with antibodies to the major outer membrane protein. Infect. Immun. 38: 745-754. 6) Caldwell, H.D., and Hitchcock, P. J. 1984. Monoclonal antibodies against a genus-specific antigen of Chlamydia species: location of the epitope on chlamydial lipopolysaccharide. Infect. Immun. 44: 306-314. 7) Chiba, N., Arikawa, J., Takashima, I., and Hashimoto, N. 1984. Isolation and serological survey of chlamydiosis in feral pigeons and crows in Hokkaido. Jpn. J. Vet. Sci. 46: 243-245. 8) Clark, R.B., Nachamkin, I., Schatzki, P.F., and Dalton, H.P. 1982. Localization of distinct surface antigens on Chlamydia trachomatisHAR-13 by immune electron microscopy with monoclonal antibodies. Infect. Immun. 38: 1273-1278. 9) Collett, B.A., Newhall V, W. J. , Jersil Jr., R.A. , and Jones, R. B. 1989. Detection of surface-exposed epitopes on Chlamydiatrachomatisby immune electron microscopy. J. Gen. Microbiol. 135: 85-94. 10) Eissenberg, L.G., Wyrick, P.B., Davis, C.H., and Rumpp, J.W. 1983. Chlamydiapsittaci elementary body envelopes: ingestion and inhibition of phagolysosome fusion. Infect. Immun. 40: 741-751. 11) Karimi, S.T., Schloemer, R.H., and Wilde III, C.E. 1989. Accumulation of chlamydial lipopolysaccharide antigen in the plasma membranes of infected cells. Infect. Immun. 57: 17801785. 12) Kuo, C.-C., and Chi, E.Y. 1987. Ultrastructural study of Chlamydiatrachomatissurface antigens by immunogold staining with monoclonal antibodies. Infect. Immun. 55: 1324-1328. 13) Matsumoto, A., Bessho, R., Soejima, R., and Hino, J. 1984. Biological properties of a Chlamydia strain isolated from a pet bird, budgerigar, which was kept by a psittacosis patient. Kawasaki Med. J. 10: 77-90. 14) Moulder, J., Hatch, T.P., Kuo, C.-C., Schachter, J., and Storz, J. 1984. Chlamydia, p. 729-739. In Krieg, N.R., and Holt, J.G. (eds), Bergey's manual of systematic bacteriology, Vol. 1. The Williams and Wilkins Co., Baltimore. 15) Newhall V, W. J., Batteiger, B., and Jones, R.B. 1982. Analysis of the human serological response to proteins of Chlamydia trachomatis. Infect. Immun. 38: 1181-1189. 16) Peeling, R., Malean, I.W., and Bruham, R.C. 1984. In vitro neutralization of Chlamydia trachomatis with monoclonal antibody to an epitope on the major outer membrane protein. Infect. Immun. 46: 484-488. 17) Richmond, S. J., and Stirling, P. 1981. Localization of chlamydial group antigen in McCoy cell monolayers infected with Chlamydiatrachomatis or Chlamydiapsittaci. Infect. Immun. 34: 516-570. 18) Seki, C., Takashima, I., Arikawa, J., and Hashimoto, N. 1988. Monoclonal antibodies to Chiamydia psittaci: characteristics and antigenic analysis. Jpn. J. Vet. Sci. 50: 383-393.
IMMUNOELECTRON
MICROSCOPY
OF
C. PSITTACI
121
19) Stephens, R.S., Tam, M.R., Kuo, C.-C., and Nowinski, R.C. 1982. Monoclonal antibodies to Chlamydia trachomatis: antibody specificities and antigen characterization. J. Immunol. 128: 1083-1089. 20) Stot, J.W., and Genze, H. J. 1984. Gold markers for single and double immunolabelling of ultrathin cryosections, p. 129-142. In Polak, J.M., and Varndell, I.M. (eds), Immunolabelling for electron microscopy, Elsevier Science Publishers B.V., Amsterdam. 21) Takahashi, T., Takashima, I., and Hashimoto, N. 1988. Immunotyping of Chlamydiapsittaci by indirect immunofluorescence antibody test with monoclonal antibodies. Microbiol. Immunol. 32: 251-263. 22) Toyofuku, H., Takashima, I., Arikawa, J., and Hashimoto, N. 1986. Monoclonal antibodies against Chlamydiapsittaci. Microbiol. Immunol. 30: 945-955. 23) Zhang, Y.-X., Stewart, S., Joseph, T., Taylor, H.R., and Caldwell, H.D. 1987. Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis.J. Immunol. 138: 575-581. (Received for publication, April 30, 1991)
Immunoelectron Microscopy of Chlamydia psittaci with Monoclonal Antibodies Shuji ANDO,* Ikuo TAKASHIMA,and Nobuo
HASHIMOTO
Department of VeterinaryPublic Health, Faculty of VeterinaryMedicine, Hokkaido University, Sapporo, Hokkaido 060, Japan
(Accepted for publication, November 8, 1991)
Abstract An immunoelectron microscopic study was performed to determine the distribution of antigenic components on particles of Chlamydiapsittaci and infected. cells using a number of monoclonal antibodies (MAbs). Of three anti-lipopolysaccharide (LPS) antibodies (4D5, A2 and 4G5), two antibodies (4D5 and A2) reacted with the surface of reticulate bodies (RBs) but not with that of elementary bodies (EBs). The other antibody (4G5) reacted with both EBs and RBs. Examination of infected cells in thin sections revealed that 4D5 and A2 combined with the membranes of both EBs and RBs. These results indicate that each LPS epitope localized at a different position in the chlamydial membrane. Most MAbs directed to protein antigens reacted on the surface of both EBs and RBs though 3E9 specific for the 90 kDa and 50 kDa protein components combined with RBs only.
Genus Chlamydia is an obligate intracellular parasite that shows a unique developmental cycle in which two distinct forms, an elementary body (EB) and a reticulate body (RB), exist (14). The outer membrane of EB is suggested to play an important role in the attachment to host cells and in the inhibition of phagosomelysosome fusion (4, 10). The surface distribution of antigenic components on the outer membrane of C. trachomatis was examined with respect to the neutralizing mechanism due to antibody and to the structural characteristics of the outer membrane (5, 8, 16, 17, 19, 23). Immunofluorescent assay, peroxidase- and ferritinlabeled immunoelectron microscopy have been used for this purpose (8, 17, 19). In recent years, the immunogold-labeling method for electron microscopy has been applied to antigenic analysis of the surface of C. trachomatisas well as to that of other micro-organisms (2, 9, 12). Kuo and Chi (12) and Collett et al (9) examined the surface distribution of epitopes in C. trachomatisusing several MAbs, and Birkelund et al (2) examined lipopolysaccharide (LPS) distribution on the C. trachomatis EB surface. These studies were performed mainly on LPS, major outer membrane protein (MOMP) and other cysteine-rich proteins, and it was suggested that the surface-exposed
MOMP
might
be responsible
for the neutralization
of C. trachomatis
infection. We previously reported the production and characterization of a number of MAbs against two C.psittaci strains (18, 22). Using these MAbs, epitope distribution in C. psittaci particles was examined by immunoelectron microscopy (IEM). 113
S. ANDO
114
MATERIALS
Organisms. was
Two
derived
from
Chlamydial tion,
organisms
and al
(15)
60
min
of
10 mm
.
at
HEPES at
30,000•~g
for
Percoll-HBS particles
30
at
min
staining
preparations To
(v/v)
Ltd.,
Monoclonal
reactivity
of by
1D4,
21, all
2F4 had
study. primary
3B5
common and
antibody.
(IgG-Gold-5, ZYMED
Immunoelectron was
carried
suspension copper grids (BSA),
out
were 1%
both
with
normal
mouse
at
applied
to
the
with
8 ƒÊl and
20
tests two
(18,
25
kDa
Izawa-1
fluid
(negative
Labs. by
the
et
al
in
The
containing (PBSAG)
used were
5 nm
For
10 min
in
purified
was bovine to
this as
diameter
Chlamydia of
membrane
(w/v)
in used
(IgG-Gold-15,
microliters
suspension
for
(2F4,
Some
P-1041,
of
0.1 %
MAbs (18).
diameter markers.
Eight
Crossstrain-
components.
particles
collodion
5 min.
1. and
control)
15 nm labeling
(9).
characterized Table
Some
and
colloidal or as
in
protein
Immunolabeling
Collett
NaN3
microand
components
strains,
U.S.A.) were used
been
21).
protein
two
PBS
trypsin a
(TAAB
subspecies-
the
IgG-gold
mm
had
subgenus-,
carbon-coated
of
the
IFU/ml.
(w/v)
provided
shown
and
for
2•~106 %
resin
are
kDa
observation. of
for
use. observation,
paraformaldehyde
MAbs
40
Inc., Calif., U.S.A.)
determined (3) and
in
instructions
MAbs
genus-,
ascitic
temperature
glycine
the
with
anti-mouse
method
room
incubated (w/v)
the
3%
g
ordinary
microscopy
centrifuged
K4M
the
antibody
reactions
of
All of
as
the
were
chla-
prepared.
reacted
microscopic by
were grids
with
of 0.1
at (v/v)
of
the
was G-250
a titer
by
30,000 •~
by
with
Lowicryl to
were
defined
F2)
E.Y. Labs. Inc., Calif.,
Labs.
in
according IEM
cells
mixture
immunoreagents.
and
Goat
a
embedding
properties
was
reacted
MAbs
with
immunofluorescent
3E9,
example,
The
band
C until microscopic
at
then
30%
electron
suspended
for
again
at
stained
at -80
organisms
HBS),
of
The
transmission
g
sedimented
ml
again
of the suspensions brilliant blue
were
EDTA.
for
The
MAbs
with
fixed
and
22).
a
Newhall
composed
7.4;
30
4 C.
HBS,
were stored immunoelectron
cells
England) sections
in
under
infected
and
at
10,000 •~
(7).
post-infec-
centrifuged in
min
at
was
was
centrifuged
suspended
infected
After
indirect
3F9,
MAbs
500•~g,
30
hr
buffer
pH
resuspended
and
48
by
of
debris
supernatant
protein content with Coomassie
(w/v)
antibodies
(18,
specific
were
5 ml
saline, cell
Izawa-1 pigeon
described
in
Host
for
HBS
examined
the
thin
previously
30,000 •~g in
as
suspended
was
protein content) sections for
Berkshire,
manufacturer,
pellet
Total assay
glutaraldehyde.
Equip.
The
were
and
purity. binding
0.02% at
the
twice
monolayers
tube
and
organisms
method
containing
min,
at
centrifuged
buffered
sec).
Strain a feral
harvested
and
were
study. from
gradient
(HEPES w,
this
derived
cells,
density
20
at
washed
post-inoculation,
centrifuge 1%
centrifuged
the
cell
hr
C.
229
in
harvested
NaCl
10
4
(200 ƒÊg/ml prepare ultrathin
229
PBS
at
HeLa
pellets
(75
for
Purified
to check protein-dye
in
min
4 C.
(TEM) by the
48
30
was
negative
At
500•~g
and
mydial
mm
settings
at
was
were
cell
145
low
used
P-1041
Percoll
cells
resultant
and
twice
were
and in
by
METHODS
psittaci
grown
infected
The
centrifugation
C.
AND
(13),
purified
Briefly,
4 C.
sonicated
of
were
partially
et
HeLa
strains
a budgerigar
ET AL
on
chlamydial
aspirated serum block
200-mesh and albumin non-specific
the
IMMUNOELECTRON Table
1.
MICROSCOPY Properties
of monoclonal
OF
C. PSITTACI
115
antibodies
binding. The grids were placed on a droplet (30 pl) of primary antibody solution in PBSA (PBSAG without glycine) for 30 min at 37 C, washed 10 times with PBSA and then put on a droplet (30 pl) of IgG-Gold-5 in PBSA for 30 min at 37 C. After careful washings with PBSA, the specimens were incubated with 0.05% (w/v) BSA in distilled water (pH 7.0) for 5 min, and then dried on a filter paper. Immunolabeling of ultrathin sections was performed as follows. The thin sections mounted on a grid were floated on a droplet (40 pl) of 1% (w/v) BSA in PBS for 2 hr at room temperature to block non-specific binding, and then placed on a droplet (40 pl) of primary antibody solution in PBS containing 1% (w/v) BSA for 2 hr at room temperature. After several washings with PBS, the grid was transferred onto a droplet (40 pl) of IgG-Gold-15 in 50 mm Tris, pH 7.4, 150 mm-NaC1 containing 1% BSA and 0.05% NaN3 and incubated for 2 hr at room temperature. After washings with PBS, the sample was fixed with 2.5% (v/v) glutaraldehyde in phosphate buffer (pH 7.4) for 10 min. The sample was washed with distilled water 5 times, air-dried, and then stained with uranyl acetate and lead nitrate solutions. The optimum concentrations of antibodies and IgG-gold were tested by box titration. Each MAb was serially diluted (1: 10, 1: 50, 1: 100, 1: 200, 1: 400, 1: 800 and 1: 1,600) and used in the IEM. IgG-gold and normal mouse ascitic fluid were diluted at 1: 20 and 1: 50 dilutions, respectively. RESULTS
Electron Microscopic Observations of Chlamydial Particles in Negative Stained and ThinSectioned Preparations Partially
purified
organisms
are
shown
in Fig.
la.
EEs
and
RBs
were
distin-
116
S. ANDO
Fig.
1.
Electron
staining HeLa
Fig.
of 229
2.
cells
Electron
antibodies. RB ascitic
micrographs
(b)
(bar,
(bars,
0.5 ƒÊm). as
a negative
organisms
P-1041
harvested
strain
(bar,
at
1 ƒÊm).
48 (b)
hr
post-infection.
Thin
section
(a)
Negative
of
P-1041-infected
after
reaction
1 ƒÊm).
micrographs Genus-specific
fluid
of
Percoll-purified
ET AL
of
Percoll-purified
anti-LPS Izawa-1 control
MAb strain (bars,
EB
chlamydial 4G5 (c)
was and
0.5 ƒÊm).
reacted RB
(d)
EBs with were
and
RBs
Izawa-1 reacted
strain with
EB
a normal
with (a)
and
mouse
IMMUNOELECTRON Table
Fig.
3. with (bars,
2.
Electron IgG-gold.
Reactivities
of monoclonal
micrographs
of chlamydial
Anti-MOMP
0.5 ƒÊm).
MICROSCOPY
MAb
3E9
MAb was
applied
OF
antibodies
EBs
4E11
was to
and reacted
Izawa-1
C. PSITTACI
in immunoelectron
RBs
after
with strain
reaction
Izawa-1 EB
(c)
microscopy
with strain
and
117
RB
MAbs EB
(a)
(d)
(bars,
visualized and
RB
(b)
0.5 ƒÊm).
guished by their size. Thin sections of infected HeLa 229 cells were prepared at 48 hr after infection. In this preparation, the EB/RB ratio in the inclusions was approximately 4: 1 (Fig. 1b). Immunocytochemistry for Pured To define
the specific
C. psittaci
reactions
between
organisms
and
antibodies,
the distribu-
118
S. ANDO
ET AL
Fig. 4. Electron micrograph of ultrathin section of P-1041-infected cells after reaction with a genus-specific anti-LPS MAb 4D5. EBs and RBs were labeled with IgG-gold (arrows) (bar, 0.5 ,um).
tion of the gold particles on isolated chlamydial particles was compared with that on the background on the chlamydial particles treated with normal mouse ascitic fluids (negative control), because gold particles conjugated with immunoglobulin tended to aggregate more often than protein-A gold (20). Eight examples of characteristic reaction patterns on the surface of EBs and RBs are shown in Figs. 2 and 3. The results of all antibodies used in this study are summarized in Table 2. The labeling distribution of genus-specific anti-LPS antibodies (4G5, 4D5 and A2) were divided into two patterns. MAb 4G5 bound over a large part of the surface of RBs but the labelings on EBs were partially localized on the surface in both strains (Fig. 2, a and b, Table 2). In contrast, 4D5 and A2 only reacted with the RB surface, but not with the EB surface. No labeling was seen on the organisms treated with normal mouse ascitic fluid (Fig. 2, c and d). All protein-directing MAbs (4F8 and others) except 3E9 apparently reacted with the surface of both EB and RB particles (Table 2, Fig. 3, a and b). In contrast, a subgenus-specific anti-Izawa-1 MAb, 3E9, showed a different reaction pattern from other protein-indirecting MAbs; 3E9 apparently combined only with the surface of RBs, but not with the surface of EBs (Fig. 3, c and d). Immunoreactionof EBs and RBs in Thin Sections Epitopes corresponding to two genus-specific anti-LPS MAbs (4D5 and A2) were not observed on the surface of purified EB particles (Table 2). Therefore, to examine the presence of LPS epitopes in in situ chlamydial particles, IEM was performed on thin sections of infected cells (Table 2, Fig. 4). The MAbs, 4D5 and A2, evidently reacted with EBs and RBs in the inclusions. Gold particles were located on the membranes of EB and RB particles and also within the particles (Fig. 4). Binding of gold particles was rarely seen in the cyto-
IMMUNOELECTRON
MICROSCOPY
OF
C. PSITTACI
119
plasm surrounding the chlamydial inclusions and no reaction was seen on the thin section treated with normal mouse ascitic fluid (data not shown). These results may indicate that the binding of gold particles to chlamydial particles is specific. DISCUSSION
In this study, the localization of chlamydial antigenic components was analyzed with immunoelectron microscopy. The LPS epitopes of C. psittaci recognized with three anti-LPS MAbs were divided into two groups depending on the labeling patterns. MAbs 4D5 and A2 reacted with RBs but not with EBs when MAbs were applied to intact organisms. In contrast, 4G5 combined with the surface of RB particles and partially with the EB surface. Observations of the infected cells in thin sections demonstrated that LPS epitopes reactive with 4D5 and A2 were present in EB particles. Specificity of the reaction on thin section was supported by the gold distribution limited in chlamydial particles and no labeling in the negative control. Therefore, these results strongly suggest that in situ EBs possess LPS perhaps in the membrane, but the epitopes reactive with 4D5 and A2 are not exposed on the EB surface. Birkelund et al (2) showed that anti-LPS MAbs bound strongly on the surface of formalin-fixed EB and that LPS readily dissociated from the surface of unfixed EBs. In contrast, Collett et al (9) did not detect the LPS antigen on the surfaces of C. trachomatisEB particles using anti-LPS MAbs and suggested that the LPS moiety was not exposed on the EB surface. This confusion, as to the localization of the LPS moiety on the EB surface, might be due to different MAbs used. Chlamydial LPS has at least three antigenic domains (6). Therefore, the MAbs used in this study might recognize different domains of the LPS moiety because the reaction patterns were divided into two groups. The epitopes corresponding to 4G5 were exposed on a limited part of the EB surface, but the epitopes bound with 4D5 and A2 were rarely detected on the EB surface. In contrast, LPS epitopes corresponding to these three MAbs (A2, 4D5 and 4G5) were detected over a wide area of the RB surface. Therefore, there are different modes for integration of the LPS moiety in the RB and EB membranes. One domain might be exposed on the EB surface, while the other might be concealed in the EB membrane. Karimi et al (11) reported the accumulation of chlamydial LPS antigen in the plasma membranes of infected cells, and suggested that LPS antigen was transported from inclusions to the host plasma membranes. However, gold particle in the cytoplasm and plasma membrane was never encountered in the thin sections in the present study. One of 11 MAbs directed to chlamydial protein, 3E9, showed a reaction pattern similar to two anti-LPS MAbs (4D5 and A2). Epitopes of 3E9 were detected on the RB surface but not on the EB surface. We examined the kinetics of the antigen appearance in the infected cells and revealed that antigens corresponding to 3E9 and LPS antigens were detected throughout the growth cycle of Izawa-1 and P-1041 strains (1). Therefore, these antigens may be essential for the chlamydial growth.
120
S. ANDO
ET AL
The protein antigens recognized by most of the anti-protein MAbs except 3E9 were exposed on the surfaces of both RB and EB particles. The neutralization of C. trachomatis was reported to be attributed to the surface-exposed epitopes on MOMP (23). However, it has not been clear how neutralization of C. psittaci infection takes place. Therefore, it would be of special interest to examine whether MAbs against surface-exposed epitopes of EB have neutralizing ability. REFERENCES
1) Ando, S., Suwa, T., Takashima, I., and Hashimoto, N. 1991. Kinetic studies on the appearance of antigens of Chlamydiapsittaci during its developmental cycle. J. Vet. Med. Sci. 53: 691-697. 2) Birkelund, S., Lundemose, A.G., and Christiansen, G. 1989. Immunoelectron microscopy of lipopolysaccharide in Chlamydiatrachomatis. Infect. Immun. 57: 3250-3253. 3) Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytic. Biochem. 72: 248-254. 4) Byrne, G.I., and Moulder, J.W. 1978. Parasite-specified phagocytosis of Chlamydiapsittaci and Chlamydia trachomatisby L and HeLa cells. Infect. Immun. 19: 598-606. 5) Caldwell, H.D., and Perry, L. J. 1982. Neutralization of Chlamydia trachomatis infectivity with antibodies to the major outer membrane protein. Infect. Immun. 38: 745-754. 6) Caldwell, H.D., and Hitchcock, P. J. 1984. Monoclonal antibodies against a genus-specific antigen of Chlamydia species: location of the epitope on chlamydial lipopolysaccharide. Infect. Immun. 44: 306-314. 7) Chiba, N., Arikawa, J., Takashima, I., and Hashimoto, N. 1984. Isolation and serological survey of chlamydiosis in feral pigeons and crows in Hokkaido. Jpn. J. Vet. Sci. 46: 243-245. 8) Clark, R.B., Nachamkin, I., Schatzki, P.F., and Dalton, H.P. 1982. Localization of distinct surface antigens on Chlamydia trachomatisHAR-13 by immune electron microscopy with monoclonal antibodies. Infect. Immun. 38: 1273-1278. 9) Collett, B.A., Newhall V, W. J. , Jersil Jr., R.A. , and Jones, R. B. 1989. Detection of surface-exposed epitopes on Chlamydiatrachomatisby immune electron microscopy. J. Gen. Microbiol. 135: 85-94. 10) Eissenberg, L.G., Wyrick, P.B., Davis, C.H., and Rumpp, J.W. 1983. Chlamydiapsittaci elementary body envelopes: ingestion and inhibition of phagolysosome fusion. Infect. Immun. 40: 741-751. 11) Karimi, S.T., Schloemer, R.H., and Wilde III, C.E. 1989. Accumulation of chlamydial lipopolysaccharide antigen in the plasma membranes of infected cells. Infect. Immun. 57: 17801785. 12) Kuo, C.-C., and Chi, E.Y. 1987. Ultrastructural study of Chlamydiatrachomatissurface antigens by immunogold staining with monoclonal antibodies. Infect. Immun. 55: 1324-1328. 13) Matsumoto, A., Bessho, R., Soejima, R., and Hino, J. 1984. Biological properties of a Chlamydia strain isolated from a pet bird, budgerigar, which was kept by a psittacosis patient. Kawasaki Med. J. 10: 77-90. 14) Moulder, J., Hatch, T.P., Kuo, C.-C., Schachter, J., and Storz, J. 1984. Chlamydia, p. 729-739. In Krieg, N.R., and Holt, J.G. (eds), Bergey's manual of systematic bacteriology, Vol. 1. The Williams and Wilkins Co., Baltimore. 15) Newhall V, W. J., Batteiger, B., and Jones, R.B. 1982. Analysis of the human serological response to proteins of Chlamydia trachomatis. Infect. Immun. 38: 1181-1189. 16) Peeling, R., Malean, I.W., and Bruham, R.C. 1984. In vitro neutralization of Chlamydia trachomatis with monoclonal antibody to an epitope on the major outer membrane protein. Infect. Immun. 46: 484-488. 17) Richmond, S. J., and Stirling, P. 1981. Localization of chlamydial group antigen in McCoy cell monolayers infected with Chlamydiatrachomatis or Chlamydiapsittaci. Infect. Immun. 34: 516-570. 18) Seki, C., Takashima, I., Arikawa, J., and Hashimoto, N. 1988. Monoclonal antibodies to Chiamydia psittaci: characteristics and antigenic analysis. Jpn. J. Vet. Sci. 50: 383-393.
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19) Stephens, R.S., Tam, M.R., Kuo, C.-C., and Nowinski, R.C. 1982. Monoclonal antibodies to Chlamydia trachomatis: antibody specificities and antigen characterization. J. Immunol. 128: 1083-1089. 20) Stot, J.W., and Genze, H. J. 1984. Gold markers for single and double immunolabelling of ultrathin cryosections, p. 129-142. In Polak, J.M., and Varndell, I.M. (eds), Immunolabelling for electron microscopy, Elsevier Science Publishers B.V., Amsterdam. 21) Takahashi, T., Takashima, I., and Hashimoto, N. 1988. Immunotyping of Chlamydiapsittaci by indirect immunofluorescence antibody test with monoclonal antibodies. Microbiol. Immunol. 32: 251-263. 22) Toyofuku, H., Takashima, I., Arikawa, J., and Hashimoto, N. 1986. Monoclonal antibodies against Chlamydiapsittaci. Microbiol. Immunol. 30: 945-955. 23) Zhang, Y.-X., Stewart, S., Joseph, T., Taylor, H.R., and Caldwell, H.D. 1987. Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis.J. Immunol. 138: 575-581. (Received for publication, April 30, 1991)
