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Journal of General Microbiology (1 990), 136, 1091-1097.
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1091
Yersinia enterocolitica immunodominant 60 kDa antigen, common to a
broad range of bacteria, is a heat-shock protein
HIROYUKI
YAMAGUCHI,
TOMOKO
YAMAMOTO,"
HARUHIKO
TAGUCHI
and SACHIO
OGATA
Department o j Microbiology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181, Japan
(Received 14 November I989 ;revised 8 February I990 ;accepted I9 February 1990)
Monoclonal antibodies(mAbs) against the Yersiniuenterocolitica immunodominant 60 kDa antigen, termed crossreacting protein antigen (CRPA), were obtained by fusion of spleen cells from mice immunized with CRPA with
murine myeloma cells. The reactivities of the mAbs were examined by Western blotting against extracts of Y.
enterocolitica and 23 other species of Gram-positiveand Gram-negativebacteria. Cross-reactions were recognized
with a wide range of bacteria, but not with Gram-positive cocci. The reactivities were different for each mAb,
suggesting that both species-specific and multiple cross-reactive epitopes were present on the CRPA molecule.
CRPA was produced under heat-shock conditions in Y. enterocolitica and was shown to correspond
immunologically to the GroEL protein in Escherichia coli, a protein involved in the morphogenesisof coliphage. In
addition to CRPA, at least nine other major heat-shock proteins were detected by two-dimensional gel
electrophoresis of extracts of heat-shocked Y. enterocolitica.
Introduction
A 60 kDa protein antigen is shared by a wide range of
bacteria (Ogata et al., 1987). This antigen, designated
cross-reacting protein antigen (CRPA), was originally
identified as an immunodominant protein which was
recognized by IgG antibodies in the sera of patients with
yersiniosis and in those of mice experimentally infected
with Yersinia enterocolitica (Ogata et al., 1978, 1987).
Anti-CRPA IgG antibodies were also detected in sera of
healthy humans and the frequency of their detection
increased with age (Ogata et al., 1987). By Western
blotting analysis, human anti-CRPA sera were shown to
react with a 60 kDa protein in crude antigen fractions
prepared from Gram-negative bacteria such as Y.
enterocolitica, Vibrio cholerae, Pseudomonas aeruginosa,
ShigeIIa sonnei, Proteus mirabilis and Klebsiella pneumoniae (Yamaguchi et al., 1986). CRPA has been purified to
homogeneity by immuno-affinity chromatography using
a specific monoclonal antibody (mAb) (Yamaguchi et
al., 1989). It has a native molecular mass of 400500 kDa with subunits of 60 kDa (Ogata et al., 1987;
Yamaguchi et al., 1989). The purified Y. enterocolitica
CRPA cross-reacts with rabbit antisera against Y .
Abbreuiations : CA, common antigen ; CRPA, cross-reacting protein
antigen; mAb, monoclonal antibody.
enterocolitica, V . cholerae, Escherichia coli, Pseudomonas
aeruginosa and S. sonnei (Yamaguchi et al., 1989).
CRPA is different from Kunin's enterobacterial
common antigen (Kunin, 1963),which is an amino sugar
heteropolymer, and from Hofstra's common antigen
(Hofstra & Dankert, 1979), which is found in the outer
membrane of Gram-negative bacteria (Yamaguchi et al.,
1986). However, the 62 kDa antigen originally identified
in Pseudomonas has been shown to be an antigen
common to Gram-negative bacteria, the so-called common antigen (CA) (Hleriby, 1975; Jensen et al., 1985;
Sompolinsky et al., 1980). Recently, the 65 kDa protein
antigen of Mycobacterium tuberculosis, which is one of
the major immunologically active mycobacterial antigens following infection and immunization, was demonstrated to correspond to CA and to the GroEL protein of
E. coli, a protein required for the morphogenesis of
coliphage (Shinnick et al., 1988). Because of the
reactivity of purified CRPA with antisera against Gramnegative bacteria, including P. aeruginosa, a common
epitope might be shared between CRPA and CA. In
order to define more closely the antigenicity of CRPA
and its cross-reactivity with protein antigens found in
other bacteria, including the GroEL protein of E. coli,
mAbs were produced and characterized in the present
study. Since the GroEL protein is a heat-shock protein,
the production of CRPA under heat-shock conditions in
Y. enterocolitica was also investigated.
0001-5931 O 1990 SGM
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H. Yamaguchi and others
Methods
Bacteria and crude antigen extracts. All bacteria (Table 1) used in this
study were from the Department of Microbiology strain collection,
Kyorin University School of Medicine. Cultures grown exponentially
were harvested and then washed with phosphate-buffered saline (PBS),
pH 7.2 (0.14 M-NaCl in 20 mM-sodium phosphate buffer, pH 7-2).Cells
were resuspended in PBS and treated with an ultrasonic disintegrator
UR-200P (Tomy Seiko Co.) for 20 min at 20 kHz and then centrifuged
at lOOOOg for 1 h. The supernatant fluids (antigen extracts) obtained
were lyophilized and stored at - 20 "C.
Media. Media used were Heart Infusion agar (Wako Co.) containing
10% (v/v) sheep blood for Streptococcus, Chocolate agar containing
10% sheep blood for Neisseria, Haemophilus and Campylobacter, 1%
(w/v) Ogawa medium (Eiken Co.) for Mycobacterium, and Heart
Infusion broth for other bacteria. M9 minimal medium for labelling of
bacteria with [35S]methioninewas prepared as described by Maniatis et
al. (1982) and was supplemented with 0.3% glucose, 2 yg thiamin ml-l,
0.03 M-MgSO,, 3 m~-MgCl,, 0.1 M-CaCI,, 3 yM-FeCl,, and all Lamino acids except cysteine and methionine.
CRPA and anti-CRPA serum. CRPA was purified from Y.
enterocolitica 0 :3 (strain ZM20) by immuno-affinity chromatography
using a mAb (lA4) as previously reported (Yamaguchi et al., 1989).
Anti-CRPA serum was obtained from rabbits immunized with purified
CRPA from Y. enterocolitica.
GroEL protein and anti-GroEL serum. The purified GroEL protein
from E. coli and rabbit anti-GroEL serum were kindly provided by C.
Georgopoulos, Department of Cellular, Viral and Molecular Biology,
College of Medicine, University of Utah, Salt Lake City, Utah, USA.
Production of mAbs. BALB/c mice were injected intraperitoneally
with partially purified CRPA mixed with Freund's complete adjuvant
(Difco). Ten days later, the mice were injected intraperitoneally with
CRPA suspended in Freund's incomplete adjuvant (Difco). This
immunization step was repeated three times at ten-day intervals.
Finally, a booster injection was given intravenously using only partially
purified CRPA. Three days after the final injection, the spleens of the
mice were removed and the spleen cells were fused with murine
myeloma cells (P3-X63-Ag8-U1) following the procedure described by
Kohler & Milstein (1975). The hybridomas producing apparently
specific mAbs against the 60 kDa CRPA were identified by enzymelinked immunosorbent assay (ELISA) as described by Engvall &
Perlmann (1972), and cloned twice by limiting dilution. The mAb
preparations used for Western blot analysis were culture supernatant
fluids of the hybridomas.
SDS-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blot
analysis. SDS-PAGE (12.5 %, w/v, acrylamide) was carried out as
described by Laemmli (1970). Crude antigen (25 pg) was loaded in each
lane. Western blot analysis was carried out as described by Towbin et
al. (1979). After electrophoresis, the separated proteins were transferred to nitrocellulose membranes (Schleicher & Schuell) at 0.25 A
overnight. After blocking with 3% (w/v) gelatin in Tris-buffered saline
(0.15 M-NaCl, 10 mM-Tris/HCl, pH 7-4),the membranes were sequentially treated for 1 h with rabbit anti-CRPA serum (1 :lOOO), rabbit
anti-GroEL serum (1 :1000) and each mAb (1 : lo), each diluted in
blocking buffer. They were then incubated with goat anti-rabbit IgG
(1 :500) or goat anti-mouse IgG, IgM and IgA peroxidase conjugate
(1 :500) (Miles Yeda) for 1 h. Dilutions were made in Tris-buffered
saline containing 0-05% Tween 20 (Sigma) and 1% (w/v) BSA (Sigma).
Immunoblots were developed with Tris-buffered saline containing
H 2 0 2and o-dianisidine (Sigma).
Two-dimensional gel electrophoresis. Isoelectric focusing in the first
dimension (1.6%, v/v, Ampholines pH 54-7-0; 0.4%, v/v, Ampholines
pH 3-5-10) was performed according to the procedure of O'Farrell
(1975) as modified by Georgopoulos et al. (1982). SDS-PAGE in the
second dimension was performed as described above.
Heat-shock response. Y . enterocolitica 0 :3 (strain ZM20) was grown
to the mid-exponential phase in M9 medium at 20°C. Cells were
harvested by centrifugation and resuspended at lo8 cells ml-l in M9
medium pre-warmed to the required temperature. Of this suspension,
1 ml each was incubated at either 20 or 42 "C for 10 min, and then
labelled with 50 pCi (18.5 MBq) of [35S]methionine for 5 min at the
respective temperature. Cells were harvested by centrifugation, and
resuspended in 100 y1 of lysis buffer [1.5 M-urea, 0.4 mhl-2-mercaptoethanol, 3.2% (v/v) Nonidet P-40, 8% (v/v) Ampholines pH 5.0-7.0,
1.6% (v/v) Ampholines pH 3.0-101, lysed by seven freeze-thaw cycles
(frozen at - 80 "C and thawed at 37 "C in a water-bath, each for 5 min)
and stored at -80 "C.
Results
Species specificity of CRPA mAbs
Screening of hybridoma supernatant fluids for reactivity
with the CRPA from Y. enterocolitica (Yamaguchi et al.,
1989) yielded four clones. Table 1 shows the reactivities
of these mAbs, termed lA4, 5C1, 5C3 and 3C8, in
Western immunoblotting against crude antigen extracts
of 24 bacterial species.mAb 3C8, which had the broadest
reactivity, recognized a band of 60-65 kDa in crude
antigen extracts from 19 other bacterial species comprising Gram-negative rods, Gram-negative cocci and
Gram-positive rods. Since this pattern of reactivity was
similar to that with anti-CRPA polyclonal antibodies,
mAb 3C8 may recognize a framework epitope on the
60 kDa CRPA. mAb lA4, which had previously been
used for immuno-affinity chromatography to purify the
CRPA (Yamaguchi et al., 1989), recognized only the
60 kDa CRPA from Y. enterocolitica, indicating that this
mAb recognized a species-specific epitope. The reactivity of mAb 5Cl saggests that this mAb recognized a
shared epitope present on CRPA of Y . enterocolitica and
its equivalent protein in V . cholerae.
Induction of CRPA under heat-shock conditions in
Y . enterocolitica
Y. enterocolitica can grow over a wide range of
temperatures (Gronberg & Kihlstrom, 1989). Balanced
growth can be sustained even at 4 "C. During the search
for optimal conditions for CRPA production prior to
protein purification, it was observed that the amount of
CRPA in Y. enterocolitica was apparently greater, as
judged by Western immunoblotting, in the crude antigen
fraction of cells grown at 37 "C or 42 "C rather than at
25 "C, which is the conventional temperature for
cultivation of Y. enterocolitica (data not shown). To
determine whether the apparently enhanced production
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Y . enterocolitica 60 k D a heat-shock protein
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Table 1. Reactivities of bacterial extracts in Western immunoblotting with mAbs against
CRPA and rabbit anti-CRPA serum
Reaction* with
mAbs:
Bacterial extract
Yersinia enterocolitica
Vibrio cholerae
Serratia marcescens
Shigella sonnei
Proteus mirabilis
Proteus idgaris
Salmonella enteritidis
Klebsiella oxytoca
Vibrio alginolyticus
Vibrio parahaemolyticus
Pseudomonas aeruginosa
Pseudomonas maltophilia
Enterobacter cloacae
Citrobacter jreundii
Escherichia coli
Enterobacter aerogenes
Neisseria gonorrhoeae
Haemophilus influenzae
Flavobacterium sp.
Mycobacterium tuberculosis
Coagulase-negative Staphylococcus
Staphylococcus aureus
Streptococcus pyogenes
Streptococcus pneumoniae
*
1A4
5C 1
5c3
3C8
Rabbit
anti-CRPA
serum
+-
+
+-
+
+
+
+
+
+
+
+
+
+
+
+
+-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+ denotes a reacting protein band of 60-65 kDa; - denotes no reacting band in this size range.
of CRPA at elevated temperatures was based on a heatshock response, the proteins induced after heat shock in
Y . enterocolitica were examined. At least ten major
proteins were detected in response to heat shock as early
as 15 min after the transfer of the cells to 42 "C (Fig. 1a ,
b). These ten heat-shock proteins ranged in size from 7-5
to 80 kDa. In Western immunoblotting, anti-CRPA
polyclonal antibodies recognized two of these heat-shock
proteins, which were identical in molecular mass (Fig.
1 c, d), suggesting that CRPA was post-translationally
modified. CRPA was present in cells grown at 20 "C, but
its synthesis was enhanced at 42 "C (Fig. 1 a , b). These
results demonstrate that CRPA is one of the heat-shock
proteins in Y . enterocolitica.
In E . coli, there is a set of 17 heat shock proteins, one of
which is the GroEL protein (Neidhardt et al., 1984;
VanBogelen et al., 1987).The fact that CRPA is found in
the same relative migratory position in two-dimensional
gels as the GroEL protein of E. coli, coupled with the fact
that the native molecular mass of both proteins is
> 500 kDa, suggested to us that they might be similar or
identical proteins (Hendrix, 1979). To confirm this
possibility, the reactivity of the purified GroEL protein
from E. coli and the purified CRPA from Y. enterocolitica
with both anti-GroEL and anti-CRPA sera in Western
immunoblotting was determined (Fig. 2). The blotting
patterns with polyclonal antibodies indicated that the
CRPA and GroEL proteins shared common epitopes.
The possible divergence of epitopes between the GroEL
protein and CRPA was also investigated using the four
mAbs produced against CRPA. The broadly reactive
mAbs 3C8 and 5C3, but not the more specific mAbs lA4
and 5C1, recognized epitopes on the GroEL protein
(results not shown).
Discussion
In the present study, species-specific and cross-reactive
epitopes have been demonstrated on the 60 kDa CRPA
molecule. The patterns of reactivities of the mAbs with
extracts of heterologous bacterial species (Table 1)
suggest that CRPA has multiple cross-reactive epitopes
shared variously by a wide range of Gram-negative and
some Gram-positive bacteria. It may be possible to
develop a test for diagnosis of infection with Y.
enterocolitica based on the identification of its CRPA by
mAb 1A4. However, the reactivity of mAb lA4 with
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H . Yamaguchi and others
Fig. 1. Two-dimensional gel electrophoresis of heat-shocked Y . enterocolitica extracts and Western immunoblot analyses with mAb
1 A4. Bacterial cells were incubated for 10 min at 20 "C (a, c) or at 42 "C (b, d ) , and then labelled with [3SS]methioninefor 5 min at the
respective temperature. Following electrophoresis of the extracts, as described in Methods, the separated proteins were transferred to
nitrocellulose filters and reacted with mAb 1A4 (c, d ) . Subsequently, the filters were exposed to Fuji X-ray film (a, b). The open arrow in
each gel points to the position of the protein which reacted with the monoclonal antibody. The filled arrows point to the positions of the
other heat-shock proteins, whose rates of synthesis are higher in the cells labelled at 42 "C.
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Y. enterocolitica 60 kDa heat-shock protein
Fig. 2. Reactivities of the CRPA of Y . enterocolitica and the GroEL
protein of E. coli with anti-CRPA and anti-GroEL polyclonal
antibodies by Western blotting analysis. After transfer to nitrocellulose, the immobilized proteins were reacted with anti-CRPA antibody
(lanes 1 and 2) or with anti-GroEL protein antibody (lanes 3 and 4). In
the initial SDS-PAGE, lanes 1 and 3 were loaded with purified CRPA
(0.6 pg protein) from Y . enterocolitica, and lanes 2 and 4 were loaded
with purified GroEL protein (2.5 pg protein) from E. coli.
CRPA from all Y. enterocolitica serotypes needs to be
established. Information is available about several crossreacting protein antigens in diverse bacterial species, viz.
the 62 kDa protein which has been called CA in
Pseudomonas aeruginosa (Hoiby, 1975; Jensen et al.,
1985; Sompolinsky et al., 1980), the 65 kDa mycobacterial protein (Shinnick, 1987),the 60 kDa Borrelia burgdorferi protein (Hansen et al., 1988), Coxiella burnetii protein
(Vodkin et al., 1988)and the 60 kDa antigen in Legionella
(Plikaytis et al., 1987). Since the most broadly reactive
mAb 3C8 recognized both a 65 kDa protein in M .
tuberculosis and a 62 kDa protein in P. aeruginosa (Table
1095
l), it is likely that CRPA is related to CA, although this
has to be proven by showing cross-reactivity between CA
and CRPA.
In Mycobacterium bouis, the 65 kDa antigen is one of
the major immunologically active antigens following
infection or immunization and is able to elicit a strong
delayed-type hypersensitivity reaction in experimental
animals (Bruyn et al., 1987). In addition, it stimulates the
proliferation of T cells in immune mice, suggesting that it
is likely to be involved in the development of cellmediated immunity (Kaufmann et al., 1987). In contrast,
Willem et al. (1985) suggested that arthritis was induced
by a T-cell clone which recognized an epitope on the
mycobacterial 65 kDa antigen.
The development of the arthritis-like Reiter’s syndrome is often preceded by various bacterial infections,
e.g. with Y . enterocolitica, Shigella jlexneri, Salmonella
typhimurium, K . pneumoniae, Campylobacter jejuni and
Chlamydia trachomatis (Calin, 1984). Ogasawara et al.
(1986) reported that the 60 kDa and 80 kDa antigens of
K . pneumoniae reacted with anti-HLA-B27 mAb and that
lymphocytes from chronic arthritis patients who were
HLA-B27-posit i ve reacted with anti-K . pneumoniae antibodies. These findings suggest that the broadly conserved bacterial 60-65 kDa antigens such as CA, CRPA
and mycobacterial65 kDa protein may play a role in the
development of auto-immune disease which is preceded
by bacterial infection.
In the present study, we have shown that CRPA is one
of the proteins induced by the heat-shock response in Y.
enterocolitica and that it corresponds immunologically to
the GroEL protein, a major heat-shock protein in E. coli
(Neidhardt et a/., 1984). The mycobacterial 65 kDa
antigen has also been shown to be related to the GroEL
protein (Shinnick et a/., 1988). Although the role, if any,
of the GroEL protein in the heat-shock response has not
been elucidated, it is known to be involved in the
morphogenesis of coliphage (Friedman et al., 1984) and
also to be essential for E. coli growth (Fayet et al., 1989).
Heat-shock proteins are synthesized in response to
various environmental stresses including a sudden
increase in temperature and are highly conserved among
prokaryotic and eukaryotic organisms (Lindquist, 1986;
Neidhardt et al., 1984). The possible induction of heat
shock in pathogens when a bacterial infection occurs and
the consequent release of cross-reacting antigens like
CRPA, CA and mycobacterial antigen may result in a
strong immune response to these proteins because of
possible previous encounters with the same epitopes.
In E. coli, approximately 17 heat-shock proteins are
found which are diverse with respect to size, net charge,
cellular abundance, and the extents of their induci bilities
by heat shock (Neidhardt et a/., 1984). Ten of these heatshock proteins are the products of known genes and have
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H . Yamaguchi and others
been characterized to a greater or lesser extent (Neidhardt & VanBogelen, 1987). We found that ten proteins
ranging in size from 7.5 to 80 kDa were induced under
heat-shock conditions in Y . enterocolitica (Fig. 1).
Yersinia spp. harbour plasmids ranging in size from 60
to 75 kb which are necessary for virulence (Gemski et al.,
1980;Ben-Gurion & Shafferman, 1981). These plasmids
are associated with a number of temperature-inducible
features of the bacteria, including production of V and W
antigens (Burrows & Bacon, 1960), autoagglutination
(Shurnik et al., 1984), and the expression of outermembrane proteins in the absence of Ca2+(Bolin et al.,
1982, 1988). It is possible that these temperatureinducible proteins are virulence attributes for pathogenic
Yersinia spp.
A detailed analysis of the heat-shock response in Y .
enterocolitica is required to identify and characterize
these gene products.
We thank C. Georgopoulos for providing purified GroEL protein
and anti-GroEL antiserum.
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