Brucellosis is an important and widespread zoonotic disease which
is caused by the genus of Brucella. Brucella melitensis and Brucella
abortus are the two most common causative agents of Brucellosis in
both human and cattle (Yang, 1995; Corbel, 1997). The pathogenicity of
B. melitensis is generally higher than B. abortus for human.
Brucellae are facultative intracellular and gram negative coccobacilli
which usually survive within phagocytic cells such as macrophages (Lin
and Ficht, 1995; Baloglu et al., 2000). They do not possess spores
or elaborate any known exotoxins (Jawets et al., 2003). Their pathogenicity
is the result of a multi-factorial phenomenon. Environmental growth and
intracellular conditions of the bacteria can significantly influence cellular
proteins level expression. Host-parasite interactions during natural infections
will expose the bacteria to many physical and biological stresses (Lin
et al., 1992). Phagocytosed and intracellular bacteria survive
by adopting themselves to the extreme conditions of lysosome and intracellular
environmental changes. This adaptation will prevent bacterial lysis by
the intracellular defense mechanisms of the phagocytic cells. When Brucella
sp. are exposed to elevated temperatures or heat shocks, they began to
synthesize heat shock proteins that not only can help the bacteria to
survive high temperatures; but also, can play a potentiating role in the
pathogenesis of the bacteria. Heat shock proteins (hsps) produced by different
bacteria are highly conserved and probably allow adaptation of the producing
bacteria to many stressful conditions. Some of these hsps, members of
Gro EL family, are readily recognized by the host immune system during
infections and the ensuing immune response can have a protective role
in the host. The most important member of the Gro EL hsp family is a 62
kDa protein (hsp 62) (Yilan et al., 1998).
In the present study, B. melitensis and B.
abortus strains isolated from human and cows were exposed to different
heat shocks. Total bacterial protein profiles of the shocked and the control
B. melitensis and B. abortus isolates were analyzed by sodium
dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Hsp60
of shocked and un-shocked bacteria were identified by western blotting
using anti-hsp60 specific antibodies. The acute phase sera of human patients,
who were naturally infected with Brucella sp., were isolated for
western blotting and serological tests such as wright, combs wright, 2ME,
Brucella IgG and Brucella IgM were performed for each sera
sample. The presence of anti-hsp60 antibodies and other anti-Brucella
protein antibodies in the acute phase sera of human patients and control
sera were analysed by western blots.
MATERIALS AND METHODS
Isolation and culture of bacterial strains: This study were performed in Microbiology Department,
School of Medicine, Iran University of Medical Sciences and Health Services,
Tehran, Iran in 2006. Five strains of B. abortus and five strains
of B. melitensis were isolated from cows and human, respectively
and were cultured on Brucella agar plates. Five milliliters blood
samples were taken from each individual and promptly cultured in Brain
Heart Infusion (BHI) broth at 37°C under 5% CO2-air. The
media were incubated for 4 week, during which 5 sub-cultures (days 3,
7, 14, 21 and 28) were made onto solid medium Brucella agar containing
5% sheep blood. The Brucella agar plates were incubated at 37°C
under 5% CO2-air for 48-72 h. Any colony seen was subjected
to a gallery of biochemical tests such as Gram stain, Methyl red, Vogous
Proskauer, TSI, urease, oxidase, catalase tests and growth on media containing
different concentration of basic fuschin and thionine.
Serum samples: Two milliliters of serum samples were taken from
brucellosis patient and control group and stored at -20°C. The serological
tests were performed for all the sera.
Heat shocks: The bacterial colonies were transferred into 4 different
tubes containing Brucella broth and incubated with shaking at 37°C
until the optical density (at 600 nm) reached to 1.0 (Gomes et al.,
2000). After incubation, the tubes were placed at 37, 39, 40 and 42°C
for 3 h. The bacterial suspensions were centrifuged at 5000 x g for 30
min. The pellets were washed twice with normal saline and once with 10
mM Tris-base buffer (pH 7.5). The washed cells were resuspended in 2 mL
Tris-base buffer and subsequently, 10 mL of cold acetone was added to
each tube on ice. The tubes were stored at -20°C for 5-7 days, after
which they were centrifuged at 5000 x g for 30 min. The supernatants were
decanted and the killed bacteria were allowed to dry and then stored at
Protein extraction: The bacterial proteins were extracted according
to the method of Rosenbakh and his Colleques (Abdolalizadeh et al.,
2002) with some modifications. The bacterial cells were washed with Tris-base-saline
buffer (20 mM Tris-base, 100 mM NaCl; pH 7.5). The wet bacterial pellets
(0.16 g) were resuspended in 640 μL of TSB (Tris buffered saline).
PMSF (Phenyl Methane Sulfonyl Fluoride) and EDTA (Ethylene Diamine Tetra
Acetic acid) were added each at final concentration of 1 mM. Subsequently,
4.8 mL of lysis buffer (Tris-base buffer containing 2% SDS, 10% glycerol
and 0.7 M 2ME at pH 7.2) was added to each tube and incubated at 60°C
for 4 h. After cooling the tubes to room temperature, lysosyme (1 mg for
each 100 mg of dry weight) was added and incubated at 37°C overnight.
The cell suspensions were plated in boiling water for 10 min and then
centrifuged at 30000 x g for 30 min. The supernatant was divided into
aliquots and kept at -20°C.
Electrophoresis: SDS-PAGE was performed in 13.5% resolving and 5%
stacking polyacrylamide slab gels (Abdolalizadeh et al., 2002).
The bacterial protein extracts were mixed with equal volume of SDS-PAGE
sample buffer and incubated for 10 min at 100°C. Fifteen microliter
of each sample was loaded into each well. Electrophoresis was performed
at constant voltage (150 volts) and the gels were stained with Coomassie
brilliant blue. The stained gels were stored in 10% acetic acid solution.
Immunoblotting: Western blotting was preformed according to the
method of Towbin (Mostafaie, 1999) using transfer buffer (Tris-base 25
mM, glycin 192 mM and methanol 15% with pH 8.3) and PVDF membrane. A total
of 10 sera collected from the patients with brucellosis who had wright
titers of ≥1:320 and 2ME titers of ≥1:160 were used. PVDF membranes
were rinsed with methanol for 5 min and then used for transfer. Transfer
of protein bands from SDS-PAGE gel into PVDF membrane was carried out
at 75 mA for 30 min, 100 mA for 30 min, 200 mA for 90 min and finally
300 mA for 90 min. Following transfer, the membranes were blocked in PBS
containing 0.5% tween20, then washed three times PBS containing tween20
0.05% (PBS-T). The membranes were incubated with anti-hsp60 antibody (1/4000)
or human sera (1/50) for 90 min at room temperature. After washing of
the membranes four times with PBS-T, the secondary antibody conjugated
with HRP (1/4000 for anti-human Igs and 1/4000 For anti-rabbit IgG) was
added for 90 min. After washing four times with PBS-T, the membranes were
incubated in enzyme substrate solution (3, 3-Diaminobenzidine and H2O2)
until reactive bands developed. The membranes were finally washed with
distilled water, dried and kept in dark.
In the present study, five strains of B. abortus
and five strains of B. melitensis were isolated from cows and human
suffering from brucellosis, respectively. In addition, ten sera with Wright
titers of = 1:320 and 2-ME titers of = 1:160 collected from patients with
brucellosis. All sera were positive for Brucella IgG and Brucella
IgM in ELISA tests.
The isolated and mass cultured bacteria were placed under heat shocks
at 39, 40 and 42°C. Thereafter the extracted proteins from the heat-shocked
as well as the non shocked controls were resolved by SDS-PAGE (Fig.
SDS-PAGE pattern of B. abortus (lane a and b)
was composed of protein bands mainly with molecular mass in the range
of 10-100 kDa. The major protein groups were in the range of 30-75 and
14 -20 kDa. The amounts of a 60 kDa protein band (hsp60) was significantly
enhanced following heat shocks compared to controls.
SDS-PAGE pattern of B. melitensis (lane c and
d) also indicated protein bands in the range of 10-100 kDa. The most significant
protein groups of these isolates were in the range of 45-75 and 14-30
kDa. In the pattern of heat shocked bacteria in 42°C, not only a few
new proteins bands appeared, but also there were a generalized increase
in the level of most protein band. A 78 kDa and another protein band in
the range of 40-45 kDa were present only in the control non-shocked cells
and not in any of the heat-shocked bacteria. The amounts of a 60 kDa protein
band (hsp60) was significantly enhanced following heat shocks in relation
to the unheated cells. The results also indicated that heat shock responses
induce more expression of a 60 kDa protein (hsp60) in B. abortus than
in B. melitensis.
of extracted proteins from the heat-shocked (42°C) and non shocked
control B. abortus
(a and b, respectively) and B. melitensis
(c and d, respectively). The right and left sides is protein size
marker on the basis of kDa
Reaction of pure
hsp60 (1), shocked and un-shocked B.
(2 and 3, respectively), un-shocked and shocked B.
(4 and 5, respectively) with anti-hsp60 specific antibody
Immunoblotting of Escherichia coli hsp60 and the extracted proteins
from heat shocked and control B. abortus and B. melitensis
with polyclonal anti-hsp60 antibody showed that this antibody detect homologous
proteins with similar molecular mass (Fig. 2). Furthermore
Reactions of shocked
B. abortus protein extracts with sera from healthy subjects
(a, b) and brucellosis patients (c, d). The middle lane is protein
size marker (78, 66, 45, 29, 18.5, 14.5 kDa, respectively)
these results indicated that heat treatment induce hsp60
in the two bacterial species (lane 2 and 5), although response of B.
abortus is more intense than B. melitensis. These results correlate
considerably with the results of SDS-PAGE and verify the effect of heat
on induction of hsp60. As immunoblotes of B. abortus and B.
melitensis showed, anti-hsp60 antibody react with an additional protein
band with molecular mass of 15-16 kDa with unknown specificity.
The shocked and un-shocked bacterial protein extracts that resolved by
SDS-PAGE and transferred to PVDF membranes, were incubated with sera from
brucellosis patients as well as healthy subjects. The sera from brucellosis
patients reacted with several protein bands of B. abortus with
molecular mass 14, 29 and 60 kDa (Fig. 3, 4).
The 60 kDa band was the most significant and showed strong reactions with
all patients sera. The mentioned protein bands showed week or no reactivity
against sera from healthy individuals.
Furthermore, reactions of B. melitensis proteins with patients
and control sera indicated that sera from patients but not from the healthy
individuals strongly react with proteins extracted from heat-shocked bacteria
in the positions 10, 60 and 100 kDa (Fig. 5, 6).
The result indicates that none of these bands showed up in the reactions
between control sera with protein extracted from un-shocked B. melitensis.
The reactions between patients and controls sera with un-shocked B.
melitensis extracted proteins are shown in Fig. 6.
Many new protein bands
Reactions of un-shocked
B. abortus proteins with sera of brucellosis patients (1,
2) and healthy subjects (3, 4)
Reactions of shocked
B. melitensis protein extracts with sera of brucellosis patients
(1, 2) and healthy people (3, 4)
show up in the interaction of un-shocked bacterial proteins
with patients sera that are absent in reaction with control sera. A common
60 kDa band is seen in reactions between shocked B. melitensis
proteins with all patients` sera. This band is less frequently detected
in the reaction between un-shocked bacterial proteins with the patient`s
The heavily stained smear seen in the upper and middle
sections of the blots correlates to the presence of high levels of anti
Brucella LPS antibodies in patient`s sera. A 14 kDa band is also
detected in the immunoblots of all samples, both patients and healthy
Reactions of un-shocked
B. melitensis protein extracts with sera of brucellosis patients
(1, 2) and healthy people (3, 4).
Significant differences in protein bands were not detected
following electrophoresis of 39 and 40 heat shocked cells in relation
to the unheated bacteria.
In order to find the effect of heat stress on expression
of hsp60, five strains of B. abortus and five strains of B.
melitensis were isolated from cows and human, respectively and shocked
at 39, 40 and 42°C. Total bacterial protein profiles of shocked and
un-shocked bacteria were resolved by SDS-PAGE and analyzed against anti-hsp60
specific antibody and the patient`s sera by immunobloting.
Comparing the protein profiles of the heat-shocked versus
un-shocked bacteria showed a general increase in protein expression levels
in the heat-shocked samples; especially in a 60 KDa protein band (hsp60)
in 42°C shocked bacteria. This is in agreement with similar study
preformed by Lin et al. (1992). In this study, they observed a
significant increase in the level of the 60 kDa protein following heat-shocking
of B. abortus. The present study which was conducted on clinical
isolates of B. melitensis and B. abortus, did not show any
significant protein level expression differences in bacteria shocked at
39 and 40°C in comparison with the un-shocked cells. In the Lin et
al. (1992) study, maximum hsp proteins levels were produced in B.
abortus cells shocked at 42-46°C. The duration of bacterial heat
shock used in this study was 3 h as the Lin investigation found the maximum
heat shock response occurring after 2-3 h. Besides cell wall antigen such
as the outer membrane antigen, there are some other cellular proteins
that can play important immunologic roles during disease process (Lindler
et al., 1996). These antigens can act as important candidate for
serological diagnosis of brucellosis and/or subunit vaccine development
(Angel et al., 2003; Juliana et al., 2007). The levels of
hsp proteins are significantly increased under heat shock (Kaufmann, 1990).
Some of these hsps are readily recognized by the host immune system during
natural infections. The immunogenicity of the shocked and un-shocked bacterial
proteins against the acute phase sera of human patients, who were naturally
infected with Brucella, were analyzed by western blots.
This study aims to elucidate the immunogenicity of the
heat-shocked and un-shocked bacterial proteins, especially hsp60, against
patients and control sera. The study`s long term objective is to find
a suitable antigenic candidate for specific serological tests as well
as for subunit vaccine studies.
Highly pure E. coli hsp60 and polyclonal anti-hsp60
antibody were used to specifically detect hsp60 protein expression levels
in the B. melitensis and B. abortus. Pure hsp60 protein
and also shocked and un-shocked extracted B. melitensis and B.
abortus protein samples which resolved and blotted, were incubated
with polyclonal anti-hsp60 antibody. As the results showed (Fig. 2), the
antibody reacted much more strong with the 60 kDa protein extracted from
the heat shocked B. melitensis and B. abortus isolates in
relation with those recovered from the un-shocked bacteria. This indicates
an increase level of hsp60 production under heat shock conditions. The
reaction of the anti-hsp60 antibodies with a 14 kDa protein band is probably
due to the general cross-reaction of the polyclonal antibodies.
The heat-shocked bacterial protein reactions with patients
sera showed striking differences with those of control sera reaction.
This result points to the immunogenic nature of hsp60 and the relatively
strong immune response induced by the human immune system during natural
As it is concluded from SDS-PAGE and Immunoblotting results, hsp60 production
in B. abortus following heat shocks was significantly greater than
B. melitensis. We initially thought that the greater virulence
of B. melitensis relative to B. abortus, was probably due
to the higher in vivo induction of hsp60 following stress shocks.
But paradoxically, we observed higher hsp60 production by B. abortus
relative to B. melitensis. It thus can be postulated that the higher
levels of hsp60 production by B. abortus and the enhanced antigenic
properties of hsp60, which leads to higher anti-hsp60 antibodies can in
effect counteract the higher hsp60 production and subsequently the pathogenicity
of B. abortus. In other words, the increased hsp production by
B. abortus can induce a potent immune response which will in effect
help to restrict bacterial virulence. On the other hands, lower hsp60
production by B. melitensis would induce a relatively much lower
immune response against the bacterium leading to its greater virulence
As Wei et al. (1999) has indicated, the mammalian
hsp60 acts as a danger signal for innate immune system such as macrophages;
therefore, another reason for lower pathogenicity of B. abortus
compared to B. melitensis may be the role of Brucella hsp60
as a danger signal. hsp60 Receptors on the surface of macrophages has
been designated as toll-like receptor-4. After binding of hsp60 to these
receptors, cytokines such as TNF-α, IL-1 and IL-6 are released. T-helper-2
(Th2) cell is shifted to Th1 by TNF-α. Th1 cells play a major role
in Cell Mediated Immunity (CMI) which is very effective against intracellular
bacteria. Therefore, low production of hsp60 produces less TNF-α
leading to more pathogenicity. On the other hands, high production of
hsp60 produces higher TNF-α leading to activation of CMI and therefore,
lower pathogenicity. At the result, the pathogenicity of B. melitensis
by producing lower levels of hsp60 is higher than B. abortus.
The routine serological tests commonly used for sero-diagnosis
of animal and human brucellosis, is based on determination of anti-LPS
antibody levels. These antibodies persist at relatively high titers for
long period of times, even after full patients recovery (Abdolalizadeh
et al., 2002; Mostafaie et al., 2005). Unfortunately, these
serological tests have many cross-reactions with other Gram negative bacteria
(Abdolalizadeh et al., 2002; Mostafaie et al., 2005). Because
of these problems, researchers have long sought to develop more specific
non-LPS based serological tests directed against proteinatious component
of the bacteria. Therefore, hsp60 can potentially be used as a suitable
candidate for specific ELISA tests and subunit vaccine development (Naoko
et al., 2002).
Abdolalizadeh et al. (2002) and Mostafaie et
al. (2005) studied the reaction between immunogens of B. abortus
with human, gout and rabbit sera. Mostafaie (1999) looked at the interactions
between major outer membrane proteins of B. abortus (S19 strain)
and B. melitensis (16M strain) with human patients sera. In another
study by Mostafaie et al. (2000), the interaction between the Brucella
LPS with human sera was investigated. Angel et al. (2003) have
used the superoxide dismutase gene of B. abortus in order to create
a DNA vaccine that was capable of inducing protective immunity in mice.
In none of these studies, the interactions of bacterial
hsps with human sera were investigated. Therefore, this is probably the
first report of Comparison of Heat Shock Response in Brucella abortus
and Brucella melitensis and interaction of human patient sera with
the hsp60 in both bacteria.
The authors like to thank: Microbiology department,
School of medicine, Iran university of medical science, Tehran, Iran;
Cellular and molecular research center of Iran University of Medical Sciences,
Tehran, Iran; Medical biology research center of Kermanshah University
of Medical Sciences, Kermanshah, Iran; and Microbiology and Immunology
department of Babol University of Medical Science.