Download Proteomic analyses of the time course responses of mice infected

RESEARCH LETTER
Proteomic analyses of the time course responses of mice
infected with Brucella abortus 544 reveal immunogenic
antigens
Jin Ju Lee1, Hannah Leah Simborio2, Alisha Wehdnesday Bernardo Reyes2, Dae Geun Kim2,
Huynh Tan Hop2, Wongi Min2, Moon Her1, Suk Chan Jung1, Han Sang Yoo3 & Suk Kim2,4
1
Animal and Plant Quarantine Agency, Anyang, Gyeonggi-do, Korea; 2Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang
National University, Jinju, Korea; 3Department of Infectious Diseases, College of Veterinary Medicine, Seoul National University, Seoul, Korea; and
4
Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Korea
Correspondence: Suk Kim, Institute of
Animal Medicine, College of Veterinary
Medicine, Gyeongsang National University,
Jinju 660-701, Korea.
Tel.: +82 55 772 2359;
fax: +82 55 772 2349;
e-mail: [email protected]
Received 15 May 2014; revised 2 June 2014;
accepted 18 June 2014. Final version
published online 22 July 2014.
DOI: 10.1111/1574-6968.12522
MICROBIOLOGY LETTERS
Editor: Akio Nakane
Keywords
Brucella abortus; immunogenic proteins;
serodiagnosis; infection period.
Abstract
Brucellosis is a major zoonotic disease caused by pathogens of the genus Brucella. The eradication of brucellosis in domestic animals, associated with the
prevention of human infection, can be attained through accurate diagnosis.
However, the conventional serological diagnosis of brucellosis has limitations,
particularly in detecting the infection period. Accordingly, the aim of this study
was to determine reliable immunogenic proteins to detect Brucella abortus
infection according to time course responses to aid in the appropriate management of this disease. Proteomic identification through two-dimensional electrophoresis (2DE), followed by immunoblotting, revealed 13, 24, and 55
immunodominant B. abortus 544 proteins that were reactive to sera from
experimentally infected mice at early (10 days), middle (30 days), and late
(60 days) infection periods, respectively. After excluding several spots reactive
to sera from Yersinia enterocolitica O:9-infected and noninfected mice, 17 of
the 67 immunodominant proteins were identified through MALDI-TOF MS.
Consequently, the identified proteins showed time course-dependent immunogenicity against Brucella infection. Thus, the results of this study suggest that
the production of immunogenic proteins during infection periods improves
the diagnosis and discovery of vaccine candidates.
Introduction
Brucella is a genus of major zoonotic pathogens that
induce chronic infections in a broad range of animals,
including livestock, wildlife, and humans (Pappas et al.,
2005). These bacteria are considered to be a major health
threat, reflecting the highly infectious nature of these
organisms and the worldwide outbreak of this disease
(Seleem et al., 2010). Importantly, these pathogens escape
immune detection, and subsequent elaborate virulence is
a fundamental aspect of the pathogenic lifestyle of these
microorganisms (Roop et al., 2009).
In the last few decades, various diagnostic test methods
have been developed for the successful surveillance and
control of Brucella infection. The standard diagnostic
techniques for brucellosis include the isolation of causative
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
organisms through bacterial culture, molecular assays for
bacterial genome detection, and serology through antibody detection. Bacterial culture, however, requires biosafety level three conditions and a skilled expert, while
genomic detection is a rapid but limited technique for the
recognition of active infection. Current serological assessments are more reliable and generally approved (Al
Dahouk & Nockler, 2011). For the serological diagnosis of
brucellosis in animals and humans, classical methods, such
as the Rose Bengal test (RBT), agglutination test, and
enzyme-linked immunosorbent assay (ELISA), have been
widely established in various countries and are preferred
in routine clinical practice (Al Dahouk et al., 2003; Nielsen & Yu, 2010) However, more enhanced sensitivity and
specificity in the confirmatory diagnosis of brucellosis are
still needed (Christopher et al., 2010; McGiven, 2013).
FEMS Microbiol Lett 357 (2014) 164–174
Immunogenic antigens for infection with B. abortus
Consistent with these assessments, the diagnostic antigen
used in classical serological tests is commonly derived
from smooth lipopolysaccharide or O-polysaccharide as
an immunodominant epitope for antibodies secreted in
the humoral immune response (McGiven et al., 2003;
Laurent et al., 2004). Despite the strong immunoreactivity
of these common antigens, the specificity of LPS-based
assays is assumed to be low, reflecting cross-reactions with
other relevant bacteria, such as Y. enterocolitica O:9, which
conserves the similar O-polysaccharide composition (Corbel, 1979; Nielsen et al., 2004). Accordingly, diverse modifications in the diagnosis of brucellosis have been
examined to overcome the limitations arising from the
immune response against Brucella infection (McGiven
et al., 2008).
Considering the increasing concerns on novel immunogens as alternatives to typical antigens, we conducted a de
novo study focusing on the reactions of specific antigens
to hosts during different infection periods. Accordingly,
we investigated reactive antigenic proteins on different
time course responses in mice experimentally infected
with B. abortus. Given that the correlation of infection
time with the diagnosis and control of this disease is
practically noteworthy, immunogenic antigens of B. abortus reactive at different infection periods can be considered as adequate diagnostic makers and effective vaccine
candidates.
Materials and methods
Bacterial strains and culture conditions
Brucella abortus 544 (ATCC 23448), a smooth, virulent
B. abortus biovar 1 strain, was used as a standard reference strain. Yersinia enterocolitica O:9 was obtained from
the Laboratory of Bacteriology Division in Animal and
Plant Quarantine Agency, Korea. Routine strain cultivation was conducted in Brucella broth (Becton Dickinson,
MD) or agar (containing 1.5% agar). The bacteria were
cultured in broth at 37 °C with shaking until reaching
the stationary stage, and subsequently, the number of viable bacteria was calculated from 10-fold serial dilutions
on Brucella agar.
Preparations of antisera
A total of 25 BALB/c mice (female 6- to 8-week-old) were
divided into three groups: B. abortus infected (n = 10),
Y. enterocolitica O:9 infected (n = 10), and uninfected
control (n = 5). Prior to infection, blood samples from
all mice were collected and assessed using the buffered
plate agglutination test (BPAT) and Rose Bengal test
(RBT) according to the OIE standard procedures of
FEMS Microbiol Lett 357 (2014) 164–174
165
serological tests for brucellosis (McGiven et al., 2006;
Ragan et al., 2013), confirming that all animals were seronegative for brucellosis. In two infection groups, each animal was infected with 2 9 104 CFUs of either B. abortus
or Y. enterocolitica through an intraperitoneal route. The
remaining five mice were injected with sterile PBS as
noninfected controls. After infection, mice sera were collected from each animal at three different stages of infection: early (10 days), middle (30 days), and late
(60 days). For the serological tests, three kinds of sera
were chosen: three noninfected sera, observed as RBTand BPAT-negative, three pooling sets of two Y. enterocolitica-infected sera observed as RBT-negative and
BPAT-positive, representing complete agglutination at 10
and 30 days postinfection, three pooling sets of three
B. abortus-infected sera observed as RBT- and BPATpositive, representing complete agglutination at 10, 30,
and 60 days postinfection. All experimental procedures
described were reviewed and approved through the Animal Ethical Committee of Gyeongsang National University (Authorization Number GNU-130423-M0031).
Preparations of whole cell proteins of
B. abortus
Brucella abortus cultures at stationary growth phase were
collected through centrifugation at 8000 g for 20 min at
4 °C and washed three times with ice-cold PBS (pH 7.6).
The resulting pellet was resuspended in 50 mM Tris-HCl
(pH 7.6) containing protease inhibitor cocktail and sonicated on ice. The sonicated suspension was centrifuged at
12 000 g for 1 h at 4 °C, and the collected pellet was
resuspended in lysis buffer (5 M urea, 2 M thiourea, 2%
CHAPS, 1% SB 3-10, 1% DTT, and protease inhibitor
cocktail), followed by incubation with vigorous stirring at
room temperature for 1 h. After lysis, the solution was
centrifuged at 100 000 g for 30 min, and the supernatant
was collected. The protein concentration was determined
using the Bio-Rad protein stain (Bio-Rad Laboratories,
Inc.) according to the Bradford method (Bradford, 1976).
IEF and 2DE
Isoelectronic focusing (IEF) and two-dimensional electrophoresis (2DE) were performed using a previously
described modified method (Shaw & Riederer, 2003; Gorg
et al., 2004). IEF was processed on a Protean IEF system
(GE Healthcare) under the following conditions: 500 V
for 1 h, gradient phase of 1000 V for 1 h, 1000 V for
3 h, gradient step of 10 000 V for 3 h, 10 000 V for 5 h,
50 V for 30 min, and a final step of 50 V for 30 min.
After IEF, all strips were equilibrated and loaded on the
top of 12% SDS-polyacrylamide gels. The proteins were
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Published by John Wiley & Sons Ltd. All rights reserved
J. J. Lee et al.
166
separated through 2DE in resolving buffer (25 mM Tris,
pH 8.8, 192 mM glycine, and 0.1% SDS) and transferred
to polyvinylidene difluoride (PVDF) membranes (Millipore) for immunoblot analysis. In addition, the replicated
gels from the same samples were subjected to silver staining. Three replicates of 2DE were conducted in independent experiments.
The digested peptides were extracted using 0.1% TFA/50%
ACN and vacuum-dried through centrifugation. The peptide mixture was resuspended in 0.5% TFA and desalted
using ZipTip plates (Millipore), followed by elution with
0.2% TFA/50% ACN. Moreover, the solution was combined with the matrix (10 mg mL 1 a-cyano-4-hydroxycinnamic acid in 50% ACN/1% TFA).
Immunoblotting with antisera
MALDI-TOF MS analysis and protein
identification
The membranes were blocked for 1 h at room temperature with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) to remove nonspecific
binding, followed by washing three times for 20 min with
TBS-T. The membrane blots were incubated overnight at
4 °C with a 1 : 500 diluted solution of antisera collected
from infected mice. Thereafter, the blots were incubated
for 1 h at room temperature in solution containing a
1 : 5000 dilution of rabbit anti-mouse IgG HRP-conjugated antibody (Sigma). After washing, the immunoreaction was observed using ECL detection reagents (GE
Healthcare). Moreover, the immunoreactive protein spots
were visualized using a ChemiDoc XRS camera equipped
with Quantity Analysis software (Bio-Rad).
Image analysis and in-gel enzymatic digestion
After silver staining, the 2D gels were scanned on an ImageScanner and cropped using IMAGEQUANT TL analysis software (GE Healthcare). Automatic spot detection, spot
matching, and gel image alignment were performed using
PROGENESIS SAMESPOTS v2.0 software (Nonlinear Dynamics)
(Silva et al., 2010). For data analysis, spot matching across
all gels without omitting values was required for spot
merging. Each gel was processed in three replicates in parallel with three independent protein preparations. A single
average gel was generated from three independent subgels
containing only protein spots included in at least two subgels. The common spots on all subgels were selected
according to shape and intensity for the normalization of
spot volumes to equalize the probable variation in the
staining trait. In addition, spot matching was conducted
between the stained spots in gel, and the immunogenic
protein spots detected through immunoblotting. The
matched spots were excised, and the gel plugs containing
single proteins were in-gel digested through an enzymatic
reaction with porcine trypsin as previously described (Gorg
et al., 2004). The target spots were treated with 50 mM
ammonium bicarbonate (NH4CO3, pH 7.8)/50% acetonitrile (ACN) for 1 h at room temperature, followed by
dehydration in ACN and vacuum-drying. Subsequently,
the spots were rehydrated for 16 h at 37 °C and digested
with trypsin (10 ng lL 1) in 50 mM NH4CO3 (pH 7.8).
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
All spectra were obtained using an ABI 4700 proteomics
analyzer and TOF–TOF Mass Spectrometer (Applied Biosystems). MS/MS data were collected using an Nd:YAG
laser (200 Hz repetition rate), generating up to 4000
shots for each spectrum. The instrument default calibration was used, without applying the internal or external
calibration, and peptide mass fingerprint data were
explored using the NCBI protein sequence and annotated
Brucella genomic database via the Mascot search engine
(Matrix Science, UK) (Perkins et al., 1999). The information for selected proteins was retrieved using the EXPASY
database (http://www.expasy.org/) (Hoogland et al.,
2008). Functional categories were created according to
the cluster of orthologous groups (COGs) protein database acquired through the comparison of predicted and
known proteins in microbial genomes from NCBI COG
(Makarova et al., 2007).
Results
Immunogenic proteins in B. abortus 544 and
comparison with cross-reacting bacteria
The whole cell proteins from B. abortus 544 were recognized using annotated 2D proteome profiles (Fig. 1a). A
total of 1181 protein spots were detected on the silverstained 2DE gels within a pI range of 4–7. To investigate
the antigenic proteins of B. abortus from definite periods
of infection in mice, sera were obtained at three different
stages of infection (10, 30, and 60 days) from mice
challenged with B. abortus and used to detect immunogenic proteins through immunoblotting. Initially, to
preclude cross-reactions, the sera from mice infected with
Y. enterocolitica O:9 (YP) were also subjected to immunoblotting. A total of 13, 24, and 55 B. abortus proteins
were detected in the sera from mice infected with
B. abortus for 10, 30, and 60 days of infection, respectively (Table 1 and Fig. 1b-d). There were 7 and 14 spots
detected from nonspecific reactions with negative control
(NC) sera and cross-reaction with YP sera, respectively
(Table 1 and Fig. 2). In comparison with all protein
spots, four of seven NC-reactive protein spots (spot no.
FEMS Microbiol Lett 357 (2014) 164–174
Immunogenic antigens for infection with B. abortus
167
(a)
(b)
(c)
(d)
Fig. 1. 2DE map of Brucella abortus 544 proteins and immunoblotting profile with antisera from mice infected with B. abortus. (a) 2DE map of
whole cell B. abortus proteins within a pI range of 4–7 identified from the silver-stained 2DE gels. The immunoblotting analysis was conducted
with antisera from mice at 10 (b), 30 (c), and 60 (d) days postinfection with B. abortus. Three replicates of 2DE were performed in independent
experiments.
6, 27, 44, and 79) and 11 of 14 YP-reactive protein spots
(spot no. 19, 33, 41, 43, 51, 52, 58, 59, 60, 63, and 79)
matched with BP-reactive protein spots (Table 2). Consequently, a total of one (spot no. 58), three (spot no. 43,
51, 79), and eight spots (spot no. 19, 33, 41, 51, 58, 59,
60, 63) reactive to BP sera at 10, 30, and 60 days, respectively, were ruled out due to nonspecific reactions as
FEMS Microbiol Lett 357 (2014) 164–174
shown with matched protein spots to either NC or YP
sera (Supporting information, Fig. S1). Therefore, a total
of 67 specific immunodominant protein spots reactive to
only at least one of the three infection periods of BP sera
were selected. The matching analysis revealed common
specific immunodominant protein spots reactive to BP
sera from two infection periods: 1 (spot no. 71), 5 (spot
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Published by John Wiley & Sons Ltd. All rights reserved
J. J. Lee et al.
168
Table 1. Comparison of the immunoreactive proteins of Brucella
abortus 544 independently reacted with BP (10, 30, and 60 days
postchallenge), except NC and YP
No. of
Antisera
immunoreactions
Period of
challenge
nonmatched
protein
Total no.
of protein
Independence
compared
(days)
spots
spots
(%)*
BP
10
7
13
53.84
30
60
13
33
24
55
54.16
60.00
3
3
7
14
42.86
21.43
NC
YP
*The percent independence was calculated as the number of nonmatched proteins to the antisera immunoreactions divided by the
total number of proteins in the antisera immunoreactions 9 100.
no. 8, 13, 28, 53, and 58), and 8 (spot no. 6, 10, 12, 22,
51, 64, 68, and 82) common specific protein spots
between 10 and 30 days, 10 and 60 days, and 30 and
60 days, respectively (Table 2 and Fig. S2a). Additionally,
a total of 7 (10 days), 13 (30 days), and 33 (60 days)
specific immunodominant protein spots reactive to BP
sera from a single infection period were detected (Table 1
and Fig. S2b–d). Correspondingly, the percent independence values for immunoreactions at an individual
infection period were 53.84, 54.16, and 60.0% at 10, 30,
and 60 days, respectively.
(a)
Identification of immunogenic proteins
relevant to infection periods
Among the 67 immunoreactive proteins, 17 proteins with
markedly greater intensity than average were identified
using MALDI-TOF MS. The results of the protein identification analysis are shown in Table 3. These data
revealed that multiple immunogenic proteins at different
locations on 2DE-immunoblotting showed diverse Mr
and pI values and were correlated with theoretical values.
Evidence of the subcellular location obtained through an
NCBI BLAST search revealed that among these 17 identified
proteins, seven proteins (41.2%) were localized to the
cytoplasmic region, two proteins (11.7%) were localized
to the outer membrane-bound periplasmic space, and
one protein (5.9%) was localized to the ribosome, while
the location of the remaining seven proteins (41.2%)
remained unknown. In particular, a total of seven proteins were associated with different enzymatic activities,
such as transferase, hydrolase, protease, kinase, and dehydrogenase, and two proteins (spot no. 8 and 28) were
involved in elongation factor activity in the cytoplasm. In
addition, there were two hypothetical proteins (spot no.
68 and 71) identified with different ORFs (BruAb1_0179
and BruAb2_0845) and Mr values of 19.2 and 18.5 kDa,
respectively.
(b)
Fig. 2. 2DE and immunoblotting analysis of Brucella abortus 544 proteins with the sera from noninfected and Yersinia enterocolitica-infected
mice. A total of 7 and 14 immunoreactions were detected through reactions with noninfected (a) and Y. enterocolitica-infected (b) mice sera,
and the corresponding protein spots are labeled on the blots. The serial numbers of the immunoreactive proteins identified from 2DE and
immunoblot analysis are shown.
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Lett 357 (2014) 164–174
Immunogenic antigens for infection with B. abortus
169
Table 2. Comparison of the immunoreactive proteins of Brucella abortus 544 reacted with BP (10, 30, and 60 days postchallenge), NC or YP
Immunoreactions of whole Brucella proteins to different
antisera
BP
10 days
‡
30 days
60 days
NC
+
+
+
+§
+
+
+
+
+
+
+
+
+
+
+
+
+
+
YP
+
+
+
+
No. of matched
protein spots*
Designated label of
matched protein spots†
Total no. of
protein spots
2
3
1
3
9
1
1
5
8
0
6, 79
6, 27, 44
58
43, 51, 79
19, 33, 41, 51, 52, 58, 59, 60, 63
79
71
8, 13, 28, 53, 58
6, 10, 12, 22, 51, 64, 68, 82
29
59
26
35
61
43
36
63
89
78
*The number of identical spots detected by commonly positive reaction.
The numeral label of spots as shown in figures of 2DE gels or blots.
‡
Negative reactions detected through immunoblotting.
§
Positive reactions detected through immunoblotting.
†
The functions of the selected proteins were determined
based on the classification of the proteins encoded in
complete genomes through COGs: 10 proteins were associated with transport and metabolism [four proteins for
amino acid transport (spot no. 10, 12, 13, and 22), three
proteins for carbohydrate transport (spot no. 14, 20, and
30), two proteins for inorganic ion transport (spot no. 48
and 74), and 1 protein for nucleotide transport (spot no.
53)]; three proteins were involved in translation, ribosomal structure, and biogenesis (spot no. 8, 28, and 82);
one protein was associated with transcription (spot no.
64); and two proteins were associated with unknown functions (spot no. 68 and 71). One ribosomal protein reactive
with BP sera at 10 days postchallenge was identified as the
50S ribosomal protein L25 (RPL25; spot no. 82). Together
with ribosomal proteins, elongation factor G (EF-G) and
elongation factor Ts (tsf), associated with protein translation, were detected at similar pIs (5.05 and 5.02) but with
different Mr values (76.2 and 31.5 kDa), and both proteins
were reactive to BP sera at 30 days postchallenge. Particularly, two proteins, likely periplasmic serine endoprotease
DegP-like (htrA) and adenylate kinase (adk), were
assigned to different B. melitensis locus tags: BMEI1330
and BMEI0778, respectively. The existence values of these
proteins were inferred through homology, as definite
orthologs exist in closely related species.
Discussion
Brucellosis is a disturbing infectious disease that certainly
needs to be solved due to chronic courses and relapses
(Vrioni et al., 2008). Accordingly, it is necessary to
strictly regulate and consistently monitor brucellosis in
FEMS Microbiol Lett 357 (2014) 164–174
many countries. To achieve successful disease control,
paramount diagnosis and effectual vaccine development
are necessary (McGiven, 2013). On a large scale, allconclusive measures are underpinned by impressive
approaches to advance the specific immunogenic antigens
of Brucella for application at all costs. Considerable parallels exist between the immune responses and immunogenic products employed through influential interactions
with these bacteria and their hosts. The renovation of
these parallels can notably improve diagnostic tools and
vaccine development against Brucella infections. In the
last few decades, the low specificity of diagnosis due to
cross-reacting bacteria and the poor efficiency of live
attenuated vaccine with Brucella strains has impeded the
development of a treatment for this painful disease (Ko
et al., 2012). In practice, diverse modifications for the
diagnosis of brucellosis have been investigated to overcome the limitations arising from the immune response
against Brucella infection (McGiven et al., 2008). Hence,
in the knowledge and strategies developed to examine the
immunogenicity of Brucella using a proteomics approach,
the Brucella immunogens that are relevant to interactions
with hosts during the infectious stage remain unknown.
Thus, it is important to address the mechanism or correlation regarding both Brucella immunogenic proteins and
infection periods. In the current study, we provide insight
into the correlation between Brucella immunogenic proteins and infection periods, highlighting the necessity to
verify both aspects to decipher diagnoses and identify
vaccine candidates for brucellosis.
Proteomic analyses using MALDI-TOF MS have been a
potent tool for bacterial identification in diagnostic
microbiology and vaccine development (Connolly et al.,
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
71
ND
ND
82
53
ND
68
ND
ND
64
28
ND
22
ND
ND
12
13
ND
10
ND
day
10 day
spot
8
30
No. of
B. abortus
day
60
ND
immunoreactions
Antisera
BruAb2_0845
adk
tsf
ahcY
fusA
BruAb1_1470
BruAb1_0179
rpoZ
dadA
htrA
BruAb1_1389
Gene name
3341366
1196489
3340671
3340511
3340968
3340810
3339118
3339864
3341955
1197041
3340946
Gene ID*
62317745
17987061
62317250
62290939
62290147
62290420
62289158
62289611
62317250
17987613
62290288
GI
BruAb2_0845
Hypothetical protein
Adenylate kinase
YP_223598.1
NP_539695.1
Q577E7
Q8YHL9
Q57CX8
177
194
305
BruAb2_0845
BMEI0778
BruAb1_1167
BruAb1_2072
BruAb1_1241
BruAb1_1470
BruAb1_0179
400
197
370
312
306
171
191
18 506
20 908
31 528
51 044
76 301
22 369
19 198
14 562
18 571
20 864
31 491
53 514
76 235
22 383
19 210
14 514
5.02
6.63
5.02
5.30
5.05
5.91
5.13
4.22
6.47
5.81
4.86
pI
43
32
41
26
18
47
31
33
20
20
10
(%)
coverage
Unknown
Cytoplasm
Cytoplasm
Cytoplasm
Cytoplasm
Ribosome
Unknown
Unknown
Unknown
space
Periplasmic
Unknown
location§
Subcellular
E: Amino acid
metabolism
transport and
E: Amino acid
metabolism
transport and
E: Amino acid
category¶
COG functional
E: Amino acid
biogenesis
structure and
ribosomal
J: Translation,
biogenesis
structure and
ribosomal
J: Translation,
unknown
S: Function
K: Transcription
unknown
S: Function
metabolism
transport and
F: Nucleotide
biogenesis
structure and
ribosomal
J: Translation,
metabolism
Elongation factor Ts
YP_221867.1
466
694
207
174
102
45 110
53 514
55 205
TMw‡
transport and
Q57AG3
Q57CQ5
Q57BY2
Q57FJ4
BruAb1_0668
45 367
53 482
55 228
EMw†
hydrolase
YP_222732.1
YP_221940.1
YP_222213.1
YP_220951.1
133
152
337
182
Score
homocysteine
S-adenosyl-L-
(EF-G)
Elongation factor G
protein L25
50S ribosomal
BruAb1_0179
Hypothetical protein
omega
polymerase subunit
Q57E91
BruAb2_0309
BMEI1330
BruAb1_1389
Locus tag*
metabolism
DNA-directed RNA
YP_221404.1
416
513
524
length
transport and
Q579E2
Q8YG32
Q57CB4
no.*
Sequence
small subunit
YP_223103.1
NP_540247.1
YP_222081.1
Protein ID
Sequence
Mw
dehydrogenase
D-amino acid
DegP-like
endoprotease
serine
Probable periplasmic
Serine protease Do
Protein identification
Accession
Table 3. Identification of selected immunoreactive proteins of B. abortus 544 reacted with BP (10, 30, and 10 days postchallenge)
170
J. J. Lee et al.
FEMS Microbiol Lett 357 (2014) 164–174
FEMS Microbiol Lett 357 (2014) 164–174
ND
ND
ND
ND
31
48
30
74
ND
ND
ND
ND
ND
ND
ND
day
60
pgk
BruAb2_0325
sodC
mdh
BruAb1_0137
BruAb1_1463
Gene name
3340208
3341905
3341416
3340925
3339167
3339591
Gene ID*
62290601
62317265
62317454
62290781
62289118
62290359
GI
kinase
Phosphoglycerate
dehydrogenase
Aldehyde
dismutase, Cu–Zn
Superoxide
dehydrogenase
Malate
YP_222394.1
YP_223118.1
YP_223307.1
YP_222574.1
Q9 L560
Q579C7
P15453
Q57AX1
Q57FN4
395
500
173
320
190
BruAb1_1714
BruAb2_0325
BruAb2_0527
BruAb1_1903
BruAb1_0137
BruAb1_1463
Locus tag*
173
249
283
76
206
87
Score
41 810
53 744
18 233
33 854
20 569
40 368
EMw†
41 796
53 435
18 131
33 704
20 468
40 337
TMw‡
5.64
5.64
6.24
5.24
4.74
6.33
pI
17
29
43
7
39
16
(%)
coverage
Cytoplasm
Unknown
space
Periplasmic
Cytoplasm
Cytoplasm
Unknown
location§
Subcellular
R: General
category¶
COG functional
metabolism
transport and
G: Carbohydrate
metabolism
transport and
G: Carbohydrate
metabolism
transport and
P: Inorganic ion
metabolism
transport and
G: Carbohydrate
metabolism
transport and
P: Inorganic ion
prediction only
NifU-like protein
YP_220911.1
367
length
function
Q57C43
no.*
Sequence
protein
YP_222152.1
Protein ID
Sequence
Mw
hydrolase family
Cholylglycine
Protein identification
Accession
*Gene ID, protein ID, accession no., and locus tag were retrieved from the NCBInr database.
†
Experimental molecular weight.
‡
Theoretical molecular weight of UniProtKB database entry.
§
Subcellular locations were predicted using PSORTB v. 2.0.4.
¶
Cluster of orthologous groups (COGs) protein database generated by comparing microbial genomes from the NCBI COG.
ND, not detected.
20
14
day
10 day
spot
ND
30
No. of
B. abortus
immunoreactions
Antisera
Table 3. Continued
Immunogenic antigens for infection with B. abortus
171
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
J. J. Lee et al.
172
2006; Sa-Correia & Teixeira, 2010). The identification of
Brucella using proteomic techniques can be more useful
than conventional diagnostic methods in rapidity, accuracy, and cost (Lista et al., 2011), but these methods have
not routinely been applied because of limitations on
access to all-conclusive protein profile databases. In the
present study, the numbers of immunogenic protein spots
at three different phases of infection, comprising early
(10 days), middle (30 days), and late (60 days) periods,
excluding matched spots from negative sera- and crossreacting spots, comprised different proportions of immunoreactants: 13 spots at 10 days, 24 spots at 30 days, and
55 spots at 60 days postchallenge. These results showed
that immunoreactive proteins increase with increasing
infection time. The matching analysis revealed 1, 5, and 8
common spots responsive to two infection periods, such
as 10 and 30 days, 10 and 60 days, and 30 and 60 days,
respectively, representing commonly immunogenic proteins at different stages of infection with B. abortus. These
data indicate that these protein spots could be potential
immunodominant proteins possessing strong sensitivity
during particular infection periods. In addition, 7, 13,
and 33 protein spots were immunoreactive with only one
group of sera from different infection periods, independent of other periods, with calculated percent independence values of 53.84%, 54.16%, and 60.00% at 10, 30,
and 60 days, respectively. Notably, these findings illustrate
that specific immunogenic proteins reactive to antisera at
a single infection period could be detected at certain
times of infection. Some of the immunogenic protein
spots strongly reactive with BP sera were identified by
MALDI-TOF MS, and the proteins encoded by different
ORFs with > 99% sequence identity to B. abortus were
verified. The COG functional classification revealed the
functions of the immunogenic proteins during a particular stage of infection. Immunoreactivity to BP sera at
10 days postchallenge showed a large proportion of proteins involved in amino acid transport and metabolic
functions, suggesting that these proteins are associated
with the early stages of B. abortus infection. Of these proteins, two types of serine proteases, Do-and DegP-like
protease (HtrA), were critical for virulence in gramnegative bacteria, such as heat-shock proteins displaying
chaperone activity (Lewis et al., 2009; Ge et al., 2014).
Similarly, several studies have shown that Brucella htrA
was critical to virulence in animal models and cultured
neutrophils (Elzer et al., 1996a, b). In addition, the specific proteins reactive to sera at 30 days were associated
with translation, ribosomal structure, and biogenesis, and
a minority of proteins was associated with the transport
and metabolism of amino acids (ahcY) and nucleotides
(adk). The typical translation activity of each protein
plays a particular role associated with several signaling
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
events, leading to protein expression. Of these, there was
the ribosomal protein, RPL25 protein, that was identified
as the LSU ribosomal protein L25P in B. melitensis
(BMEI0481) (Wagner et al., 2002) and B. abortus
(BAB1_1551) (Connolly et al., 2006). However, the proteins that were immunoreactive with sera at 60 days were
predicted to participate in carbohydrate transport and
metabolism, despite common reactions with sera at 10 or
30 days. These proteins, including phosphoglycerate
kinase (pgk), aldehyde dehydrogenase, and malate dehydrogenase (mdh), have been identified in the B. melitensis
proteome using global proteomic analysis (Wagner et al.,
2002), and immunogenic candidate proteins for vaccine
development have been selected through the proteomic
analysis of the B. abortus cell envelope (Connolly et al.,
2006). Particularly, one of the proteins that showed
immunoreactivity to BP sera at 10 and 60 days is Cu–Zn
superoxide dismutase (sodC). In previous studies, this
enzyme has been defined as a heat-shock protein and an
antioxidant that participates in superoxide dismutase
activity, binding metal ions to destroy radicals (Onate
et al., 2003; Gee et al., 2005; Saez et al., 2008). Although
the functions of these proteins have well described, we
emphatically note the implication on the suitable period
for facilitating the superlative functions of these proteins.
Indeed, although the likelihood of a significant correlation between infection periods and proteomic functions is
only presumptive, it is certain that the proteins identified
in this study might be specific immunogenic markers or
vaccine candidates of infection activity according to the
disease stage.
Given that the serological diagnosis is based on antigen–antibody reactions that depend on diverse factors,
such as antibody type and infection time, the accuracy of
antibody detection remains questionable. Nevertheless,
the low awareness regarding the effects of differences in
immunogenicity on infection stage is of critical importance. At present, novel diagnostic immunogens should
be upgraded to monitor Brucella infections at any stage
of disease. The most important concern regarding brucellosis diagnosis is identifying faultless immunogenic antigens of universal use whenever the infection is initiated
and progressed. Therefore, the results of the present study
suggest highly efficient immunogenic proteins from
B. abortus, which modulate key aspects of diagnosis and
subsequently can be used as reliable vaccine candidates
for disease control.
Acknowledgements
The work was supported by iPET (112012-3); Ministry of
Agriculture, Food and Rural Affairs, and Animal and
Plant Quarantine Agency, Korea.
FEMS Microbiol Lett 357 (2014) 164–174
Immunogenic antigens for infection with B. abortus
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Comparative analysis between non-specific and
specific reactions of B. abortus proteins.
Fig. S2. Comparative analysis among specific reactions of
B. abortus proteins.
FEMS Microbiol Lett 357 (2014) 164–174