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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 ª 2014 Federation of European Microbiological Societies. 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 ª 2014 Federation of European Microbiological Societies. 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. 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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