Identification of a protective protein from stationary-phase exoproteome of Brucella abortus

Authors


  • In this paper the authors use a 2DE–MS and MALDI-TOF/TOF tandem mass spectrometry approach to describe the exoproteome of Brucella abortus. Of the 26 proteins identified, one is a novel, highly immunogenic protein that elicits improved protection in the mouse model. This has significance for the development of Brucella subunit vaccine.

Abstract

Brucellosis is a worldwide zoonotic disease. No Brucella vaccine is available for use in humans, and existing animal vaccines have limitations. To search the putative vaccine candidates, we studied the exoproteome of Brucella abortus NCTC 10093 using 2-DE–MS approach. Twenty-six proteins were identified using MALDI-TOF/TOF tandem mass spectrometry. Outer membrane protein 25, d-galactose periplasmic-binding protein, oligopeptide ABC transporter protein and isopropylmalate synthase were found to be the most abundant proteins. Most proteins (6, 23%) were predicted to be involved in amino acid transport and metabolism followed by carbohydrate transport and metabolism (4, 15%). Outer membrane protein 25, Omp2b porin and one hypothetical protein were predicted as outer membrane proteins. In addition, Omp28, Omp31 and one ribosomal protein (L9) were also identified. The ribosomal protein L9 was produced as a recombinant protein and was studied in mouse model for vaccine potential. It was found to be immunogenic in terms of generating serum antibody response and release of IFN-γ from mice spleen cells. Recombinant L9-immunized mice were protected against challenge with virulent B. abortus strain 544, suggesting usefulness of ribosomal protein L9 as a good vaccine candidate against brucellosis.

Introduction

Brucellae are Gram-negative intracellular bacteria that cause an important zoonotic disease called brucellosis. Brucellosis accounts for more than 500 000 new cases annually. Infection with Brucella abortus, a species that primarily affects bovines, often results in abortions and infertility in domestic and wild mammals. In humans, the major cause of disease is Brucella melitensis, although several cases have also been attributed to B. abortus (Corbel, 1997; Ashford et al., 2004). Brucellosis in humans manifests itself as a chronic infection with undulant fever and general malaise. Brucellosis is a weakening disease and requires prolonged antibiotic treatment. Even after treatment, relapses have been detected in 5–30% of cases (Vrioni et al., 2008). Brucella spp. are considered potential bioterror agent and have been classified as NIAID category B priority pathogens (Pappas et al., 2006). Attenuated, live Brucella strains such as B. abortus RB51 and RB19 and B. melitensis Rev1 are being used as vaccines to control brucellosis in domestic animals (Schurig et al., 2002). However, these vaccines are not suitable for humans because they can cause disease in healthy individuals (Perkins et al., 2010). These vaccines also have limitations for veterinary use, as they may induce abortions when administered to pregnant animals (Schurig et al., 1991) and show cross-reaction with natural infection during serodiagnosis (Stevens et al., 1994), suggesting an urgent need to develop new Brucella vaccine.

The postgenomic era holds the possibility of rational design of novel vaccines for important human pathogens. The discovery and development of these new vaccines are likely to be accomplished through integrated proteomic strategies (Sengupta et al., 2010). Immunoproteomic approaches such as cell envelope immunoproteome of B. abortus (Connolly et al., 2006) and soluble proteins immunoproteome of B. melitensis (Yang et al., 2011) have revealed a number of vaccine candidates, for example, 60 kDa GroEL, Omp31 and adoHcyase (Leclerq et al., 2002; Cassataro et al., 2005; Yang et al., 2011). All living microorganisms interface with their surroundings through proteins that are located on cell envelope, displayed on cell surface or are released into extracellular milieu. The term ‘exoproteome’ of a microorganism describes the protein content that can be found in its extracellular proximity. These proteins may arise from secretion, other protein export mechanisms or cell lysis, but only the most stable proteins in this environment would remain most abundant (Armengaud et al., 2012). These extracellular proteins are most likely to interact with the host cells, some of which may also have vaccine potential. Based on this premise, we investigated the exoproteome of B. abortus with the following objectives: (1) identification of extracellular proteins of B. abortus, (2) in silico analysis of the identified proteins and (3) validation of a selected candidate with respect to vaccine potential in mouse model.

Materials and methods

Animals

Female BALB/c mice (6–8 weeks old) were obtained from the Animal Facility of DRDE and were given water and food ad libitum. The mice were maintained and used in accordance with the relevant federal guidelines and institutional policies (protocol no. MB-02/10/SK). The challenged mice were housed in animal biocontainment safety level-3 (ABSL-3) facility.

Bacterial strains and growth conditions

Brucella abortus strain NCTC 10093 (544) was obtained from Central Public Health laboratory, Colindale, London, England. Brucella abortus vaccine strain S19 was retrieved from our own culture collection. For exoproteome work, a semi-synthetic medium [TSB/RPMI] containing RPMI, 1% yeast extract and 5% dialysed tryptic soy broth was prepared as described by Delpino et al. (2009). For all other studies, Brucella cultures were grown in Brucella broth (HiMedia, India) at 37 °C with 5% CO2 for 2–3 days. All live B. abortus manipulations were performed in BSL-3 facility.

Isolation of extracellular protein and two-dimensional gel electrophoresis (2-DE)

The TSB/RPMI semi-synthetic medium (1000 mL) was inoculated with 1% inoculum of 48-h grown culture of B. abortus strain NCTC 10093 in Brucella broth and incubated at 37 °C and 5% CO2 under static conditions. The growth was continued until 7 days, that is, until late stationary phase of bacteria. Bacterial culture was centrifuged at 18 000 g. for 15 min at 4 °C, and supernatant was collected and filtered with 0.45-μm filter. Filtrate was concentrated approximately 25 times and was processed for 2DE analysis by previously described procedure (Sengupta et al., 2010).

Identification of protein spots by tandem mass spectrometry and bioinformatics analysis

Gel pieces excised from 2-DE gels were destained at room temperature with 200 μL 50% ACN/50 mM NH4HCO3 for 1 h. Gel pieces were dried, and 100 ng trypsin (Promega) in 50 mM NH4HCO3 was added to each piece. Tryptic digestion was carried out overnight at 37 °C. Peptides were extracted with 60% acetonitrile and 0.1% trifluoroacetic acid (TFA), dried and resuspended in 0.5% TFA before MS analysis. Excised and digested proteins were identified by Applied Biosystem 4800 plus MALDI-TOF/TOF Analyzer (AB Sciex) using conditions as previously described (Mao et al., 2010; Dhanwani et al., 2011). Bioinformatics analysis of the identified proteins was carried out using different online tools as described previously (Sengupta et al., 2010).

Production of recombinant protein

Full-length open reading frames of rplI gene were amplified by PCR using primers L9F: 5′-tatgcacatatggaagtcattcttctgga-3′ and L9R: 5′-tagcagctcgagtcaagcctgatcttcagct-3′, having NdeI and XhoI restriction sites (underlined) at their 5′ end. The PCR product was cloned in pET28a+ vector and transformed into BL21 (DE3) cells by standard protocol (Sambrook & Russell, 2001). The recombinant protein (rL9) was expressed by induction with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). rL9 protein expressed in soluble fractions and was purified by Ni+-NTA resin. The purified protein was dialysed against buffer containing 50 mM NaH2PO4 (pH 8.0) to remove imidazole. The endotoxin level in purified rL9 was determined by a chromogenic LAL endotoxin assay kit (Lonza, Switzerland).

Determination of immunogenicity of L9 protein

A group of BALB/c mice (n = 15) was injected with three doses (day 0, 14 and 21) of rL9 protein formulated with aluminium hydroxide gel (rL9 + Al) by subcutaneous route. Each animal was injected 25 μg (100 μL) of rL9 protein per dose. Another two groups of mice were injected PBS (0.01 M, pH 7.2) or PBS with Al (PBS + Al) subcutaneously and were included as negative controls. Five mice were randomly selected from each immunized group, and blood samples were collected from the retro-orbital sinuses at 7, 17, 28, 49, 57, 63 and 71 days postinoculation. Levels of anti-rL9 antibodies were determined by enzyme-linked immunosorbent assay (ELISA) as previously described (Kumar et al., 2009). For cytokine determination, four mice from each group were sacrificed on 28 days after last immunization; their splenocytes were prepared, grown at 37 °C for 72 h in 5% CO2 and stimulated with 10 μg mL−1 of rL9 protein (Kumar et al., 2009). Supernatants were harvested and stored −70 °C until assayed for various cytokines by ELISA (BD OptEIA). Appropriate positive (Con A, 5 μg mL−1) and negative controls (without antigen) were also included in the experiment.

Protection assay

A group (n = 6) of mice (vaccine group) was inoculated intraperitoneally with B. abortus strain S19 (5 × 104CFU per mouse) on day 21 and was included as positive control for protection studies. On day 51 (30 days after last injection), six immunized mice from all groups were challenged with 2 × 105 CFU per mouse of B. abortus strain 544 by intraperitoneal injection. The challenged animals were sacrificed on day 81. The spleen of animals was removed, homogenized, serially diluted in PBS, plated on Brucella agar and incubated at 37 °C with 5% CO2. The numbers of colonies were counted after 48–72 h, and the results were expressed as the mean log CFU/spleen ± SD per group.

Statistical analysis

The experimental data between two groups were analysed using student's t-test, and data among several groups were analysed by one-way anova by SigmaStat (Jandel Scientific). A P value < 0.05 was considered significantly different.

Results

Identification of extracellular proteins

Extracellular proteins obtained after 4 days (early stationary phase) and 7 days (late stationary phase) of growth in TSB/RPMI semi-synthetic medium were analysed by 12% SDS-PAGE. A number of protein bands were observed in the extracellular fraction of 7 days old culture, and because the late stationary-phase exoproteome has not been described earlier for Brucella, this fraction was further analysed by 2-DE. A total of 49 spots were marked (Fig. 1) from which 26 proteins were identified (Table 1). For most of the proteins, identification was based on MS–MS analysis of ≥ 5 peptides from the MS spectrum of trypsin digested proteins. The MASCOT search results indicated the best match in B. abortus for almost all the identified proteins. Most of the experimental values of MW and pI for protein spots on 2-DE gel are close to the corresponding theoretical values, indicating unambiguous identification.

Table 1. Pattern/profile, post-translational modifications and topology search results for secreted proteins of Brucella abortus 10093, indicating theoretical localization
B. abortus proteinProtein spotsaPROSITEb signalp c secretomep d tmhmm e lipop f psortb g
  1. a

    Spot numbers refer to Fig. 1.

  2. b

    Scans a sequence against PROSITE or a pattern against the UniProt Knowledgebase (Swiss-Prot and TrEMBL).

  3. c

    The presence of signal peptide predicted by signalp 3.0.

  4. d

    Nonclassical, that is, nonsignal peptide-triggered protein secretion by secretomep. For each input sequence, the server predicts the possibility of nonclassical secretion. For bacteria, four scores are generated by the secretomep server for each input sequence. The determining score is the ‘SecP score’, for which a value above 0.5 indicates possible secretion. Values in parenthesis are SecP scores.

  5. e

    Number of predicted transmembrane helices in proteins using tmhmm server, version 2.0.

  6. f

    Prediction of lipoproteins and signal peptides using lipop 1.0.

  7. g

    Prediction of protein subcellular localization using psort version 2.0.

  8. OM, outer membrane; CytM, cytoplasmic membrane; Peri, periplasmic; Cyt, cytoplasmic.

Information storage and processing
 Translation, ribosomal structure and biogenesis (J)
50S ribosomal protein L916Ribosomal protein L9 signatureNoNo0Cyt (9.97)
Translation elongation factor Tu42GTP-binding elongation factors signatureNoNo0Cyt (9.97)
Cellular processes
Cell envelope biogenesis, outer membrane (M)
Hypothetical protein10No hitYesYes1SpIOM (9.92)
Penicillin acylase precursor38, 39No hitNoYes1Unknown
Cell motility and secretion (N)
Translocation protein TolB50, 51No hitYesYes1SpIPeri (9.44)
Post-translational modification, protein turnover, chaperones (O)
Twin-arginine translocation signal domain-containing protein12, 15Twin-arginine translocation (Tat) signal profile, Thioredoxin domain profileNoNo0SpIUnknown
Protease DO31PDZ domain profileNoYes1SpIPeri (9.76)
HlyD family secretion protein47No hitYesYes0SpICytM (9.82)
Inorganic ion transport and metabolism (P)
Molybdenum ABC transporter protein11No hitYesNo0SpIPeri (9.44)
Iron compound ABC transporter periplasmic iron37Iron siderophore/cobalamin periplasmic-binding domain profileYesYes0SpIPeri (9.44)
Metabolism
Amino acid transport and metabolism (E)
Glycine betaine/l-proline ABC transporter18, 34No hitYesYes0SpIUnknown
Glycine betaine/l-proline ABC transporter35No hitYesNo0SpIUnknown (expected multiple location)
2-isopropylmalate synthase26, 27, 28, 29, 30Pyruvate carboxyltransferase domain, alpha-isopropylmalate and homocitrate synthases signature 1, 2NoNo0Cyt (8.96)
Branched-chain amino acid ABC transporter21, 44No hitYesYes0SpIPeri (9.44)
Branched-chain amino acid ABC transporter22, 45No hitYesYes0SpIUnknown
Oligopeptide ABC transporter protein23, 24, 25Bacterial extracellular solute-binding proteins, family 5 signatureYesYes0SpIPeri (9.99)
Carbohydrate transport and metabolism (G)
d-galactose-binding periplasmic protein precursor19No hitYesYes0SpIUnknown
Sugar ABC transporter protein41No hitYesYes0SpIUnknown
Glycerol-3-phosphate ABC transporter protein52No hitYesYes0SpIPeri (10.0)
Omp2b porin43No hitYesYes0SpIOM (9.93)
Poorly characterized proteins
General function prediction only (R)
YaeC family lipoprotein33TAT twin-arginine translocation (Tat) signal profileYesNo0SpICytM (7.88)
31-kDa cell surface protein17, 36No hitNoYes0Unknown
Function unknown (S)
ABC transporter periplasmic substrate-binding protein20No hitYesYes0SpIUnknown
ABC transporter periplasmic substrate-binding protein40Bacterial extracellular solute-binding proteins, family 5 signatureYesYes0SpIPeri (10.0)
Omp28 precursor32No hitYesYes0SpIPeri (9.44)
NO known COG
Outer membrane protein Omp251, 2, 3, 4, 5, 6, 8, 13, 14, 46, 48No hitYesYes0SpIOM (10.0)
Figure 1.

Extracellular proteins of Brucella abortus. Two-dimensional Coomassie-stained gel of proteins in the culture supernatant of B. abortus strain NCTC 10093 in TSB/RPMI medium.

A number of ABC transporter proteins, outer membrane proteins, protease, ribosomal protein and hypothetical protein were among other proteins identified in exoproteome of B. abortus. Outer membrane protein 25, d-galactose periplasmic-binding protein, oligopeptide ABC transporter protein and isopropylmalate synthase were found to be the most abundant proteins. Clusters of orthologous groups (COG) were assigned to the proteins. Leaving apart proteins for which a COG could not been assigned, most (23%) proteins were predicted to be involved in amino acid transport and metabolism followed by carbohydrate transport and metabolism (15%; Table 1).

In silico analysis of protein similarity and features

A total of 19 proteins were predicted by signalp to be secreted in the classical Sec pathway, which is characterized by the presence of signal peptide. However, four proteins were predicted to have transmembrane helices, indicating an extra cytoplasmic, but membrane-associated location. psort predicted nine periplasmic, three outer membrane, three cytoplasmic, two cytoplasmic membrane-associated proteins and nine proteins of unknown localization. One of the proteins, glycine betaine/l-proline ABC transporter, predicted for unknown localization, was expected to be present in multiple locations (Table 1).

Production of purified rL9

Ribosomal protein L9 expressed an expected 23-kDa protein and was identified by SDS-PAGE and Western blotting (Fig. 2a–c). Expression level of rL9 was found to be 26 mg L−1 of medium and contained < 0.01 EU μg−1 of protein.

Figure 2.

Expression and purification of rL9. (a) Protein profile showing the expression of rL9 in Escherichia coli BL21 (DE3) cells. Lane 1: protein molecular mass marker (in kDa), Lane 2: uninduced E. coli BL21 (DE3) cell pellet, Lane 3: induced E. coli BL21 (DE3) cell pellet. (b) Western blots showing the specific expression of rL9 using monoclonal antipolyhistidine conjugate. Lane 1: induced E. coli BL21 (DE3) cell pellet, Lane 2: protein molecular mass marker. (c) SDS-PAGE analysis showing the affinity-purified rL9 protein. Lane 1: protein molecular mass marker, Lane 2: purified protein by immobilized metal affinity chromatography.

Immunogenicity of purified rL9

The generation of antigen-specific antibodies was measured by ELISA. rL9 elicited vigorous antibody response. The titre of IgG and IgG1 isotype increased steadily and remained elevated until the time of study (Fig. 3a and b). IgG2a titres after peaking 1 week after the last dose declined steadily (Fig. 3c). Stimulation of splenocytes with rL9 induced a significant (< 0.05) production of IFN-γ (Fig. 4a) and IL-4 (Fig. 4b) in spleen cells of rL9-immunized mice as compared to controls. No significant IL-2 or IL-10 production was observed. ConA mitogen induced production of cytokines in all groups (data not shown).

Figure 3.

Kinetics of humoral response: IgG antibody (a) and IgG1 (b), IgG2a (c) isotype response to rL9 protein in serum samples of five animals. The arrows below the x-axis show the day of immunization.

Figure 4.

Cytokine response: IFN-γ (a), IL-4 (b) production in the spleen cells of rL9 immunized mice and controls. Each sample was assayed in triplicate wells from four mice in each group. Cytokines in the culture supernatants were measured by sandwich ELISA. Significant differences of comparison (< 0.05) between antigen group and PBS or PBS + Al group are indicated by asterisks (*) and (o), respectively.

Recombinant L9 protects BALB/c mice against B. abortus infection

The potential of rL9 to confer protection was evaluated on the basis of their ability to reduce the bacterial load of virulent B. abortus in spleens of immunized mice. Mice inoculated with rL9 with Al exhibited about two-log reduction (protection units 2.11) in bacterial counts than PBS control (Table 2); S19 vaccine conferred 2.61 units of protection.

Table 2. Protection against Brucella abortus strain 544 provided to BALB/c mice by vaccination with rL9 protein (representative data from two separate experiments)
Treatment group (n = 6)AdjuvantLog10 CFU of B. abortus/spleenaUnits of protectionb
  1. a

    The number of bacteria in spleens (CFU/spleen) is represented as the mean log CFU ± SD per group.

  2. b

    Units of protection were obtained by subtracting the mean log CFU/spleen of the vaccinated group from the mean log CFU/spleen of the control (PBS)-immunized group.

  3. Significantly different (< 0.05) compared with value of controls (PBS group)a or (PBS + Al)b, ns = nonsignificant compared with PBS control as determined by one-way anova multiple comparison (Bonferroni t-test).

PBSNone5.78 ± 0.32
PBSAluminium hydroxide5.53 ± 0.380.25ns
rL9Aluminium hydroxide3.67 ± 0.272.11a,b
B. abortus S19None3.17 ± 0.542.61a,b

Discussion

The identification of extracellular proteins of many pathogenic bacteria has been subject of many recent studies (Tjalsma et al., 2004; Dumas et al., 2009; Sengupta et al., 2010; Barh et al., 2013). In the present work, 26 proteins were identified in late stationary-phase exoproteome of B. abortus. Some spots corresponded to the same protein with slight difference of pH or MW. Of the identified proteins, majority were predicted to be periplasmic (34.6%), outer membrane-/cytoplasmic membrane-associated (19%) or cytoplasmic (11.5%). A significant number (34.6%) was predicted to have unknown subcellular localization.

One of the major groups of extracellular proteins reported in the present work were identified as ABC transporter proteins and predicted to have periplasmic subcellular localization. Delpino et al. (2009); in their study on the differential proteome of early growth phase exoproteome of wild-type B. abortus and its isogenic virB mutant, also showed many sugar ABC transporters and outer membrane protein homologous to TRAP transporter conserved between both types of strains. They also identified 11 other protein, for example, chaperon protein DnaK (Hsp70), aspartate aminotransferase, polyribonucleotide nucleotidyltransferase, choloylglycine hydrolase, dihydrolipoamide dehydrogenase, which were only observed in WT B. abortus, were not observed in the present work. It seems that these proteins are secreted only in the early stationary phase of B. abortus. ATP-binding cassette (ABC) transporters are wide spread among living organisms and comprise one of largest protein families and are typically required for import and export of allocrites, for example, amino acids, carbohydrates, peptides and metal ions, across the cytoplasmic membrane. ABC transporters have been shown to have role in virulence in many bacterial species including Yersinia pestis, Salmonella typhimurium and Streptococcus pneumoniae (Garmory & Titball, 2004); hence, they may be suitable targets for generating deletion mutants for development of live attenuated antibacterial vaccines. Brucella abortus mutant having a deletion in virulence gene encoding the ABC transporter protein EsxA exhibited superior protection in mice against challenge with virulent B. abortus than the commercial vaccine (Rosinha et al., 2002). Role of ABC transporter in Brucella ovis pathogenesis has also been studied, and an ABC transporter deletion mutant was found attenuated than the wild-type strain in spleens and liver of BALB/c mice (Silva et al., 2011). It would be interesting to find out, in future, the role of ABC transporters identified in the present study in virulence or immunogenicity of Brucella.

Another group of proteins identified in the present work are outer membrane or cytoplasmic membrane proteins. Omp25, Omp2b and a hypothetical protein were predicted as outer membrane proteins. Omp25 has been reported to have vaccine potential (Goel & Bhatnagar, 2012). One of the predicted unknown proteins of this study, 31-kDa surface protein, has been shown to be protective in nature (Cassataro et al., 2005). Another protein identified in the present work, Omp28 (also called BP26), has been described to have the diagnostic potential in bovine brucellosis by our group earlier (Tiwari et al., 2011).

Three proteins in this study were predicted as cytoplasmic proteins. One of these proteins was identified as ribosomal proteins. Ribosomal protein L9 has previously been shown to be part of cell envelop proteome of B. abortus (Connolly et al., 2006). The results of present study are not sufficient enough to rule out the cell lysis; however, growing body of evidence suggests that the ribosomal proteins are associated with the inner membrane of prokaryotes (Sinha et al., 2005). Some cytoplasmic proteins have been actually shown to moonlight on bacterial cell surface or have more than one role in an organism (Henderson & Martin, 2011). Though, we cannot say anything about the moonlighting function of any of the proteins identified in the present work.

As many of the proteins identified in the present study have earlier been shown to confer protection in mouse model, for example Omp31 and Omp25, we selected one outer membrane protein ‘Omp2b’ and one predicted cytoplasmic protein ‘ribosomal protein L9’ to take up for their vaccine potential. The selection for Omp2b was straightforward for the reason it being an outer membrane protein; we were in addition tempted to explore the vaccine potential of ribosomal protein L9 because ribosomal fractions of Brucella were found to be immunogenic in an earlier study (Corbel, 1976), and extensive research has been carried out on ribosomal protein L7/L12 of Brucella sp. for its vaccine potential (Oliveira & Splitter, 1996; Mallick et al., 2007) where it was found to confer significant protection. Our initial experiments indicated that recombinant Omp2b protein did not protect mice against B. abortus infection; hence, it was not taken up further (results not shown).

In this work, human compatible aluminium hydroxide adjuvant was used to study the immunogenicity and protective potential of L9 protein. The IgG antibody and IgG1 isotype levels sustained over a period of time; IgG2a titres, however, declined after peaking on week 1 postinoculation. This may be because of Al adjuvant use, which is known to skew the antibody response to Th2 type. The immune response characterized by IFN-γ is associated with protection against brucellosis (Murphy et al., 2001; Paranavitana et al., 2005). IFN-γ causes upregulation of macrophage anti-Brucella activity (Oliveira et al., 1998), which is the main component of protective response. High levels of IFN-γ were produced in restimulated splenocytes when compared to PBS + Al and PBS control animals. Significant levels of IL-4 were also observed rL9 + Al group when compared with control animals. Similar to results of this study, many previous studies on recombinant Brucella vaccine candidates, for example, L7/L12, BLS, Omp31, Omp16 and Omp19 and AdoHcyase have earlier shown to elicit IgG antibody response in serum and production of IFN-γ in spleen cells of mouse model (Oliveira et al., 1994; Velikovsky et al., 2003; Cassataro et al., 2005; Pasquevich et al., 2009; Yang et al., 2011). Some of these and other studies also reported the production of IL-4 (BLS, SurA) or IL-2 (Omp31, BLS, SurA, AdoHcyase) cytokines (Velikovsky et al., 2003; Cassataro et al., 2005; Delpino et al., 2007; Yang et al., 2011).

Protective ability of rL9 formulation was studied in BALB/c mice. Vaccination with rL9 conferred significant protection (2.11 units) against challenge with virulent B. abortus strain 544 when compared with control group animals. The protection provided by L9 protein is comparable to or even better than some of earlier reported protein of Brucella, for example, L7/L12, BLS, Omp31, Omp16 and Omp19 (Oliveira & Splitter, 1996; Velikovsky et al., 2003; Cassataro et al., 2005; Pasquevich et al., 2009). It would be interesting to find in future that immunization of ribosomal protein L9 with adjuvants that skew responses to Th1 type or plasmid DNA vaccination may confer better protection.

In conclusion, the results of present study suggest that identification of extracellular proteins from Brucella may aid in discovery of better vaccines or diagnostic molecules, and ribosomal protein L9 in the form of recombinant protein formulation could be potential effective vaccine against B. abortus infection.

Acknowledgements

The authors are thankful to Director, DRDE, for providing all facilities and support required for this study.

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