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Brucella abortus is an important zoonotic pathogen that causes severe economic loss to husbandry and poses a threat to human health. The B. abortus A19 live vaccine has been extensively used to prevent bovine brucellosis in China. However, it is difficult to distinguish the serological response induced by A19 from that induced by natural infection. In this study, a novel genetically marked vaccine, A19ΔvirB12, was generated and evaluated. The results indicated that A19ΔvirB12 was able to provide effective protection against B. abortus 2308 (S2308) challenge in mice. Furthermore, the safety and protective efficacy of A19ΔvirB12 have been confirmed in natural host cattle. Additionally, the VirB12 protein allowed for serological differentiation between the S2308 challenge/natural infection and A19ΔvirB12 vaccination. However, previous studies have found that the accuracy of the serological detection based on VirB12 needs to be improved. Therefore, we attempted to identify potential supplementary antigens with differential diagnostic functions by combining label-free quantitative proteomics and protein chip technology. Twenty-six proteins identified only in S2308 were screened; among them, five proteins were considered as potential supplementary antigens. Thus, the accuracy of the differential diagnosis between A19ΔvirB12 immunization and field infection may be improved through multi-antigen detection. In addition, we explored the possible attenuation factors of Brucella vaccine strain. Nine virulence factors were downregulated in A19ΔvirB12. The downregulation pathways of A19ΔvirB12 were significantly enriched in quorum sensing, ATP-binding cassette transporter, and metabolism. Several proteins related to cell division were significantly downregulated, while some proteins involved in transcription were upregulated in S2308. In conclusion, our results contribute to the control and eradication of brucellosis and provide insights into the mechanisms underlying the attenuation of A19ΔvirB12.

Brucellosis, an important bacterial zoonosis caused by Brucella species, has drawn increasing attention worldwide. As an intracellular pathogen, the ability of Brucella to deal with stress within the host cell is closely related to its virulence. Due to the similarity between the survival pressure on Brucella within host cells and that during the stationary phase, a label-free proteomics approach was used to study the adaptive response of Brucella abortus in the stationary stage to reveal the possible intracellular adaptation mechanism in this study. A total of 182 downregulated and 140 upregulated proteins were found in the stationary-phase B. abortus. B. abortus adapted to adverse environmental changes by regulating virulence, reproduction, transcription, translation, stress response, and energy production. In addition, both exponential- and stationary-phase B. abortus were treated with short-term starvation. The exponential B. abortus restricted cell reproduction and energy utilization and enhanced material transport in response to nutritional stress. Compared with the exponential phase, stationary Brucella adjusted their protein expression to a lesser extent under starvation. Therefore, B. abortus in the two growth stages significantly differed in the regulation of protein expression in response to the same stress. Overall, we outlined the adaptive mechanisms that B. abortus may employ during growth and compared the differences between exponential- and stationary-phase B. abortus in response to starvation.

Background Brucella spp. is an important zoonotic pathogen responsible for brucellosis in humans and animals. Brucella abortus A19 strain is a widespread vaccine in China. However, it has a drawback of residual virulence in animals and humans. Methods In this study, the BALB/c mice were inoculated with either 100 μL PBS(control group, C group), 109 CFU/mL inactivated B. abortus A19 strain (I group), 105 CFU/mL (low-dose group, L group) 106 CFU/mL live B. abortus A19 strain (high-dose group, H group), or 105 CFU/mL live B. abortus A19 strain combined with 109 CFU/mL inactivated B. abortus A19 strain (LI group). Mice were challenged with B. abortus strain 2308 at 7 week post vaccination. Subsequently, the immune and protective efficacy of the vaccines were evaluated by measuring splenic bacterial burden, spleen weight, serum IgG, interferon-gamma (IFN-γ), interleukin-4 (IL-4) percentage of CD4 + and CD8 + T cells of mice via bacterial isolation, weighing, ELISA and flow cytometry, respectively. Results The splenic bacterial burden and spleen weight of the mice in group LI were mostly equivalent to the mice of group H. Moreover, Brucella-specific serum IgG, IFN-γ, IL-4, and the percentage of CD4+ and CD8+ T cells of the LI group mice were similar to those of the H group. In the subsequent challenge test, both vaccines conferred protective immunity to wild-type (WT) 2308 strain. In addition, the levels of IL-4 and IFN-γ, CD4+ and CD8+ T cells in these mice were similar to those of the mice in the H group. Conclusions Combined immunization with low dose live vaccine and inactivated vaccine allowed to reduce the live B. abortus A19 vaccine, dose with an equivalent protection of the high-dose live vaccine.

The myriad of posttranslational modifications (PTMs) of proteins that occur in all living cells are crucial to all kinds of biological processes. Brucella is an intracellular parasitic bacterium that can cause chronic diseases in both humans and livestock. To reveal the relationship between PTMs and the virulence and survival of Brucella, we described the first comprehensive multiple PTM-omics atlas of B. abortus 2308. Five PTMs involving lysine, namely 2-hydroxyisobutyrylation, succinylation, crotonylation, acetylation, and malonylation were identified. Nearly 2,000 modified proteins were observed, and these proteins took part in many biological processes, with a variety of molecular functions. In addition, we detected many significant virulence factors of Brucella among the modified proteins. 10 of the 15 T4SS effector proteins were detected with one or more PTMs. Moreover, abundant PTMs were detected in other typical virulence factors. Considering the role of PTMs in various biological processes of Brucella virulence and survival, we propose that the virulence of Brucella is associated with the PTMs of proteins. Taken together, this study provides the first global survey of PTMs in Brucella. This is a prospective starting point for further functional analysis of PTMs during the survival of Brucella in hosts, interpretation of the function of Brucella proteins, and elucidation of the pathogenic mechanism of Brucella.

Brucellosis is an important zoonotic disease that causes great economic losses. Vaccine immunisation is the main strategy for the prevention and control of brucellosis. Although live attenuated vaccines play important roles in the prevention of this disease, they also have several limitations, such as residual virulence and difficulty in the differentiation of immunisation and infection. We developed and evaluated a new bacterial ghost vaccine of Brucella abortus A19 by a new double inactivation method. The results showed that the bacterial ghost vaccine of Brucella represents a more safe and efficient vaccine for brucellosis. We further characterised the antigenic components and signatures of the vaccine candidate A19BG. Here, we utilised a mass spectrometry-based label-free relative quantitative proteomics approach to investigate the global proteomics changes in A19BGs compared to its parental A19. The proteomic analysis identified 2014 proteins, 1116 of which were differentially expressed compared with those in A19. The common immunological proteins of OMPs (Bcsp31, Omp25, Omp10, Omp19, Omp28, and Omp2a), HSPs (DnaK, GroS, and GroL), and SodC were enriched in the proteome of A19BG. By protein micro array-based antibody profiling, significant differences were observed between A19BG and A19 immune response, and a number of signature immunogenic proteins were identified. Two of these proteins, the BMEII0032 and BMEI0892 proteins were significantly different (P < 0.01) in distinguishing between A19 and A19BG immune sera and were identified as differential diagnostic antigens for the A19BG vaccine candidate. In conclusion, using comparative proteomics and antibody profiling, protein components and signature antigens were identified for the ghost vaccine candidate A19BG, which are valuable for further developing the vaccine and its monitoring assays.

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