Project description:Brucellosis is a serious zoonotic disease caused by the genus Brucella, a group of Gram-negative and facultative intracellular bacteria. At present, although live attenuated vaccine such as Rev.1, S19 and RB51 have been widely used for bovine brucellosis, these vaccines have some drawbacks, and safe and effective alternatives have been demanded. Previously, our group showed that several Brucella proteins malate dehydrogenase (rMDH) are effective in eliciting IgG responses in murine model following intraperitoneal injection. The Brucella infections are usually transmitted through mucosal membrane via oral or aerosol exposure. Therefore, induction of mucosal immunity is promising contribute in development of vaccine. In the study, rMDH was loaded with chitosan nanoparticles (CNs) which is mucoadhesive, biocompatible, nontoxic, and immune-enhancing adjuvant to induce both systemic and mucosal immunity. Then, in order to evaluate immunogenicity of the antigen in BALB/C mouse, total RNA from nasal-associated lymphoid tissues at 1 h, 6 h, 12 h after intranasal immunization was analyzed using RNA-seq.
Project description:Brucellosis, an important bacterial zoonosis caused by Brucella species, has drawn increased attention around the world. As an intracellular pathogen, the ability of Brucella to deal with stress within the host cell is closely related to its virulence. The survival pressure on Brucella within a phagosome is considered similar to that during the stationary phase. Here, label-free proteomics approach was used to study the adaptive response of Brucella abortus (B. abortus) in the stationary stage. 182 down-regulated and 140 up-regulated 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 logarithmic and stationary-phase B. abortus were treated with short-term starvation. The logarithmic-phase B. abortus restricted cell reproduction and energy utilization in response to nutritional stress. Additionally, the expression levels of some virulence-related proteins were identified as being significantly regulated during the transition from logarithmic to stationary phase or under starvation treatment, such as Type IV secretion system protein (T4SS), VjbR, and integration host factor (IHF). Altogether, we outlined adaptive mechanisms that B. abortus could employ during the growth and compared the differences between logarithmic and stationary-phase B. abortus in response to starvation.
Project description:Brucellosis is a serious infectious disease and continues to be an important cause of morbidity. It can be seen almost anywhere in the world and at any age. Acute phase heals or becomes chronic form. Infection of 10-30% of patients becomes chronic, despite early diagnosis and treatment. Although our knowledge about Brucella virulence factors and the host response increase rapidly, how they can hidden from the immune system and cause chronic disease are still unknown. We aimed to investigate the immunological factors which belong to CD4+ T cells and their roles in the transition of brucellosis from acute to chronic infection. Using miRNA microarray, more than 2000 miRNAs were screened in CD4 + T cells of patients with acute or chronic brucellosis and healthy controls that were sorted from peripheral blood with flow cytometry and validated by RT-qPCR. Findings were evaluated using GeneSpring GX (Agilent) 13.0 software and KEGG pathway analysis. In comparison to acute cases, expression levels of 28 miRNAs were significantly altered in chronic cases. All of miRNAs were up-regulated in chronic cases in comparison with the control group. Apart from a miRNA (miR-4649-3p), 27 miRNAs were not expressed in the acute cases (p <0.05, fold change> 2). These miRNAs have the potential to be markers for chronic cases. The differential expressed miRNAs and their predicted target genes that involved in previously MAPK signaling pathway, regulation of actin cytoskeleton, endocytosis, and protein processing in endoplasmic reticulum indicating their potential roles in chronic brucellosis and its progression. It is the first study of miRNA expression analyzes of human CD4+ T cells to clarify the mechanism of inveteracy in brucellosis.
Project description: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 bacteria 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, and a number of signature immunogenic proteins were identified. Two of these proteins, BMEII0032 and BMEI0892, were confirmed to be 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.
Project description:Infection of sheep with Brucella ovis results in ovine brucellosis, a disease characterized by infertility in rams, abortion in ewes and increased perinatal mortality in lambs. During the course of the infection both the ovine immune response and host cell gene expression are modified. The objective of this research was to conduct a preliminary characterization of differential gene expression in rams experimentally infected with B. ovis by microarray hybridization and real-time RT-PCR.
Project description:Brucellosis is one of the most common zoonotic epidemics worldwide. Vaccination against Brucellosis is an important control strategy to prevent the disease in many high-prevalence regions. At present, Brucella vaccine strain S2 is the most widely used vaccine in China. In this study, to uncover the related mechanisms underlie virulence attenuation of S2, we characterized the transcriptional profile of S2 and 1330 infected macrophages by transcriptome analysis. The results revealed that expressions of 440 genes were significantly different between macrophages infected by 1330 and S2. Data analysis showed that in the gene ontology term, the different expressed genes involved in innate immune response, phagoctyosis, recognition, and inflammatory response were significantly enriched. Pathway enrichment analysis indicated that the genes involved in transcriptional misregulation in cancer, staphylococcus aureus infection pathways and NF-kappa B signaling pathway were significantly affected. To reveal the molecular mechanisms related to different expression profiles of infected macrophages, the transcription levels of the different genes between the two bacterial genomes were also detected. In total, the transcription of 29 different genes was significantly changed in either culture medium or infected microphages. The results of current study can be conducive to the promotion of better understanding of the related mechanisms underlie virulence attenuation of S2 and interactions between host cells and brucella strains.
Project description:Many pathogenic bacteria use a regulatory process termed Quorum Sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection and response to these molecules depend on transcriptional regulators belonging to the LuxR family. Such a system have been discovered in the intracellular pathogen Brucella melitensis, a Gram-negative bacteria responsible for brucellosis, a word-wide zoonosis remaining a serious public health concern in endemic countries. Two LuxR-type regulators, VjbR and BabR, have been identified in the genome of this pathogen. The vjbR mutant is highly attenuated in all tested models suggesting a crucial role of QS in the virulence of Brucella. This attenuation is at least due to the involvement of VjbR in the activation of the virB operon coding for a type four secretion system essential for Brucella to reach its intracellular replication compartment. At present, no function has been attributed to BabR. To assess the role of both Brucella QS-regulators, we performed in tandem comparative transcriptomic and proteomic analyses of vjbR and babR mutants. These experiments revealed that 10% of Brucella genome is regulated through those regulators, revealing that QS is a global regulatory system in this intracellular pathogen. The overlapping between BabR and VjbR targets suggest an unexpected cross-talk between these two regulators. Moreover, our results demonstrate that VjbR and BabR regulate many gene and/or proteins involved in stress response, metabolism and virulence. These targets are potentially involved in the adaptation of Brucella to the oxidative, pH and nutritional stresses encountered within the host. These findings highlight the involvement of QS in the virulence of Brucella and led us to suggest that this regulatory system could be implied in the spatial and sequential adaptation of Brucella to the host environment. Keywords: Quorum Sensing, Comparative gene expression, Brucella melitensis
Project description:Brucella abortus (B. abortus), an intracellular bacterium, is the causative agent of Brucellosis. This organism invades into macrophages and then survives through its abilities to modulate host cells functions. The biggest problem caused by B. abortus is that it prevents macrophage elimination and makes it difficult to remove B. abortus from the host body. Therefore, it is essential to identify the bacterial genes involved in virulence factor as a first step to understanding the bacterial pathogenicity and controlling Brucellosis. To identify these genes, B. abortus mutant strains were generated using transposon mutagenesis and transcriptomic profile during macrophage infection were analyzed. The gene expression level was analyzed using total RNA obtained from THP-1 cells infected with B. abortus wild type and mutant strains and cellular immunity during the infections were compared to wild type infected cell to identify the role of genes in B. abortus pathogenicity. Transcriptomic profiling showed that two mutant strains having disrupted genes related to 4-hydrobenzoate 3-monooxygenase (PHBH) of C1 strain and heme exporter protein cytochrome C (CcmC) of C10 strain, induced suppression of cytokine expression during infection in human macrophages. Conversely, two other mutant strains of exopolyphosphatase (PPX)of C27 and Peptidase M24 of C32 induced activation of cytokine expression in the THP-1 macrophage cells.