Project description:Gene expression analysis of wild-type and STING knock-out mouse bone marrow-derived macrophages (mBMDM) infected with Brucella abortus or transfected with Brucella abortus DNA. Genes whose expression are affected by Brucella abortus in a STING-dependent manner will be identified and signaling pathways regulated by STING will be elucidated.
Project description:In a time course study, we characterized global gene expression profile of B. abortus-infected macrophages from cattle naturally resistant (R) and susceptible (S) to brucellosis. B. abortus infection causes early down-regulation of transcript levels in Mø from R cattle at 4 h p.i. (22 up- and 126 down-regulated genes) which is reversed by 12 h post-infection (31 up- and 25 down-regulated genes), compared to uninfected control. On the other hand, B. abortus-infected S bovine macrophages exhibited a down-regulated expression profile at 4 (45 up- and 65 down-regulated genes) and 12 h p.i. (47 up- and 193 down-regulated genes). The analysis of the results indicates that B. abortus – infected Mø from cattle naturally R and S to brucellosis display different transcriptional profiles. Specific genes and biological processes identified in this study will further help elucidate how different macrophages from resistant and susceptible animals interact with Brucella during the early infectious process. Keywords: Expression profiling by microarray
Project description:We focused on whether transposon mutagenesis in Brucella abortus could induce difference in the trascriptional responses of RAW 264.7 cell infection model compared to the wild strain infected RAW 264.7 cells. The function of genes in Brucella abortus was analyzed through the identified differences in gene expression between RAW 264.7 cell infected with wild and mutant strains.
Project description:We focused on whether transposon mutagenesis in Brucella abortus could induce difference in the trascriptional responses of RAW 264.7 cell infection model compared to the wild strain infected RAW 264.7 cells. The function of genes in Brucella abortus was analyzed through the identified differences in gene expression between RAW 264.7 cell infected with wild and mutant strains. We analyzed altered transcription in RAW 264.7 cells at 0, 6, 12, and 24 h following the infection with 10 MOI of Brucella abortus wild and mutant strains.
Project description:Microarray analysis determined that 7.82% (244/3334) of Brucella abortus genes were up-regulated and 5.4% (180/3334) were down-regulated in RAW264.7 macrophages, compared to free-living bacteria in TSB In the study, Brucela abortus was isolated from infected macrophages at 24 post-infection, DNA microarray was used to analysis the differentially expressed genes between intracellular bacteria and free-living ones in TSB
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.
Project description:Identification of host responses at the gene transcription level provides a molecular profile of the events that occur following infection. Brucella abortus is a facultative intracellular pathogen of macrophages that induces chronic infection in humans and domestic animals. Using microarray technology, the response of macrophages 4 hours following B. abortus infection was analyzed to identify early intracellular infection events that occur in macrophages. Of the more than 6,000 genes, we identified over 140 genes that were reproducibly differentially transcribed. First, an increase in the transcription of a number of pro-inflammatory cytokines and chemokines, such as TNF-α, IL-1β, IL-1α, and members of the SCY family of proteins, was evident that may constitute a general host recruitment of antibacterial defenses. Alternatively, Brucella may subvert newly arriving macrophages for additional intracellular infection. Second, transcription of receptors and cytokines associated with antigen presentation, e.g., MHC class II and IL-12p40, were not evident at this 4 hour period of infection. Third, Brucella inhibited transcription of various host genes involved in apoptosis, cell cycling, and intracellular vesicular trafficking. Identification of macrophage genes whose transcription was inhibited suggests that Brucella utilizes specific mechanisms to target certain cell pathways. In conclusion, these data suggest that B. abortus can alter macrophage pathways to recruit additional macrophages for future infection while simultaneously inhibiting apoptosis and innate immune mechanisms within the macrophage permitting intracellular survival of the bacterium. These results provide insights into the pathogenic strategies used by Brucella to survive long-term within a hostile environment. Keywords: Macrophage, intracellular pathogen, Brucella abortus, inflammatory immune response
Project description:Brucellosis is one of the most widespread bacterial zoonoses worldwide. Here, our aim was to identify the effector mechanisms controlling the early stages of intranasal infection with Brucella in C57BL/6 mice. During the first 48 hours of infection, alveolar macrophages (AMs) are the main cells infected in the lungs. Using RNA sequencing, we identified the aconitate decarboxylase 1 gene (Acod1; also known as Immune responsive gene 1), as one of the genes most upregulated in murine AMs in response to B. melitensis infection at 24 hours post-infection. Upregulation of Acod1 was confirmed by RT-qPCR in lungs infected with B. melitensis and B. abortus. We observed that Acod1-/- C57BL/6 mice display a higher bacterial load in their lungs than wild-type (wt) mice following B. melitensis or B. abortus infection, demonstrating that Acod1 participates in the control of pulmonary Brucella infection. The ACOD1 enzyme is mostly produced in mitochondria of macrophages, and converts cis-aconitate, a metabolite in the Krebs cycle, into itaconate. Dimethyl itaconate (DMI), a chemically-modified membrane permeable form of itaconate, has a dose-dependent inhibitory effect on Brucella growth in vitro. Interestingly, modelling studies suggest the binding of itaconate into the binding site of isocitrate lyase. DMI does not inhibit multiplication of the isocitrate lyase deletion mutant ∆aceA B. abortus in vitro. Finally, we observed that, unlike the wt strain, the ∆aceA B. abortus strain multiplies similarly in wt and Acod1-/- C57BL/6 mice. These data suggest that bacterial isocitrate lyase might be a target of itaconate in AMs.
Project description:We describe the isolation of sufficient Brucella abortus RNA from primary host cell environment using modified reported methods for RNA-seq analysis, and simultaneously characterize the transcriptional profiles of intracellular B. abortus and bone marrow-derived macrophages (BMM) from BALB/c mice at 24 h (replicative phase) post-infection.