Project description:Goal: High-throughput sequencing-by-synthesis (Illumina) RNA sequencing technology was carried out with an aim to gain deeper understanding of immune host protective mechanisms. Here, RNA-seq was applied to understand the differential gene expression profile of mice spleen following immunization with Brucella abortus S19∆per mutant (perosamine synthetase gene mutant of Brucella abortus S19) in comparison to mock immunized mice spleen (PBS inoculated). Methods: RNA-seq data of 15th day post immunized mice spleen (with Brucella abortus S19∆per) and PBS control mice were generated by deep sequencing ( in duplicate) using IlluminaNextSeq 500 . The sequence reads that passed quality filters were analyzed for transcript abundance using RSEM package (RNA-Seq by Expectation Maximization) (Li and Dewey, 2011). Breifly, the RSEM package generated a reference sequence based on given mouse transcript annotations (Mus_musculus.GrCm38.83.chr.gtf.gz). The Bowtie allignmet tool available within the package was used to calculate expected counts (number of mapped reads) using quality trimmed reads and reference sequence. Finally, the expected counts estimated by RSEM were fed into different DE package tools, such as DESeq2 (Love et al., 2014), edgeR (Robinson et al., 2010) and EBSeq (Leng et al., 2013) in order to identify differentially expressed genes across spleen samples (B. abortus S19∆per versus (vs) PBS control. Functional annotation of differently expressed genes were carried out using g:Profiler (Reimandet al., 2016; http://biit.cs.ut.ee/gprofiler/). Results: A mean of 37.58 million processed reads (range: 30.51 million to 51.79 million reads per individual RNA-seq library) were generated during the experiment. The expected counts generated by the RSEM package followed by differential analysis calculation using different DE packages identified a total of 1917 differentially expressed genes (DEGs), of which 968 and 949 genes were up- and down-regulated respectively. Functional annotation revealed 545 significantly enriched genes to be associated with immune system processes within the total 1917 differentially expressed genes. Further analysis revealed 21 genes showing significant expression were also in MHC-I and MHC-II antigen processing and presentation pathway during S19∆per immunization. Conclusions:The RNA-seq data revealed the coordinated up-regulation of MHC-I and MHC-II processing pathways providing insights into the molecular mechanism of immune protection conferred by B. abortus S19∆per in mice at day 15 post immunization and might aid in the development of new attenuated vaccine strains with improved efficacy.
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: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.
Project description:Isogenic deletion and truncation of specific genes encoding RNases in Brucella abortus were analyzed for changes in gene expression. The main goal of this work is to determine the mRNAs that exhibit dysregulation when small regulatory RNAs (i.e., Bsr8) or RNases (i.e., RNaseE and RNaseJ) are invactivated in Brucella abortus. Small regulatory RNAs often control gene expression by binding directly to mRNAs to block translation or induce their degradation, and RNA from a deletion of one sRNA gene, bsr8, was analyzed to uncover the mRNAs that may be controlled by BsrB. RNases are enzymes that cleave RNAs during processing, turnover, and regulatory events, and RNaseE and RNaseJ appear to be important for B. abortus virulence. Therefore, to determine the mRNAs potentially targetd by these RNases, RNA from a strain harboring a RNaseE truncation and a strain carrying a deletion of rnaseJ were analyzed. In the end, the objective of this study was to gain insight into the regulatory patterns of specific B. abortus sRNAs and RNases.
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:Brucella type strains (B. abortus 544 and B. melitensis 16 M), field-isolates (B. abortus T and B. melitensis C) were independently cultivated six times on Tryptic Soy Broth at 37 °C in the presence of 5% CO2 until mid-logarithmic phase. The cells were harvested by brief centrifugation and the bacteria was inactivated using recommended ethanol precipitation procedure. Following which the cells were lysed, proteins quantified and the differentially expressed proteins among B. abortus and B. melitensis were identified using liquid chromatography-mass spectrometry (LC/MS) based label free quantitative proteomics analysis.
Project description:Brucellosis is still a widespread zoonotic disease. Very little is known about the interaction between B. abortus and trophoblastic cells, which is essential for better understanding the pathogenesis of the Brucella-induced placentitis and abortion, a key event for transmission of the disease. The goal of this study was to evaluate the profile of gene expression by bovine trophoblastic cells during infection with B abortus. Explants of chorioallantoic membranes were inoculated with B. abortus strain 2308. Microarray analysis was performed at 4 h after infection, and expression of cytokines and chemokines by trophoblastic cells was assessed by real time RT-PCR at 6 and 12 h after inoculation. In addition, cytokine and chemokine expression was evaluated in placentomes from experimentally infected cows. Expression of pro-inflammatory genes by trophoblastic cells was suppressed at 4 h after inoculation, whereas a significant up-regulation of CXC chemokines, namely CXCL6 (GCP-2) and CXCL8 (IL-8), was observed at 12, but not at 6 h after inoculation. Placentomes of experimentally infected cows had a similar profile of chemokine expression, with upregulation of CXCL6 and CXCL8. Our data indicate that B. abortus modulates the innate immune response by trophoblastic cells, suppressing expression of pro-inflammatory mediators during the early stages of infection that is followed by a delayed and mild expression of pro-inflammatory chemokines, which is similar to the profile of chemokine expression in the placentomes of experimentally infected cows. This trophoblastic response is likely to contribute to the pathogenesis of B. abortus-induced placentitis. Keywords: trophoblast response to Brucella
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.