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:MavR is a 160 nucleotide regulatory RNA molecule that is produced during B. abortus strain 2308 growth in nutrient-replete broth. We used custom-made Affymetrix B. abortus strain 2308 derived GeneChips to compare the gene expression properties of wild type and isogenic mavR mutant cells.
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:MucR is one of the few transcriptional regulatory proteins that has been linked to Brucella pathogenesis. We used custom-made Affymetrix B. abortus strain 2308 derived GeneChips to copare the gene expression properties of wild type and isogenic mucR mutant cells.
