Project description:Intracellular proteolysis via ATP-dependent proteases is a conserved biological process. The ATP-dependent Clp protease is made up of peptidase ClpP and ATP-dependent chaperones and plays an important role in the proteolysis process. In several pathogenic bacteria, the Clp protease is implicated in the stress reponses and contributes to the bacterial virulence. In brucella abortus, the ΔclpP mutant strain exhibited a pronounced growth defect in TSB medium and the results showed that the ΔclpP mutant was sensitive to high temperature, high osmotic environment and iron deficient environment. In addition, deletion of the clpP significantly affected Brucella virulence in macrophage and mice infection models. Based on the iTRAQ analysis, the different expressed proteins were mainly involved in amino acid transport and metabolism, energy production and conversion, and secondary metabolites biosynthesis, transport and catabolism. Overall, our study revealed preliminary molecular mechanism between Clp protease, bacterial growth, stress response and bacterial virulence in Brucella strains
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.
Project description:Purpose:To gain insight into the spectrum of Brucella gene affected by the ClpP protease, as well as the genetic basis for the distinctive phenotypic properties exhibited by the clpP mutant strain. Methods: Three B. abortus 2308 and the three clpP mutant were grown in TSB at 37℃ until the log phase was reached, total RNA was isolated using the TRIzol according to the manufacturer’s instructions.The sequencing library of each RNA sample was prepared by using NEB Next Ultra Directional RNA Library Prep Kit for Illumina as recommended by the manufacturer.The HiSeq 4000 platform was used to perform the transcriptome sequencing. Results: In total, 772 genes that exhibited different expression (Fold change>4, and FDR<0.05) were identified in the clpP mutant. In the clpP mutant strain, 376 genes were up-regulated and 396 genes were down-regulated. Conclusions: According to the result of COG analysis, the different expressed genes were mainly involved in Energy production and conversion, Cell wall/membrane/envelope biogenesis, and Carbohydrate transport and metabolism. KEGG pathway analysis demonstrated that the genes exhibited significant difference were primarily enriched in ABC transporters, beta-Lactam resistance, Oxidative phosphorylation, Cationic antimicrobial peptide (CAMP) resistance, Limonene and pinene degradation, Lysine degradation, Valine, leucine and isoleucine degradation, Chloroalkane and chloroalkene degradation, Ascorbate and aldarate metabolism, Tryptophan metabolism, Citrate cycle (TCA cycle), and Histidine metabolism.
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: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: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. B. abortus strain 2308 or mucR mutant cells were grown to stationary phase growth, total bacterial RNA was isolated and subjected to GeneChip hybridization and analysis. We sought to determine the regulatory effects of MucR.
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.
