Project description:We used B. melitensis M5-90 wild type to construct gene-deleted strains of B. melitensis ∆per, RAW264.7 cells infected with B. melitensis M5-90 and B. melitensis M5-90 ∆per for 4h, respectively. miRNA microarray and mRNA array experiments were performed, qRT-PCR validation for miRNAs and mRNAs. We performed a joint analysis of differentially expressed miRNAs and mRNAs, and proved that the target gene of miRNA-146b is Tbc1d14. It further confirmed that miR-146b targeting tbc1d14 regulates brucella-mediated autophagy of RAW264.7 cells. Finally, the molecular mechanism of tbc1d14 influencing brucella-mediated autophagy of RAW264.7 cells was preliminarily revealed by DGE sequencing.
Project description:We used B. melitensis M5-90 wild type to construct gene-deleted strains of B. melitensis ∆per, RAW264.7 cells infected with B. melitensis M5-90 and B. melitensis M5-90 ∆per for 4h, respectively. miRNA microarray and mRNA array experiments were performed, qRT-PCR validation for miRNAs and mRNAs. We performed a joint analysis of differentially expressed miRNAs and mRNAs, and proved that the target gene of miRNA-146b is Tbc1d14. It further confirmed that miR-146b targeting tbc1d14 regulates brucella-mediated autophagy of RAW264.7 cells. Finally, the molecular mechanism of tbc1d14 influencing brucella-mediated autophagy of RAW264.7 cells was preliminarily revealed by DGE sequencing.
Project description:Investigation of whole genome gene expression level changes in a Brucella melitensis delta prlr mutant compared to the wild type strain. The mutants analyzed in this study are further described in A. Mirabella, R-M Yanez, R.M. Delrue, S. Uzureau, M.S. Zygmunt, A. Cloeckaert, X. De Bolle, J.J. Letesson (2012). The two component system PrlS/PrlR of Brucella melitensis is required for persistence in mice and appears to respond to ionic strength. Microbiology
Project description:Investigation of whole genome gene expression level changes in a Brucella melitensis delta prlr mutant compared to the wild type strain. The mutants analyzed in this study are further described in A. Mirabella, R-M Yanez, R.M. Delrue, S. Uzureau, M.S. Zygmunt, A. Cloeckaert, X. De Bolle, J.J. Letesson (2012). The two component system PrlS/PrlR of Brucella melitensis is required for persistence in mice and appears to respond to ionic strength. Microbiology A six chip study using total RNA recovered from three separate wild-type cultures of Brucella melitensis 16M and three separate cultures of a prlR mutant strain. Each chip measures the expression level of 3,198 genes from Brucella melitensis 16M with nineteen 60 mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
Project description:Brucella spp. is an intracellular pathogen in vivo. The intracellular B. melitensis transcriptome was determined by initially enriched and then amplified B. melitensis RNA from total RNA of B. melitensis-infected HeLa cells. Analysis of microarray results identified 161 and 115 genes differentially expressed at 4 and 12 h p.i., respectively. Most of the genes (78%) differentially expressed were down-regulated at the earliest time point, but up-regulated (75%) at 12 h p.i. The analysis of the results indicates that Brucella undergo an adaptation period during the first 4 h p.i. that is overcome by 12 h p.i., permitting Brucella to replicate intracellularly. Specific genes and biological processes identified in this study will further help elucidate how Brucella act during the early infectious process to their eventual benefit and to the detriment of the naïve host. Keywords: Time course study of intracellular B. melitensis gene expression
Project description:Brucella spp. is an intracellular pathogen in vivo. The intracellular B. melitensis transcriptome was determined by initially enriched and then amplified B. melitensis RNA from total RNA of B. melitensis-infected HeLa cells. Analysis of microarray results identified 161 and 115 genes differentially expressed at 4 and 12 h p.i., respectively. Most of the genes (78%) differentially expressed were down-regulated at the earliest time point, but up-regulated (75%) at 12 h p.i. The analysis of the results indicates that Brucella undergo an adaptation period during the first 4 h p.i. that is overcome by 12 h p.i., permitting Brucella to replicate intracellularly. Specific genes and biological processes identified in this study will further help elucidate how Brucella act during the early infectious process to their eventual benefit and to the detriment of the naM-CM-/ve host. Keywords: Time course study of intracellular B. melitensis gene expression Gene expression of the intracellular Brucella melitensis was determined at 4 and 12 h p.i. We generated the following samples: A) B. melitensis total RNA enriched and amplified from total RNA of B. melitensis-infected HeLa cells at 4 h p.i.; B) Total RNA isolated from B. melitensis-infected HeLa cells at 4 h p.i.; C) B. melitensis total RNA enriched and amplified from total RNA of B. melitensis-infected HeLa cells at 12 h p.i.; D) Total RNA isolated from B. melitensis-infected HeLa cells at 12 h p.i. B. melitensis total RNA was initially enriched and then amplified from total RNA of B. melitensis-infected HeLa cells at 4 and 12 h p.i. in quadruplicate, indirectly labeled and co-hybridized against B. melitensis gDNA to a custom 3.2K B. melitensis oligo-array (n = 8). As there was a possibility that some HeLa transcripts cross-hybridize with probes on B. melitensis microarrays, the original total RNA from B. melitensis-infected HeLa cells were also co-hybridized against B. melitensis gDNA to B. melitensis oligo-arrays (n = 8), and any oligospots with signals were considered non-specific and eliminated from all analysis to avoid false positive gene detection. The intracellular B. melitensis gene expression was compared to the gene expression of the inoculum (n = 2). Every Brucella melitensis open reading frame was printed in triplicate on each microarray, thereby providing three technical replicates for each biological replicates. Each replicate was normalized against labeled Brucella melitensis genomic DNA.
Project description:In this study we report that B. melitensis at the late logarithmic phase of growth are more invasive for HeLa cells than at mid logarithmic or stationary growth phases. Microarray analysis of B. melitensis gene expression identified 414 up- and 40 down-regulated genes in late-log growth phase compared to the stationary growth phase. The vast majority of the up-regulated genes in late-log cultures were those associated with DNA replication, transcription and translation, intermediate metabolism, energy production and conversion, membrane transport and cell envelope, biogenesis and outer membrane, while the down-regulated genes were distributed among several functional categories. This first Brucella global gene expression study provides novel information on growth phase-specific gene regulation important not only for understanding Brucella physiology but also the initial molecular interactions between Brucella and its host. Keywords: Comparison bacterial growth phase normalized to genomic DNA There are two kind of samples consisting of RNA isolated from Brucella melitensis grown logarithmically or at stationary phase. There are four biological replicates of each sample. Every Brucella melitensis open reading frame was printed in triplicate on each microarray, thereby providing three technical replicates for each biological replicates. Each replicate was normalized against labeled Brucella melitensis genomic DNA.