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:Brucella, a notorious intracellular pathogen, causes chronic infections in many mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane; protein substrates translocated by Brucella include ABC transporters, oxidoreductases, and cell envelope biosynthesis proteins. Previously, we showed that a Tat mutant of Brucella melitensis M28 exhibits reduced survival within murine macrophages. In this study, we compared the host responses elicited by wild-type M28 and its Tat-mutant strains ex vivo. We utilized label-free quantitative proteomics to assess proteomic changes in RAW264.7 macrophages after infection with M28 and its Tat mutants.
Project description:In a time course study, we characterized global gene expression profile of B. melitensis-infected epithelium-like cells at the onset of infection. B. melitensis-infected HeLa cells exhibited a down-regulated expression profile at 4 h (48 up- and 109 down-regulated genes) that transitioned to an activated transcriptional profile at 12 h post-infection (733 up- and 224 down-regulated genes). The analysis of the results indicates that infected cells undergo an adaptation period during the first 4 h p.i. that is overcome by 12 h p.i., permitting Brucella to replicate intracellularly while minimally affecting host physiological processes. Specific genes and biological processes identified in this study will further help elucidate how both host and Brucella interact during the early infectious process to the eventual benefit of the pathogen and to the detriment of the naïve host. Keywords: Time course study of gene expression profile of Brucella melitensis-infected HeLa cells
Project description:In a time course study, we characterized global gene expression profile of B. melitensis-infected epithelium-like cells at the onset of infection. B. melitensis-infected HeLa cells exhibited a down-regulated expression profile at 4 h (48 up- and 109 down-regulated genes) that transitioned to an activated transcriptional profile at 12 h post-infection (733 up- and 224 down-regulated genes). The analysis of the results indicates that infected cells undergo an adaptation period during the first 4 h p.i. that is overcome by 12 h p.i., permitting Brucella to replicate intracellularly while minimally affecting host physiological processes. Specific genes and biological processes identified in this study will further help elucidate how both host and Brucella interact during the early infectious process to the eventual benefit of the pathogen and to the detriment of the naM-CM-/ve host. Keywords: Time course study of gene expression profile of Brucella melitensis-infected HeLa cells We generated 4 different samples: A) RNA isolated from Brucella melitensis-infected HeLa cells at 4 h p.i.; B) RNA isolated from non-infected HeLa cells at 4 h p.i. of the A samples; C) RNA isolated from Brucella melitensis-infected HeLa cells at 12 h p.i., and D) RNA isolated from non-infected HeLa cells at 12 h p.i. of the D samples. The B. melitensis-infected HeLa cells gene expression was compared with gene expression from non-infected HeLa cells treated similarly. There are four biological replicates of each sample and time point (n=16). ORFs were single spotted on each microarray. Each replicate was normalized against labeled universal human reference RNA.
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: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.