Project description:In a time course study, we characterized global gene expression profile of B. melitensis-infected bovine Peyer patches in the first 4 h p.i. Microarray analysis revealed that 2,916 bovine genes were detected as differentially expressed (z-score p < 0.025) in loops inoculated with virulent B. melitensis 16M compared with controls between 15 min and 4 h post-infection. From these genes, 2,286 (78%) were up- and 630 (22%) were down-regulated. 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. Microarrays were used to examine the transcriptional profiles of bovine intestinal Peyer patches infected with wild type Brucella melitensis 16M across five time points (15 min, 30 min, 1, 2 and 4 hours). Intestinal loops inoculated with cell culture medium were used as a control. Experiments were performed in quadruplicate (bovine ligated ileal loops surgeries were performed with four calves), generating a total of 40 arrays.
Project description:In a time course study, we characterized global gene expression profile of B. melitensis infection in bovine Peyer patches in the first 4 h p.i. Statistical analysis of microarray results reveal that 2,356 B. melitensis genes were detected as differentially expressed (z-score p < 0.025) compared with control between 15 min and 4 h post-infection. A group of 1,740 genes that were differentially expressed in at least 4 out of 5 time points was considered the core set of genes that reflect the major changes in B. melitensis gene expression during the early in vivo bovine Peyer’s patch infection and therefore important in understanding key events in the modulation of host response. From this set of 1,740 differentially expressed genes, 925 (53%) were up-regulated and 815 (47%) were down-regulated compared with the in vitro grown culture. These results reflect significant Brucella metabolic modifications and adaptation from an extracellular to an intracellular lifestyle. 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.
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 a time course study, we characterized global gene expression profile of B. melitensis-infected bovine Peyer patches in the first 4 h p.i. Microarray analysis revealed that 2,916 bovine genes were detected as differentially expressed (z-score p < 0.025) in loops inoculated with virulent B. melitensis 16M compared with controls between 15 min and 4 h post-infection. From these genes, 2,286 (78%) were up- and 630 (22%) were down-regulated. 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.
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: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