Unknown,Transcriptomics,Genomics,Proteomics

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Differential Expression of Iron Acquisition Genes by Brucella melitensis and Brucella canis During Macrophage Infection (part 1)

ABSTRACT: Brucella melitensis and Brucella canis differ by ~75 genes yet B. melitensis is highly virulent for humans while B. canis is considered rarely pathogenic. No identified bacterial factors or mechanisms account for this difference in virulence. To identify functional differences of these two bacteria, gene transcription was examined during infection of murine macrophages and compared to bacteria grown in broth. Our analysis identified transcriptional differences in macrophage infection between B. melitensis and B. canis genes involved in iron transport. Increased transcription of the TonB, enterobactin, and ferric anguibactin transport systems were observed in B. canis but not B. melitensis during infection of macrophages. Therefore, iron appears as an important requirement during the first 24h of infection by B. canis but not for B. melitensis and provides strategies for controlling these pathogens. Comparison of total bacterial RNA from Brucella melitensis infected murine macrophages to broth grown bacteria

ORGANISM(S): Brucella melitensis bv. 1 str. 16M

SUBMITTER: Gary Splitter

PROVIDER: E-GEOD-34495 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.