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Transcriptional profile of the intracellular pathogen Brucella melitensis following HeLa cells infection.

ABSTRACT: Brucella spp. infect hosts primarily by adhering and penetrating mucosal surfaces; however the initial molecular phenomena of this host:pathogen interaction remain poorly understood. Using cDNA microarray analysis, we characterized the transcriptional profile of the intracellular pathogen Brucella melitensis at 4 h (adaptational period) and 12 h (replicative phase) following HeLa cells infection. The intracellular pathogen transcriptome was determined using 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 pathogen genes differentially expressed at 4 and 12 h p.i., respectively. In concordance with phenotypic studies, most of the genes expressed were involved in pathogen growth and metabolism, and were down-regulated at the earliest time point (78%), but up-regulated at 12 h p.i. (75%). Further characterization of specific genes identified in this study will elucidate biological processes and pathways to help understand 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.

SUBMITTER: Rossetti CA

PROVIDER: S-EPMC3170451 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Transcriptional profile of the intracellular pathogen Brucella melitensis following HeLa cells infection.

Rossetti Carlos A CA Galindo Cristi L CL Garner Harold R HR Adams L Garry LG

Microbial pathogenesis 20110720 5

Brucella spp. infect hosts primarily by adhering and penetrating mucosal surfaces; however the initial molecular phenomena of this host:pathogen interaction remain poorly understood. Using cDNA microarray analysis, we characterized the transcriptional profile of the intracellular pathogen Brucella melitensis at 4 h (adaptational period) and 12 h (replicative phase) following HeLa cells infection. The intracellular pathogen transcriptome was determined using initially enriched and then amplified ...[more]

PMID: 21798337

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Project description:BackgroundIn many bacteria, the phosphotransferase system (PTS) is a key player in the regulation of the assimilation of alternative carbon sources notably through catabolic repression. The intracellular pathogens Brucella spp. possess four PTS proteins (EINtr, NPr, EIIANtr and an EIIA of the mannose family) but no PTS permease suggesting that this PTS might serve only regulatory functions.Methodology/principal findingsIn vitro biochemical analyses and in vivo detection of two forms of EIIANtr (phosphorylated or not) established that the four PTS proteins of Brucella melitensis form a functional phosphorelay. Moreover, in vitro the protein kinase HprK/P phosphorylates NPr on a conserved serine residue, providing an additional level of regulation to the B. melitensis PTS. This kinase activity was inhibited by inorganic phosphate and stimulated by fructose-1,6 bisphosphate. The genes encoding HprK/P, an EIIAMan-like protein and NPr are clustered in a locus conserved among ?-proteobacteria and also contain the genes for the crucial two-component system BvrR-BvrS. RT-PCR revealed a transcriptional link between these genes suggesting an interaction between PTS and BvrR-BvrS. Mutations leading to the inactivation of EINtr or NPr significantly lowered the synthesis of VirB proteins, which form a type IV secretion system. These two mutants also exhibit a small colony phenotype on solid media. Finally, interaction partners of PTS proteins were identified using a yeast two hybrid screen against the whole B. melitensis ORFeome. Both NPr and HprK/P were shown to interact with an inorganic pyrophosphatase and the EIIAMan-like protein with the E1 component (SucA) of 2-oxoglutarate dehydrogenase.Conclusions/significanceThe B. melitensis can transfer the phosphoryl group from PEP to the EIIAs and a link between the PTS and the virulence of this organism could be established. Based on the protein interaction data a preliminary model is proposed in which this regulatory PTS coordinates also C and N metabolism.

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.

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Transcriptional profile of the intracellular pathogen Brucella melitensis following HeLa cells infection.

Publications

Transcriptional profile of the intracellular pathogen Brucella melitensis following HeLa cells infection.

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