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Selective amplification of Brucella melitensis mRNA from a mixed host-pathogen total RNA.

ABSTRACT: BACKGROUND: Brucellosis is a worldwide anthropozoonotic disease caused by an in vivo intracellular pathogen belonging to genus Brucella. The characterization of brucelae transcriptome's during host-pathogen interaction has been limited due to the difficulty of obtaining an adequate quantity of good quality eukaryotic RNA-free pathogen RNA for downstream applications. FINDINGS: Here, we describe a combined protocol to prepare RNA from intracellular B. melitensis in a quantity and quality suitable for pathogen gene expression analysis. Initially, B. melitensis total RNA was enriched from a host:pathogen mixed RNA sample by reducing the eukaryotic RNA..Then, to increase the Brucella RNA concentration and simultaneously minimize the contaminated host RNA in the mixed sample, a specific primer set designed to anneal to all B. melitensis ORF allows the selective linear amplification of sense-strand prokaryotic transcripts in a previously enriched RNA sample. CONCLUSION: The novelty of the method we present here allows analysis of the gene expression profile of B. melitensis when limited amounts of pathogen RNA are present, and is potentially applicable to both in vivo and in vitro models of infection, even at early infection time points.

SUBMITTER: Rossetti CA

PROVIDER: S-EPMC2954846 | biostudies-other | 2010

REPOSITORIES: biostudies-other

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Selective amplification of Brucella melitensis mRNA from a mixed host-pathogen total RNA.

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

BMC research notes 20100928

<h4>Background</h4>Brucellosis is a worldwide anthropozoonotic disease caused by an in vivo intracellular pathogen belonging to genus Brucella. The characterization of brucelae transcriptome's during host-pathogen interaction has been limited due to the difficulty of obtaining an adequate quantity of good quality eukaryotic RNA-free pathogen RNA for downstream applications.<h4>Findings</h4>Here, we describe a combined protocol to prepare RNA from intracellular B. melitensis in a quantity and qua ...[more]

PMID: 20920187

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Project description:To date, fewer than 200 gene-products have been identified as Brucella virulence factors, and most were characterized individually without considering how they are temporally and coordinately expressed or secreted during the infection process. Here, we describe and analyze the in vivo temporal transcriptional profile of Brucella melitensis during the initial 4 h interaction with cattle. Pathway analysis revealed an activation of the "Two component system" providing evidence that the in vivo Brucella sense and actively regulate their metabolism through the transition to an intracellular lifestyle. Contrarily, other Brucella pathways involved in virulence such as "ABC transporters" and "T4SS system" were repressed suggesting a silencing strategy to avoid stimulation of the host innate immune response very early in the infection process. Also, three flagellum-encoded loci (BMEII0150-0168, BMEII1080-1089, and BMEII1105-1114), the "flagellar assembly" pathway and the cell components "bacterial-type flagellum hook" and "bacterial-type flagellum" were repressed in the tissue-associated B. melitensis, while RopE1 sigma factor, a flagellar repressor, was activated throughout the experiment. These results support the idea that Brucella employ a stealthy strategy at the onset of the infection of susceptible hosts. Further, through systems-level in silico host:pathogen protein-protein interactions simulation and correlation of pathogen gene expression with the host gene perturbations, we identified unanticipated interactions such as VirB11::MAPK8IP1; BtaE::NFKBIA, and 22 kDa OMP precursor::BAD and MAP2K3. These findings are suggestive of new virulence factors and mechanisms responsible for Brucella evasion of the host's protective immune response and the capability to maintain a dormant state. The predicted protein-protein interactions and the points of disruption provide novel insights that will stimulate advanced hypothesis-driven approaches toward revealing a clearer understanding of new virulence factors and mechanisms influencing the pathogenesis of brucellosis.

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:BACKGROUND:Brucella melitensis bacteria cause persistent, intracellular infections in small ruminants as well as in humans, leading to significant morbidity and economic loss worldwide. The majority of experiments on the transcriptional responses of Brucella to conditions inside the host have been performed following invasion of cultured mammalian cells, and do not address gene expression patterns during long-term infection. RESULTS:Here, we examine the application of the previously developed coincidence cloning methodology to recover and characterize B. melitensis RNA from the supramammary lymph node of experimentally-infected goats. Using coincidence cloning, we successfully recovered Brucella RNA from supramammary lymph nodes of B. melitensis-infected goats at both short-term (4 weeks) and long-term (38 weeks) infection time points. Amplified nucleic acid levels were sufficient for analysis of Brucella gene expression patterns by RNA-sequencing, providing evidence of metabolic activity in both the short-term and the long-term samples. We developed a workflow for the use of sequence polymorphism analysis to confirm recovery of the inoculated strain in the recovered reads, and utilized clustering analysis to demonstrate a distinct transcriptional profile present in samples recovered in long-term infection. In this first look at B. melitensis gene expression patterns in vivo, the subset of Brucella genes that was highly upregulated in long-term as compared to short-term infection included genes linked to roles in murine infection, such as genes involved in proline utilization and signal transduction. Finally, we demonstrated the challenges of qPCR validation of samples with very low ratios of pathogen:host RNA, as is the case during in vivo brucellosis, and alternatively characterized intermediate products of the coincidence cloning reaction. CONCLUSIONS:Overall, this study provides the first example of recovery plus characterization of B. melitensis RNA from in vivo lymph node infection, and demonstrates that the coincidence cloning technique is a useful tool for characterizing in vivo transcriptional changes in Brucella species. Genes upregulated in long-term infection in this data set, including many genes not previously demonstrated to be virulence factors in mice or macrophage experiments, are candidates of future interest for potential roles in Brucella persistence in natural host systems.

Dataset Information

Selective amplification of Brucella melitensis mRNA from a mixed host-pathogen total RNA.

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Selective amplification of Brucella melitensis mRNA from a mixed host-pathogen total RNA.

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