Dataset Information

The Acidic Stress Response of the Intracellular Pathogen Brucella melitensis: New Insights from a Comparative, Genome-Wide Transcriptome Analysis.

ABSTRACT: The intracellular pathogenic bacteria belonging to the genus Brucella must cope with acidic stress as they penetrate the host via the gastrointestinal route, and again during the initial stages of intracellular infection. A transcription-level regulation has been proposed to explain this but the specific molecular mechanisms are yet to be determined. We recently reported a comparative transcriptomic analysis of the attenuated vaccine Brucella melitensis strain Rev.1 against the virulent strain 16M in cultures grown under either neutral or acidic conditions. Here, we re-analyze the RNA-seq data of 16M from our previous study and compare it to published transcriptomic data of this strain from both an in cellulo and an in vivo model. We identify 588 genes that are exclusively differentially expressed in 16M grown under acidic versus neutral pH conditions, including 286 upregulated genes and 302 downregulated genes that are not differentially expressed in either the in cellulo or the in vivo model. Of these, we highlight 13 key genes that are known to be associated with a bacterial response to acidic stress and, in our study, were highly upregulated under acidic conditions. These genes provide new molecular insights into the mechanisms underlying the acid-resistance of Brucella within its host.

SUBMITTER: Kornspan D

PROVIDER: S-EPMC7563570 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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The Acidic Stress Response of the Intracellular Pathogen Brucella melitensis: New Insights from a Comparative, Genome-Wide Transcriptome Analysis.

Kornspan David D Zahavi Tamar T Salmon-Divon Mali M

Genes 20200828 9

The intracellular pathogenic bacteria belonging to the genus Brucella must cope with acidic stress as they penetrate the host via the gastrointestinal route, and again during the initial stages of intracellular infection. A transcription-level regulation has been proposed to explain this but the specific molecular mechanisms are yet to be determined. We recently reported a comparative transcriptomic analysis of the attenuated vaccine Brucella melitensis strain Rev.1 against the vir ...[more]

PMID: 32872264

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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.

Dataset Information

The Acidic Stress Response of the Intracellular Pathogen Brucella melitensis: New Insights from a Comparative, Genome-Wide Transcriptome Analysis.

Publications

The Acidic Stress Response of the Intracellular Pathogen <i>Brucella melitensis</i>: New Insights from a Comparative, Genome-Wide Transcriptome Analysis.

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