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Molecular characterization and identification of proteins regulated by Hfq in Neisseria meningitidis.

ABSTRACT: Hfq is a highly conserved pleiotropically acting prokaryotic RNA-binding protein involved in the post-transcriptional regulation of many stress-responsive genes by small RNAs. In this study, we show that Hfq of the strictly human pathogen Neisseria meningitidis is involved in the regulation of expression of components involved in general metabolic pathways, iron metabolism and virulence. A meningococcal hfq deletion strain (H44/76Deltahfq) is impaired in growth in nutrient-rich media and does not grow at all in nutrient-limiting medium. The growth defect was complemented by expression of hfq in trans. Using proteomics, the expression of 28 proteins was found to be significantly affected upon deletion of hfq. Of these, 20 proteins are involved in general metabolism, among them seven iron-responsive genes. Two proteins (PilE, TspA) are involved in adherence to human cells, a step crucial for the onset of disease. One of the differentially expressed proteins, GdhA, was identified as an essential virulence factor for establishment of sepsis in an animal model, studied earlier. These results show that in N. meningitidis Hfq is involved in the regulation of a variety of components contributing to the survival and establishment of meningococcal disease.

SUBMITTER: Pannekoek Y

PROVIDER: S-EPMC2734931 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Molecular characterization and identification of proteins regulated by Hfq in Neisseria meningitidis.

Pannekoek Yvonne Y Huis in 't Veld Robert R Hopman Carla Th P CT Langerak Ankie A J AA Speijer Dave D van der Ende Arie A

FEMS microbiology letters 20090403 2

Hfq is a highly conserved pleiotropically acting prokaryotic RNA-binding protein involved in the post-transcriptional regulation of many stress-responsive genes by small RNAs. In this study, we show that Hfq of the strictly human pathogen Neisseria meningitidis is involved in the regulation of expression of components involved in general metabolic pathways, iron metabolism and virulence. A meningococcal hfq deletion strain (H44/76Deltahfq) is impaired in growth in nutrient-rich media and does no ...[more]

PMID: 19374669

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Project description:Neisseria meningitidis is a human commensal that occasionally causes life-threatening infections such as bacterial meningitis and septicemia. Despite experimental evidence that gene regulation as well as the expression of small non-coding RNAs (sRNAs) affect meningococcal virulence, the organization of its transcriptome, including in particular the biogenesis of sRNAs and their mode of action, is only poorly understood. Here, we addressed these issues using a combination of high-throughput technologies. We applied differential RNA-seq (dRNA-seq) to produce a single-nucleotide resolution map of the primary transcriptome of N. meningitidis strain 8103. Our dRNA-seq analysis predicted 1,625 transcriptional start sites (TSS) including 65 non-coding RNA transcripts, of which 20 were further validated by Northern analysis. This allowed for the discovery of a novel CRISPR-associated sRNA with a Cas9-independent biogenesis. Genome-wide mapping of σ 70-dependent and independent promoters revealed that classical Escherichia coli-like σ70 promoter are absent in most of the protein coding genes in meningococci. The majority of the 706 primary TSSs (pTSSs) were associated with coding sequences, including 382 pTSS obtained for single genes and 240 pTSSs obtained for genes located in operons. By Hfq RNA immunoprecipitation sequencing (RIP-seq) we identified a large Hfq-centered post-transcriptional regulatory network comprising 24 sRNAs and 407 potential mRNA targets, and rifampicin stability assays demonstrated that Hfq binding confers enhanced stability on sRNAs. We finally confirmed the interactions of two sRNAs and their cognate target mRNA in vivo. Both directly repress prpB encoding a methylisocitrate lyse which was previously shown to be involved in meningococcal colonization of the human nasopharynx.The combination of both high-throughput approaches thus creates a compendium that not only provides a valuable data resource, but also allows for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.

Dataset Information

Molecular characterization and identification of proteins regulated by Hfq in Neisseria meningitidis.

Publications

Molecular characterization and identification of proteins regulated by Hfq in Neisseria meningitidis.

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