Dataset Information

Cell-based assay to identify inhibitors of the Hfq-sRNA regulatory pathway.

ABSTRACT: Noncoding small RNAs (sRNAs) act in conjunction with the RNA chaperone Hfq to regulate gene expression in bacteria. Because Hfq is required for virulence in several bacterial pathogens, the Hfq-sRNA system is an attractive target for antibiotic development. A reporter strain in which the expression of yellow fluorescent protein (YFP) is controlled by Hfq-sRNA was engineered to identify inhibitors of this system. A reporter that is targeted by Hfq in conjunction with the RybB sRNA was used in a genetic screen to identify inhibitors from a library of cyclic peptides produced in Escherichia coli using split-intein circular ligation of peptides and proteins (SICLOPPS), an intein-based technology. One cyclic peptide identified in this screen, RI20, inhibited Hfq-mediated repression of gene expression in conjunction with both RybB and an unrelated sRNA, MicF. Gel mobility shift assays showed that RI20 inhibited binding of Hfq to RybB and MicF with similar Ki values. These data suggest that RI20 inhibits Hfq activity by blocking interactions with sRNAs and provide a paradigm for inhibiting virulence genes in Gram-negative pathogens.

SUBMITTER: El-Mowafi SA

PROVIDER: S-EPMC4135888 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Cell-based assay to identify inhibitors of the Hfq-sRNA regulatory pathway.

El-Mowafi Shaima A SA Alumasa John N JN Ades Sarah E SE Keiler Kenneth C KC

Antimicrobial agents and chemotherapy 20140707 9

Noncoding small RNAs (sRNAs) act in conjunction with the RNA chaperone Hfq to regulate gene expression in bacteria. Because Hfq is required for virulence in several bacterial pathogens, the Hfq-sRNA system is an attractive target for antibiotic development. A reporter strain in which the expression of yellow fluorescent protein (YFP) is controlled by Hfq-sRNA was engineered to identify inhibitors of this system. A reporter that is targeted by Hfq in conjunction with the RybB sRNA was used in a g ...[more]

PMID: 25001303

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Project description:Significance Hfq is a key posttranscriptional regulator that modulates base-pairing interactions between sRNAs and their target mRNAs. Although there are more than 100 different sRNAs in Pseudomonas aeruginosa, information on their regulatory targets was available for only a few. Here, we identify the targets of 89 Hfq-associated sRNAs in this important opportunistic pathogen. Furthermore, we show that a single sRNA called PhrS dominates the RNA–RNA interaction landscape in this organism by pairing with approximately 800 targets. Our study therefore provides insight into the potential regulatory roles for many sRNAs in P. aeruginosa and identifies an sRNA with an acutely outsized influence on the RNA–RNA interactions occurring on Hfq in this bacterium. The RNA chaperone Hfq plays important regulatory roles in many bacteria by facilitating the base pairing between small RNAs (sRNAs) and their cognate mRNA targets. In the gram-negative opportunistic pathogen Pseudomonas aeruginosa, over a hundred putative sRNAs have been identified but for most, their regulatory targets remained unknown. Using RIL-seq with Hfq in P. aeruginosa, we identified the mRNA targets for dozens of previously known and unknown sRNAs. Strikingly, hundreds of the RNA–RNA interactions we discovered involved PhrS. This sRNA was thought to mediate its effects by pairing with a single target mRNA and regulating the abundance of the transcription regulator MvfR required for the synthesis of the quorum sensing signal PQS. We present evidence that PhrS controls many transcripts by pairing with them directly and employs a two-tiered mechanism for governing PQS synthesis that involves control of an additional transcription regulator called AntR. Our findings in P. aeruginosa expand the repertoire of targets for previously known sRNAs, reveal potential regulatory targets for previously unknown sRNAs, and suggest that PhrS may be a keystone sRNA with the ability to pair with an unusually large number of transcripts in this organism.

Dataset Information

Cell-based assay to identify inhibitors of the Hfq-sRNA regulatory pathway.

Publications

Cell-based assay to identify inhibitors of the Hfq-sRNA regulatory pathway.

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