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A modular high-throughput in vivo screening platform based upon chimeric bacterial receptors

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Multidrug resistance (MDR) is a globally relevant problem that requires novel approaches. Two-component systems are a promising, yet untapped target for novel antibacterials. They are prevalent in bacteria and absent in mammals, and their activity can be modulated upon perception of various stimuli. Screening pre-existing compound libraries could reveal small molecules that inhibit stimulus-perception by virulence-modulating receptors, reduce signal output from essential receptors or identify artificial stimulatory ligands for novel SHKs that are involved in virulence. Those small molecules could possess desirable therapeutic properties to combat MDR. We propose that a modular screening platform in which the periplasmic domain of the targeted receptors are fused to the cytoplasmic domain of a well-characterized receptor that governs fluorescence reporter genes could be employed to rapidly screen currently existing small molecule libraries. Here, we have examined two previously created Tar-EnvZ chimeras and a novel NarX-EnvZ chimera. We demonstrate that it is possible to couple periplasmic stimulus-perceiving domains to an invariable cytoplasmic domain that governs transcription of a dynamic fluorescent reporter system. Furthermore, we show that aromatic tuning, or repositioning the aromatic residues at the end of the second transmembrane helix (TM2), modulates baseline signal output from the tested chimeras and even restores output from a non-functional NarX-EnvZ chimera. Finally, we observe an inverse correlation between baseline signal output and the degree of response to cognate stimuli. In summary, we propose that the platform described here, a fluorescent Escherichia coli reporter strain with plasmid-based expression of the aromatically tuned chimeric receptors, represents a synthetic biology approach to rapidly screen pre-existing compound libraries for receptor-modulating activities.
Original languageEnglish
Pages (from-to)1315-1326
Number of pages12
JournalACS Synthetic Biology
Volume6
Issue number7
Early online date3 Apr 2017
DOIs
Publication statusPublished - 21 Jul 2017

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  • A modular high-throughput in vivo screening platform based on chimeric bacterial receptors

    Rights statement: This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Synthetic Biology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see doi: 10.1021/acssynbio.6b00288.

    Accepted author manuscript (Post-print), 1.17 MB, PDF document

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