title: A Modular High-Throughput In Vivo Screening Platform Based on Chimeric Bacterial Receptors.
abstract: 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 nonfunctional 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.
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Building 220, Kemitorvet, DK-2800, Kgs. Lyngby, Denmark.
authors.forename: Christina E
authors.initials: CE
authors.lastname: Lehning
authors.affiliation: Institute of Molecular Biology , Ackermannweg 4, D-55128, Mainz, Germany.
authors.forename: Jan B
authors.initials: JB
authors.lastname: Heidelberger
authors.affiliation: Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt , Max-von-Laue-Straße 15, D-60438, Frankfurt, Germany.
authors.forename: John
authors.initials: J
authors.lastname: Reinhard
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Building 220, Kemitorvet, DK-2800, Kgs. Lyngby, Denmark.
authors.forename: Morten H H
authors.initials: MHH
authors.lastname: Nørholm
authors.forename: Roger R
authors.identifier: 0000-0002-7118-1297
authors.initials: RR
authors.lastname: Draheim
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins
keywords: aromatic tuning; biological platforms; chimeric receptors; high-throughput screening; receptor engineering