title: Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.
abstract: Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: El-Mowafi
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: E
authors.identifier: http://orcid.org/0000-0003-2307-0581
authors.initials: E
authors.lastname: Sineva
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: J N
authors.initials: JN
authors.lastname: Alumasa
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: H
authors.initials: H
authors.lastname: Nicoloff
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Tomsho
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: S E
authors.initials: SE
authors.lastname: Ades
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA kkeiler@psu.edu.
authors.forename: K C
authors.initials: KC
authors.lastname: Keiler
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D057166:High-Throughput Screening Assays / Q000379:methods*; D047668:Inteins / Q000187:drug effects / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008239:Lysine; D053858:Metabolic Networks and Pathways / Q000187:drug effects; D010456:Peptides, Cyclic / Q000235:genetics / Q000378:metabolism; D019154:Protein Splicing; D015203:Reproducibility of Results; D012808:Sigma Factor / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D010456:Peptides, Cyclic; D012808:Sigma Factor; D054852:Small Molecule Libraries; C054562:yellow fluorescent protein, Bacteria; D008239:Lysine