Identification of Inhibitors of a Bacterial Sigma Factor Using a New High-Throughput Screening Assay

Abstract

ABSTRACTGram-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 σEis critical for maintenance of the cell envelope. Because σEis 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 σEpathway, and to permit high-throughput screening for antibiotic lead compounds, a σEactivity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. AnEscherichia colistrain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σEpathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σEpathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σE, inhibits RNA polymerase holoenzyme formation, and inhibits σE-dependent transcriptionin vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σEpathway.