An Rcs Stress-Based High-Throughput Screen Reveals Novel Gyrase Inhibitors as Indirect Inducers of Cell Envelope Stress in Gram-Negative Bacteria

The highly impermeable cell envelope of Gram-negative bacteria is an important hurdle to the development of novel antibacterials. However, compounds that disrupt the integrity of the cell envelope can act as potent antibiotics by directly inhibiting cell growth and viability or by enhancing the penetration of other, larger antibiotics otherwise unable to pass this barrier. To identify such novel compounds, we used the European Lead Factory compound libraries to screen >500,000 small molecules for inducing the Rcs cell envelope stress response in <i><i>Escherichia coli</i></i>. We identified a series of novel 2-quinolones and 4-quinolones that target gyrase and topoisomerase IV, suggesting unforeseen effects of such compounds on the bacterial cell envelope. Here, we show that the quinolones induce a structure-dependent profile of specific cell envelope stress responses. These response profiles were observed not only for quinolone-type but also for structurally unrelated gyrase inhibitors. Importantly, DNA damage and SOS response activation alone were insufficient to explain the high levels of cell envelope stress, underscoring gaps in our understanding of the interplay between gyrase function and maintenance of cell envelope integrity. Microscopy showed structural changes that are likely related to the observed stress. Importantly, cell elongation, associated with quinolone-induced SOS stress response, also occurred in SOS-deficient bacteria. These serendipitous findings highlight both the complexity of gyrase-associated bactericidal mechanisms and the challenges in antibiotic discovery. Nevertheless, this study supports the utility of stress-based assays as sensitive phenotypic tools for identifying new antimicrobial agents.