Genome-wide Screening of the Escherichia coli Keio Knockout Collection Identifies Genetic Determinants of Epetraborole Hypersusceptibility.

Purpose: The escalating threat of antibiotic resistance necessitates innovative strategies to enhance the efficacy of emerging antimicrobials. Epetraborole (EP) is a boron-containing antibiotic targeting leucyl-tRNA synthetase (LeuRS) and has attracted interest for its novel mechanism of action and potential to treat infections caused by multidrug-resistant (MDR) Gram-negative pathogens.

Methods: To elucidate the genetic determinants of EP susceptibility, we conducted a genome-wide screen (GWS) of the Escherichia coli Keio knockout collection, which comprises ~4,000 single-gene deletion mutants. Mutants exhibiting increased susceptibility to epetraborole were identified and validated via complementation assays.

Results: Disrupted genes included those involved in leucine biosynthesis (leuD), RNA turnover (rnb), tRNA modification (trmU), ubiquinone biosynthesis (ubiG), NAD salvage pathway (pncA), arginine transport (artJ), transcriptional regulator (yddM) and ribosome biogenesis (yhbY), suggesting that epetraborole's primary inhibition of LeuRS synergizes with defects in these pathways. Bioinformatic analyses (Omics Dashboard, DAVID, STRING) linked these genes to tRNA homeostasis, stress response networks, and central dogma processes, implicating tRNA dysregulation as a critical vulnerability under epetraborole-induced stress.

Conclusion: This study identifies novel genetic contributors to epetraborole susceptibility and provides a framework for exploring adjuvant therapies or resistance mechanisms to enhance its clinical utility against MDR infections.