Gain-of-Function Mutations in Acid Stress Response (<i>evgS</i>) Protect Escherichia coli from Killing by Gallium Nitrate, an Antimicrobial Candidate

Abstract

Widespread antimicrobial resistance encourages repurposing/refining of nonantimicrobial drugs for antimicrobial indications. Gallium nitrate (GaNt), an FDA-approved medication for cancer-related hypercalcemia, recently showed good activity against several clinically significant bacteria. However, the mechanism of GaNt antibacterial action is still poorly understood. In the present work, resistant and tolerant mutants of <i>Escherichia coli</i> were sought via multiple rounds of killing by GaNt. Multiround-enrichment yielded no resistant mutant; whole-genome sequencing of one representative GaNt-tolerant mutant uncovered mutations in three genes (<i>evgS</i>, <i>arpA</i>, and <i>kdpD</i>) potentially linked to protection from GaNt-mediated killing. Subsequent genetic analysis ruled out a role for <i>arpA</i> and <i>kdpD</i>, but two gain-of-function mutations in <i>evgS</i> conferred tolerance. The <i>evgS</i> mutation-mediated GaNt tolerance depended on EvgS-to-EvgA phosphotransfer; EvgA-mediated upregulation of GadE. YdeO, and SarfA also contributed to tolerance, the latter two likely through their regulation of GadE. GaNt-mediated killing of wild-type cells correlated with increased intracellular reactive oxygen species (ROS) accumulation that was abolished by the <i>evgS</i>-tolerant mutation. Moreover, GaNt-mediated killing was mitigated by dimethyl sulfoxide, and the <i>evgS</i>-tolerant mutation upregulated genes encoding enzymes involved in ROS detoxification and in the glyoxylate shunt of the tricarboxylic acid (TCA) cycle. Collectively, these findings indicate that GaNt kills bacteria through elevation of ROS; gain-of-function mutations in <i>evgS</i> confer tolerance by constitutively activating the EvgA-YdeO/GadE cascade of acid resistance pathways and by preventing GaNt-stimulated ROS accumulation by upregulating ROS detoxification and shifting TCA cycle carbon flux. The striking lethal activity of GaNt suggests that clinical use of the agent may not quickly lead to resistance.