Quinolone tolerance in Escherichia coli due to defects in the adenosine ribonucleotides de novo biosynthesis pathway

Central carbon metabolism is thought to link reactive oxygen species (ROS) with antibiotic-mediated bacterial death. During enrichment screening of Escherichia coli with the first-generation quinolone oxolinic acid, unstable antibiotic-tolerant mutants containing deficiencies in purB were obtained. Examination of a stable deletion mutant of purA, a gene functionally related to purB, revealed reduced lethality of oxolinic acid and ciprofloxacin. This deletion mutation had little effect on the minimal inhibitory concentration (MIC) of quinolones, thereby demonstrating that the observed protection from killing was attributable to antibiotic tolerance. AMP synthesis was blocked by the ΔpurA mutation, and ciprofloxacin tolerance was reversed by exogenous AMP supplementation. Because AMP is a precursor of ATP, interference with ATP synthesis occurs in the ΔpurA mutant. RNA-Seq analysis showed that, prior to antibiotic stress, transcript levels of NADH:quinone oxidoreductase genes were reduced by the purA deficiency, thereby predisposing E. coli to antibiotic tolerance through reduced respiration. During ciprofloxacin exposure, the purA deficiency also suppressed the surge in expression of tricarboxylic acid (TCA) cycle and ATP synthesis genes, as well as the accumulation of intracellular ATP and ROS. Thus, wild-type PurA, and by extension the downstream enzyme PurB, directs AMP toward an antibiotic-mediated, ROS-dependent death pathway. Overall, defects in PurA/PurB-mediated adenosine ribonucleotides de novo biosynthesis reveal a novel quinolone tolerance mechanism that is initiated outside central carbon metabolism; tolerance is likely attributable to a limited supply of AMP, resulting in reduced ATP synthesis and suppression of ROS accumulation.