Global response to antibiotic exposure reveals a critical role for nucleotide metabolism in high-level β-lactam tolerance

Antibiotic tolerance, the ability to survive lethal antibiotics for a prolonged period of time is a rising threat due to its role as a steppingstone towards antibiotic resistance. While tolerance has been recognized as a severe clinical threat, little is known about the mechanisms promoting tolerance. Here, we delineated the physiology of antibiotic tolerance to the classic β-lactam antibiotic, penicillin, to discover the metabolic underpinnings of how tolerant bacteria survive ordinarily lethal antibiotic exposure. We used transcriptomics and metabolomics in the hypertolerant Gram-negative cholera pathogen, Vibrio cholerae, to identify the global regulatory and metabolic response to antibiotic exposure. Key pathways like central carbon metabolism, cell wall synthesis, heat shock, two-component systems, and particularly nucleotide synthesis were significantly altered in response to penicillin. Most notably, nucleotide levels were depleted upon antibiotic exposure, concomitant with upregulation of both purine and pyrimidine synthesis functions. Consistent with a crucial role for nucleotide synthesis in tolerance, we find that targeting nucleotide synthesis synergizes with β-lactam-mediated killing in hypertolerant V. cholerae, Klebsiella pneumoniae and E. coli. These datasets thus reveal new vulnerabilities in tolerant bacteria that can serve as conceptual scaffolds for drug development and for improving antibiotic efficacy.