ATP burst is the dominant driver of antibiotic lethality in Mycobacteria

Abstract

Antibiotic-tolerant bacteria, due to their unique physiology, are refractory to antimicrobial killing and pose challenges for successful therapy.M. tuberculosis, the causative agent of tuberculosis (TB), is exceptionally difficult to control due to antibiotic tolerance. Incomplete knowledge of how bactericidal antibiotics work has impeded our understanding of the development of antibiotic tolerance in mycobacteria. In this study, using quantitative proteomics,13C-isotopomer analysis, and biochemical assays, we investigated the physiological response ofM. smegmatischallenged with sub-lethal doses of aminoglycoside and fluoroquinolone antibiotics. Two distinct classes of antibiotics elicited remarkably similar bacterial responses in central dogma, cell division, and central carbon metabolic processes. Both antibiotics increased TCA cycle flux, resulting in enhanced respiration, reactive oxygen species (ROS) production, and ATP burst. While both ROS and ATP burst were found to contribute to antibiotic lethality, our findings indicate that ATP burst, and not ROS, is the dominant cause of antibiotic-induced cell death in mycobacteria.13C isotope tracing experiments indicated metabolic bifurcation of the TCA cycle flux from the oxidative arm into glutamate-glutamine-GABA as a potential bacterial adaptive mechanism to reduce the production of reactive oxygen species (ROS) and ATP burst. In addition, antibiotic-adapted mycobacteria have an activated intrinsic drug resistance mechanism, a higher mutation rate, and are prone to developing antibiotic resistance. Our study provides a novel understanding of the intricate mechanisms of antibiotic-induced cell death, and has significance for the design of combinatorial interventions and adjuvant therapy against one of the most successful infectious diseases.