Toxin-mediated depletion of NAD and NADP drives persister formation in a human pathogen

Toxin–antitoxin (TA) systems are widespread in bacteria and implicated in genome stability, virulence, phage defense, and persistence. TA systems have diverse activities and cellular targets, but their physiological roles and regulatory mechanisms are often unclear. Here, we show that the NatR–NatT TA system, which is part of the core genome of the human pathogen Pseudomonas aeruginosa , generates drug-tolerant persisters by specifically depleting nicotinamide dinucleotides. While actively growing P. aeruginosa cells compensate for NatT-mediated NAD + deficiency by inducing the NAD + salvage pathway, NAD depletion generates drug-tolerant persisters under nutrient-limited conditions. Our structural and biochemical analyses propose a model for NatT toxin activation and autoregulation and indicate that NatT activity is subject to powerful metabolic feedback control by the NAD + precursor nicotinamide. Based on the identification of natT gain-of-function alleles in patient isolates and on the observation that NatT increases P. aeruginosa virulence, we postulate that NatT modulates pathogen fitness during infections. These findings pave the way for detailed investigations into how a toxin–antitoxin system can promote pathogen persistence by disrupting essential metabolic pathways. Synopsis The toxin-antitoxin system NatR-NatT promotes persister formation in Pseudomonas aeruginosa . This study shows that NatT acts by depleting the essential co-factors NAD and NADP and promotes drug tolerance in this human pathogen. The RES domain of NatT degrades NAD + into nicotinamide and ADP-ribose-1”-phosphate. Actively growing bacteria can counteract NAD + depletion by inducing the NAD + salvage pathway. Under nutrient-limited conditions, bacteria are unable to compensate for NatT-mediated NAD + depletion, resulting in dormancy and persistence. NatT toxin activation is inhibited by the NAD + precursor nicotinamide. The toxin NatT promotes drug tolerance in Pseudomonas aeruginosa under nutrient-limited conditions and is activated in clinical isolates.