Toxin-mediated depletion of nicotinamide dinucleotides drives persister formation in a human pathogen

Toxin-antitoxin (TA) systems are widespread in bacteria and are implicated in genome stability, virulence, phage defense and persistence. Although TA systems encompass a large variety of molecular activities and cellular targets, their physiological role and regulatory mechanisms are often unclear. Here, we show that a RES domain TA system increases the survival of the human pathogen P. aeruginosa during antibiotic treatment by generating a subpopulation of highly drug-tolerant persisters. The NatT toxin is an NAD phosphorylase, which leads to strong depletion of NAD and NADP in a subpopulation of cells. Actively growing P. aeruginosa cells effectively compensate for toxin-mediated NAD deficiency by inducing the NAD salvage path-way. In contrast, under nutrient-limited conditions, NatT generates NAD-depleted cells that give rise to drug tolerant persisters during outgrowth. Structural and biochemical analyses of active and inactive NatR-NatT complexes reveal how changes in NatR-NatT interaction controls toxin activity and autoregulation. Finally, we show that the NAD precursor nicotinamide blocks NatT activity and eliminates persister formation, exposing powerful metabolic feedback control of toxin activity. The findings that patient isolates contain natT gain-of-function alleles and that NatT increases P. aeruginosa virulence, argue that NatT contributes to P. aeruginosa fitness during infections. These studies provide mechanistic insight into how a TA system promotes pathogen persistence by disrupting essential metabolic pathways during nutrient stress.