A Genetic Determinant of Persister Cell Formation in Bacterial Pathogens

Persister cells are a small subpopulation of bacteria within a culture that exhibit tolerance to antibiotic treatment. These cells are implicated in chronic infections caused by various bacterial pathogens, including Pseudomonas aeruginosa. A well-known example is the persistent airway infections in patients with cystic fibrosis, which are notoriously difficult to treat. Despite their clinical significance, the genetic factors underlying persister formation in P. aeruginosa remain poorly understood.
To address this, we created a high-density transposon insertion library in P. aeruginosa strain PAO1 and used transposon sequencing (Tn-seq) to measure the relative frequency of each mutant following fluoroquinolone treatment. Among the 4,411 disrupted genes screened, 137 exhibited at least a 10-fold effect on survival. Notably, disruption of carB, which encodes the large subunit of carbamoyl-phosphate synthetase (CPSase), resulted in the most significant reduction in survival—up to 2,500-fold—after antibiotic exposure. CPSase is a key enzyme in pyrimidine and arginine biosynthesis, and the addition of uracil rescued the survival phenotype of the carB mutant, underscoring the importance of de novo pyrimidine biosynthesis in persister cell formation.
Further analysis revealed that disruption of carB led to intracellular ATP accumulation. Restoring low ATP levels in the mutant with arsenate treatment reinstated its antibiotic tolerance to levels comparable to the wild type. These findings suggest that reduced ATP levels enhance antibiotic tolerance by decreasing target activity and promoting persister formation.
Importance
Antibiotic treatment of P. aeruginosa infections in cystic H3B-120 fibrosis patients often fails, in part due to the presence of antibiotic-tolerant persister cells. Understanding the mechanisms underlying persister formation is critical for developing more effective treatments. In this study, we identified carB, encoding the large subunit of CPSase, as a key gene involved in persister cell formation and multidrug tolerance in P. aeruginosa. Disruption of carB induced metabolic changes that elevated ATP levels, thereby reducing persister formation, while lowering ATP in the mutant restored antibiotic tolerance. These findings support the hypothesis that intracellular ATP depletion is a general mechanism driving persister formation in bacteria and provide a potential avenue for therapeutic intervention.