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Establishment of a method to rapidly assay bacterial persister metabolism.

ABSTRACT: Bacterial persisters exhibit an extraordinary tolerance to antibiotics that is dependent on their metabolic state. Although persister metabolism promises to be a rich source of antipersister strategies, there is relatively little known about the metabolism of these rare and transient phenotypic variants. To address this knowledge gap, we explored the use of several techniques, and we found that only one measured persister metabolism. This assay was based on the phenomenon of metabolite-enabled aminoglycoside killing of persisters, and we used it to characterize the metabolic heterogeneity of different persister populations. From these investigations, we determined that glycerol and glucose are the most ubiquitously used carbon sources by various types of Escherichia coli persisters, suggesting that these metabolites might prove beneficial to deliver in conjunction with aminoglycosides for the treatment of chronic and recurrent infections. In addition, we demonstrated that the persister metabolic assay developed here is amenable to high-throughput screening with the use of phenotype arrays.

SUBMITTER: Orman MA

PROVIDER: S-EPMC3754326 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Establishment of a method to rapidly assay bacterial persister metabolism.

Orman Mehmet A MA Brynildsen Mark P MP

Antimicrobial agents and chemotherapy 20130701 9

Bacterial persisters exhibit an extraordinary tolerance to antibiotics that is dependent on their metabolic state. Although persister metabolism promises to be a rich source of antipersister strategies, there is relatively little known about the metabolism of these rare and transient phenotypic variants. To address this knowledge gap, we explored the use of several techniques, and we found that only one measured persister metabolism. This assay was based on the phenomenon of metabolite-enabled a ...[more]

PMID: 23817376

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Project description:Persisters represent a small subpopulation of cells within a bacterial culture that are tolerant to killing by antibiotics. Persisters have been linked to recalcitrant infections caused by numerous bacterial pathogens, including Pseudomonas aeruginosa A classic example is the incurable infection of the airways for patients with cystic fibrosis. The genetic mediators of persister formation for P. aeruginosa are poorly understood. We generated a high-density transposon insertion library of P. aeruginosa PAO1 and determined the relative frequency of each insertion following fluoroquinolone treatment using transposon sequencing (Tn-seq). Of the 4,411 disrupted genes included in the screen, 137 had a ≥10-fold impact on survival. The gene disruption that resulted in the lowest survival rate was disruption of carB, which codes for the large subunit of carbamoyl phosphate synthetase (CPSase). CPSase is a metabolic enzyme that is involved in pyrimidine and arginine synthesis. Disruption of carB resulted in survival rates that were reduced by up to 2,500-fold following antibiotic treatment, and this phenotype was abolished by the addition of uracil, highlighting the importance of de novo pyrimidine biosynthesis for persister formation. Disruption of carB resulted in intracellular ATP accumulation, and lowering ATP levels using arsenate restored the antibiotic tolerance profile of the mutant to levels similar to those seen with the wild type. A decrease in ATP would lead to reduced antibiotic target activity and increased survival.IMPORTANCE Antibiotic treatment of P. aeruginosa residing in the lung of cystic fibrosis patients is ineffective. Treatment failure is attributed in part to antibiotic-tolerant phenotypic variants known as persister cells. Understanding how these cells emerge will likely inform future therapeutic strategies. In the current study, we identified carB, which codes for the large subunit of carbamoyl-phosphate synthetase, as a persister gene that contributes to multidrug tolerance in P. aeruginosa Disruption of carB resulted in a metabolic perturbation that increased cellular ATP and reduced persister formation. Conversely, lowering ATP in the mutant restored antibiotic tolerance. Our data support the hypothesis that a drop in intracellular ATP is a general mechanism of persister formation in bacteria.

Dataset Information

Establishment of a method to rapidly assay bacterial persister metabolism.

Publications

Establishment of a method to rapidly assay bacterial persister metabolism.

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