A Genetic Determinant of Persister Cell Formation in Bacterial Pathogens

Abstract

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 A classic example is the incurable infection of the airways for patients with cystic fibrosis. The genetic mediators of persister formation for are poorly understood. We generated a high-density transposon insertion library of 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, 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 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 pyrimidine biosynthesis for persister formation. Disruption of 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. Antibiotic treatment of 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, which codes for the large subunit of carbamoyl-phosphate synthetase, as a persister gene that contributes to multidrug tolerance in Disruption of 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.

A Novel LysR-Type Global Regulator RpvA Controls Persister Formation and Virulence inStaphylococcus aureus

“…Carbamoyl phosphate synthetase (CPSase) is a metabolic enzyme involved in the synthesis of P. aeruginosa pyrimidine and arginine, and mutation of carB encoding the large subunit of CPSase resulted in a 2500-fold reduction in survival after antibiotic treatment, while addition of uracil could abolish this phenotype. This indicates that the biosynthesis of arginine and uracil is closely related to the formation of persister (73). Faced with reduced sugar availability in the environment, S. aureus could catabolize multiple amino acids as a secondary carbon source to generate pyruvate (alanine, serine, glycine, threonine, cysteine), 2-oxoglutarate (glutamate, glutamine, histidine, arginine, proline), and oxaloacetate (aspartate, asparagine) (74).…”

Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics

“…Mutations in the efflux system repressors identified in our Tn-seq screen are commonly found in clinical isolates that are resistant to fluoroquinolone antibiotics, as the efflux systems regulated by these repressors efficiently export this class of drugs 12,13 . Likewise, these repressors were strong hits in a recent Tn-seq screen for genes that affect P. aeruginosa survival in the presence of the broad-spectrum fluoroquinolone ciprofloxacin 14 (Fig. 1C, Fig.…”

“…To compare the results of this Tn-seq analysis with a previously published study 14 analyzing fitness determinants for survival during ciprofloxacin treatment in the P. aeruginosa PAO1 strain background (Fig. 1C, S1C and Table S1), the data from that study's supplemental Table S1 were used.…”

Polymicrobial interactions of Candida albicans and its role in oral carcinogenesis

“…Persister cells possess a resistant phenotype that minimises the susceptibility of polymicrobial community within biofilm to antimicrobial agents. 25 In addition, the biomass and the percentage surface colonisation of C albicans is significantly reduced in polymicrobial biofilms of C albicans, A naeslundii and S mutans, in comparison with the biomass of C albicans in mono-cultured biofilms when grown in flow cell environment 24 (Figure 2). This condition is due to the metabolic products of A naeslundii that have been reported to both inhibit and stimulate the biofilm formation of C albicans, depending on the experimental methods employed and the strains of C albicans used.…”

“…C albicans with low metabolic activity is more invasive, which may be linked with oral carcinogenesis, while conversely, those with high activity are non‐invasive . Slower growing microorganisms can represent “persister cells.” Persister cells possess a resistant phenotype that minimises the susceptibility of polymicrobial community within biofilm to antimicrobial agents …”