Role of oxidative stress in persister tolerance

Abstract

Persisters are dormant phenotypic variants of regular cells that are tolerant to antibiotics and play an important role in recalcitrance of chronic infections to therapy. Persisters can be produced stochastically in a population untreated with antibiotics. At the same time, a deterministic component of persister formation has also been documented in a population of cells with DNA damaged by fluoroquinolone treatment. Expression of the SOS response under these conditions induces formation of persisters by increasing expression of the TisB toxin. This suggests that other stress responses may also contribute to persister formation. Of particular interest is oxidative stress that pathogens encounter during infection. Activated macrophages produce reactive oxygen and nitrogen species which induce the SoxRS and OxyR regulons. Genes controlled by these regulons deactivate the oxidants and promote repair. We examined the ability of oxidative stress induced by paraquat (PQ) to affect persister formation. Preincubation of cells with PQ produced a dramatic increase in the number of persisters surviving challenge with fluoroquinolone antibiotics. PQ did not affect killing by kanamycin or ampicillin. Persisters in a culture treated with PQ that survived a challenge with a fluoroquinolone were also highly tolerant to other antibiotics. PQ induces SoxRS, which in turn induces expression of the AcrAB-TolC multidrug-resistant (MDR) pump. Fluoroquinolones are extruded by this MDR pump, and the effect of PQ on antibiotic tolerance was largely abolished in a mutant that was defective in the pump. It appears that PQ, acting through AcrAB-TolC, reduces the concentration of fluoroquinolones in the cells. This allows a larger fraction of cells to become persisters in the presence of a fluoroquinolone. Analysis of a lexA3 mutant indeed showed a dependence of persister induction under these conditions on SOS. These findings show that induction of a classical resistance mechanism, MDR efflux, by oxidative stress leads to an increase in multidrug-tolerant persister cells.

Fig 1

The transcriptional and metabolic cascade induced by paraquat.

Fig 2

The influence of PQ on drug tolerance of E. coli BW25113. (A) Time-dependent killing by ampicillin (50 μg/ml), ofloxacin (1 μg/ml), and kanamycin (50 μg/ml). (B) Time-dependent killing by different fluoroquinolones: ofloxacin (1 μg/ml), norfloxacin (1 μg/ml), and ciprofloxacin (0.15 μg/ml). The growing population of BW25113 was treated with 0.8 mM PQ for 30 min before antibiotics were applied.

Fig 3

Multidrug tolerance of persisters induced by PQ treatment. The growing population of BW25113 was treated with 0.8 mM PQ for 30 min and then challenged with 1 μg/ml ofloxacin (the zero time point in the figure). After 6 h, the PQ treated culture was split into four aliquots: no extra antibiotic added (PQ oflo 1), 4 μg/ml ofloxacin added (PQ oflo 1 + 4), 100 μg/ml of ampicillin added (PQ oflo 1 + Amp 100), and 30 μg/ml of tobramycin added (PQ oflo 1 + Tobra 30).

Fig 4

Induction of soxS and tolC by paraquat and ofloxacin. Ofloxacin (1 μg/ml) was added to the growing cultures of MG1655 PsoxS-GFP (A) and MG1655 PtolC-GFP (B) after a 30-min incubation with 0.8 mM PQ. The level of GFP fluorescence indicates the level of promoter activity.

Fig 5

The influence of PQ on ofloxacin tolerance of wild-type E. coli BW25113 and the MDR efflux pump mutant acrB. The growing cultures were treated with 0.8 mM PQ for 30 min before a challenge by 1 μg/ml ofloxacin to BW25113 and 0.2 μg/ml to acrB, respectively.

Fig 6

The SOS dependence of the PQ-induced fluoroquinolone tolerance. Ofloxacin (1 μg/ml) was added to the growing populations of wild-type BW25113 and lexA3, a mutant lacking SOS induction, following a 30-min treatment of 0.8 mM PQ.