Contribution of reactive oxygen species to pathways of quinolone-mediated bacterial cell death

Abstract

Background: Quinolone-mediated death of Escherichia coli has been proposed to occur by two pathways. One is blocked by inhibitors of protein synthesis; the other is not. It is currently unknown how these two pathways fit with the recent observation that hydroxyl radical accumulation is associated with quinolone lethality.

Methods: E. coli was treated with thiourea plus 2,2'-bipyridyl to block hydroxyl radical accumulation, and the effect on quinolone lethality was measured for quinolones that distinguished the two lethal pathways: oxolinic acid requires protein synthesis to kill E. coli, while PD161144, a C-8-methoxy fluoroquinolone, does not. The lethal activity of another fluoroquinolone, moxifloxacin, was partially blocked by the presence of chloramphenicol, an inhibitor of protein synthesis. That feature made it possible to determine whether the effects of chloramphenicol and thiourea plus 2,2'-bipyridyl were additive.

Results: Lethal activity of oxolinic acid was completely blocked by thiourea plus 2,2'-bipyridyl and by chloramphenicol. In contrast, PD161144 lethality was unaffected by these treatments. With moxifloxacin, both chloramphenicol and thiourea plus 2,2'-bipyridyl separately exhibited the same partial inhibition of quinolone lethality. No additivity in protection from moxifloxacin lethality was observed when thiourea, 2,2'-bipyridyl and chloramphenicol were combined and compared with the effect of chloramphenicol or thiourea plus 2,2'-bipyridyl used separately.

Conclusions: Inhibitor studies indicated that hydroxyl radical action contributes to quinolone-mediated cell death occurring via the chloramphenicol-sensitive lethal pathway but not via the chloramphenicol-insensitive pathway.

Figure 1

Effect of thiourea plus 2,2′-bipyridyl and chloramphenicol on quinolone lethality. Exponentially growing E. coli cultures were treated with either chloramphenicol or thiourea plus 2,2′-bipyridyl for 10 min, and then quinolone was added. After incubation, the percentage survival was determined as described in the text. (a) Oxolinic acid (OXO). Oxolinic acid alone (MIC = 0.25 mg/L), with 20 mg/L chloramphenicol (CHL; MIC = 2 mg/L) or with 100 mM thiourea (THI; MIC = 200 mM) plus 0.25 mM 2,2′-bipyridyl (BIP; MIC = 0.5 mM) was added at the indicated concentrations to strain SD104 (KD447) followed by incubation for 120 min. (b) PD161144. As in (a) except that PD161144 (MIC = 0.08 mg/L) replaced oxolinic acid and the incubation time was 45 min. (c) Rate of quinolone-mediated killing. Strain SD104 was treated with oxolinic acid or PD161144 at 10× the MIC for the indicated times, after which percentage survival was determined. (d) Moxifloxacin (MOX). Strain SD104 was treated with moxifloxacin (MIC = 0.06 mg/L) for 45 min, with or without pre-treatment as in (a). In addition, cells were pre-treated with chloramphenicol and thiourea plus 2,2′-bipyridyl for 10 min. Error bars represent standard deviations from the mean; similar results were obtained in replicate experiments.

Figure 2

Schematic representation of quinolone-mediated lethality. (a) Complex formed by gyrase interacting with DNA. (b) Quinolones trapping gyrase on DNA; DNA is broken with its ends constrained, which causes inhibition of DNA replication and bacterial growth. (c) First-generation quinolones (nalidixic and oxolinic acids) cause chromosome fragmentation that is blocked by chloramphenicol (CHL), a protein synthesis inhibitor. (d) Chromosome fragmentation stimulates a surge of ROS with hydroxyl radicals as the terminal product. (e) Formation of highly toxic hydroxyl radicals, the end product of an ROS cascade, is blocked by 2,2′-bipyridyl, an iron chelator, and thiourea, a hydroxyl radical scavenger, which prevent cell death. (f) Fluoroquinolones also kill bacterial cells by a protein synthesis-insensitive, ROS-independent chromosome fragmentation pathway that is postulated to occur by gyrase subunit dissociation.