Abstract
It is well known that loss of aerobic respiration in Gram-negative bacteria can diminish the efficacy of a variety of bactericidal antibiotics, which has lead to subsequent demonstrations that the formation of reactive oxygen species (ROS) and the proton motive force (PMF) can both play a role in antibiotic toxicity. The susceptibility of Gram-negative bacteria to aminoglycoside antibiotics, particularly gentamicin, has previously been linked to both the production of ROS and the rate of antibiotic uptake that is mediated by the PMF, although the relative contributions of ROS and PMF to aminoglycoside toxicity has remained poorly understood. Herein, gentamicin was shown to elicit a very modest increase in ROS levels in an aerobically grown Escherichia coli clinical isolate. The well-characterised uncoupler 2,4-dinitrophenol (DNP) was used to disrupt the PMF, which resulted in a significant decrease in gentamicin lethality towards E. coli. DNP did not significantly alter respiratory oxygen consumption, supporting the hypothesis that this uncoupler does not increase ROS production via elevated respiratory oxidase activity. These observations support the hypothesis that maintenance of PMF rather than induction of ROS production underpins the mechanism for how the respiratory chain potentiates the toxicity of aminoglycosides. This was further supported by the demonstration that the uncoupler DNP elicits a dramatic decrease in gentamicin lethality under anaerobic conditions. Together, these data strongly suggest that maintenance of the PMF is the dominant mechanism for the respiratory chain in potentiating the toxic effects of aminoglycosides.
Highlights
It is well known that the abolition of aerobic respiration dramatically reduces the toxic effects of bactericidal antibiotics ((Lobritz et al 2015); reviewed in (Stokes et al 2019))
It is well-known that respiratory activity can promote the lethality of many antibiotics, and there is unequivocal evidence to support the role of reactive oxygen species (ROS) generation by the aerobic respiratory chain in the lethality of a number of different antibiotic classes (reviewed in (Stokes et al 2019))
The interplay between ROS, respiratory activity and aminoglycoside lethality has remained less clear, and the maintenance of proton motive force (PMF) provides an alternative mechanism for the respiratory chain to potentiate the lethality of aminoglycosides
Summary
It is well known that the abolition of aerobic respiration dramatically reduces the toxic effects of bactericidal antibiotics ((Lobritz et al 2015); reviewed in (Stokes et al 2019)). Evidence has suggested that mistranslated proteins brought to the membrane activate the two-component stress-response sensor Cpx which may activate ArcA a redox-responsive two-component transcription factor leading to respiratory and metabolic changes (Kohanski et al 2008). These changes are proposed to lead to oxidative stress, production of hydroxyl radicals and cell death (Kohanski et al 2008). For aminoglycosides, such as gentamicin, there is strong evidence for a link between antibiotic uptake provided by the PMF
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