Post-stress bacterial cell death mediated by reactive oxygen species

Abstract

Antimicrobial efficacy, which is central to many aspects of medicine, is being rapidly eroded by bacterial resistance. Since new resistance can be induced by antimicrobial action, highly lethal agents that rapidly reduce bacterial burden during infection should help restrict the emergence of resistance. To improve lethal activity, recent work has focused on toxic reactive oxygen species (ROS) as part of the bactericidal activity of diverse antimicrobials. We report that when Escherichia coli was subjected to antimicrobial stress and the stressor was subsequently removed, both ROS accumulation and cell death continued to occur. Blocking ROS accumulation by exogenous mitigating agents slowed or inhibited poststressor death. Similar results were obtained with a temperature-sensitive mutational inhibition of DNA replication. Thus, bacteria exposed to lethal stressors may not die during treatment, as has long been thought; instead, death can occur after plating on drug-free agar due to poststress ROS-mediated toxicity. Examples are described in which (i) primary stress-mediated damage was insufficient to kill bacteria due to repair; (ii) ROS overcame repair (i.e., protection from anti-ROS agents was reduced by repair deficiencies); and (iii) killing was reduced by anti-oxidative stress genes acting before stress exposure. Enzymatic suppression of poststress ROS-mediated lethality by exogenous catalase supports a causal rather than a coincidental role for ROS in stress-mediated lethality, thereby countering challenges to ROS involvement in antimicrobial killing. We conclude that for a variety of stressors, lethal action derives, at least in part, from stimulation of a self-amplifying accumulation of ROS that overwhelms the repair of primary damage.reactive oxygen species | poststress cellular response | antimicrobial | antioxidant | damage repair D iscovering ways to manage antimicrobial resistance is among the most important medical challenges of our time (1). Since many antimicrobials can stimulate the production of resistant mutants, often via the SOS response (2-8), one way to limit the emergence of new resistance is to more rapidly and extensively reduce pathogen populations during infection. Toward that end, we and others have been studying how antimicrobials and other lethal stressors kill bacteria. Recent work has drawn attention to the contribution of stress-stimulated accumulation of toxic reactive oxygen species (ROS) (9-21). Finding ways to stimulate ROS-mediated killing could in principle enhance the efficacy of a broad range of antimicrobials. However, an ROS contribution to antimicrobial killing became controversial when the original observation (9) was challenged (22-24). Subsequent work countered many of the challenges (13)(14)(15)25) and extended the phenomenon to thymineless death (26), phage infection (27), the type VI secretion system (27), and overexpression of a MalE-LacZ fusion (28). Moreover, nitric oxide and hydrogen sulfide interfere with antimicrobial killing by suppressing ROS generation/ac...

Non-lethal exposure to H2O2 boosts bacterial survival and evolvability against oxidative stress

“…Recently, a report showed that bacteria lacking the AhpF/C system suffer a severe post-stress recovery. The absence of the AhpF/C system also contributes to the lethality of the mutants probably by the inability of the cell to cope with a low level of ROS after severe oxidative stress [34]. Another protein that illustrates an important influence on priming response is RecA, with the mutant showing a decreased priming response to H 2 O 2 .…”

Reactive oxygen species as the long arm of bactericidal antibiotics

“…However, promptly after the nalidixic acid is washed away, DNA synthesis resumes (12). Based on mass spectrometry quantification, Hong et al (10) measure a decrease in intracellular nalidixic acid concentration to 0.002 MIC after washing and diluting treated cells. The second feature of the system is crucial for determining whether cells die when the antibiotic remains inside the cell or, alternatively, when cells are free of the antibiotic.…”

“…For UV irradiation, the repair of primary damage by photolyase proved instrumental for this important distinction. For nalidixic acid treatment, Hong et al (10) discover that the sequestration of a secondary damaging agent, rather than repair of the primary damage, is essential. Specifically, a higher tolerance level is measured when compounds preventing the accumulation of reactive oxygen species (ROS) are added to the recovery plate ( Fig.…”

“…1). Indeed, when Hong et al (10) use a fluorescent dye to monitor intracellular ROS on the recovery plate 1 h after plating, they could detect ROS in nalidixic acid treated cells, but not in control cells. Continuous generation of ROS is unlikely to stem from stable primary damage, at least in the case of nalidixic acid treatment.…”

“…While UV tolerance inherently lends itself to the separation in time between the lethal agent and subsequent damage recovery, a similar system for the study of antibiotic tolerance has thus far been unavailable. In PNAS, Hong et al (10) describe an elegant experimental system for the separation of stress from recovery with respect to antibiotic tolerance in Escherichia coli. Using this system, they test in isolation the role of secondary damage in cell killing.…”