Antibiotic mode-of-action classification is based upon drug-target interaction and whether the resultant inhibition of cellular function is lethal to bacteria. Here we show that the three major classes of bactericidal antibiotics, regardless of drug-target interaction, stimulate the production of highly deleterious hydroxyl radicals in Gram-negative and Gram-positive bacteria, which ultimately contribute to cell death. We also show, in contrast, that bacteriostatic drugs do not produce hydroxyl radicals. We demonstrate that the mechanism of hydroxyl radical formation induced by bactericidal antibiotics is the end product of an oxidative damage cellular death pathway involving the tricarboxylic acid cycle, a transient depletion of NADH, destabilization of iron-sulfur clusters, and stimulation of the Fenton reaction. Our results suggest that all three major classes of bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage, including proteins involved in triggering the DNA damage response, e.g., RecA.

PrefaceAntibiotic drug-target interactions, and their respective direct effects, are generally wellcharacterized. In contrast, the bacterial responses to antibiotic drug treatments that contribute to cell death are not as well understood and have proven to be quite complex, involving multiple genetic and biochemical pathways. Here, we review the multi-layered effects of drug-target interactions, including the essential cellular processes inhibited by bactericidal antibiotics and the associated cellular response mechanisms that contribute to killing by bactericidal antibiotics. We also discuss new insights into these mechanisms that have been revealed through the study of biological networks, and describe how these insights, together with related developments in synthetic biology, may be exploited to create novel antibacterial therapies. IntroductionOur understanding of how antibiotics induce bacterial cell death is centered on the essential cellular function inhibited by the primary drug-target interaction. Antibiotics can be classified based on the cellular component or system they affect, in addition to whether they induce cell death (bactericidal drugs) or merely inhibit cell growth (bacteriostatic drugs). Most current bactericidal antimicrobials, which are the focus of this review, inhibit DNA synthesis, RNA synthesis, cell wall synthesis, or protein synthesis 1 .Since the discovery of penicillin was reported in 1929 2 , other, more effective antimicrobials have been discovered and developed by elucidation of drug-target interactions, and by drug molecule modification. These efforts have significantly enhanced our clinical armamentarium. Antibiotic-mediated cell death, however, is a complex process that begins with the physical interaction between a drug molecule and its bacterial-specific target, and involves alterations to the affected bacterium at the biochemical, molecular and ultrastructural levels. The increasing prevalence of drug-resistant bacteria 3 , as well as the means of gaining resistance, has made it crucial that we better understand the multilayered mechanisms by which currently available antibiotics kill bacteria, as well as explore and find alternative antibacterial therapies.Antibiotic-induced cell death has been associated with the formation of double-stranded DNA breaks following treatment with DNA gyrase inhibitors 4 , with the arrest of DNA-dependent RNA synthesis following treatment with rifamycins 5 , with cell envelope damage and loss of structural integrity following treatment with cell-wall synthesis inhibitors 6 , and with cellular energetics, ribosome binding and protein mistranslation following treatment with protein * Corresponding author: James Collins, jcollins@bu.edu, phone: (617) 353-0390. NIH Public Access Author ManuscriptNat Rev Microbiol. Author manuscript; available in PMC 2010 December 1. Published in final edited form as:Nat Rev Microbiol. 2010 June ; 8(6): 423-435. doi:10.1038/nrmicro2333. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript sy...

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H 2 O 2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H 2 O 2 . We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.reactive oxygen species | DNA repair | mutagenesis T he increasing incidence of antibiotic-resistant infections coupled with a declining antibiotic pipeline has created a global public health threat (1-6). Therefore there is a pressing need to expand our conceptual understanding of how antibiotics act and to use insights gained from such efforts to enhance our antibiotic arsenal. It has been proposed that different classes of bactericidal antibiotics, regardless of their drug-target interactions, generate varying levels of deleterious reactive oxygen species (ROS) that contribute to cell killing (7,8). This unanticipated notion, built upon important prior work (9-11), has been extended and supported by multiple laboratories investigating wide-ranging drug classes (e.g., β-lactams, aminoglycosides, and fluoroquinolones) and bacterial species (e.g., Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baumannii, Burkholderia cepecia, Streptococcus pneumonia, Enterococcus faecalis) using independent lines of evidence (12-39). Importantly,...