A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics

A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics

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.

Current antimicrobial therapies, which cover a wide array of targets , fall into two general categories: bactericidal drugs, which kill bacteria with an efficiency of >99.9%, and bacteriostatic drugs, which merely inhibit growth.

Antibacterial drug-target interactions are well studied and predominantly fall into three classes: inhibition of DNA replication and repair, inhibition of protein synthesis, and inhibition of cell-wall turnover.

Bacteriostatic drugs predominantly inhibit ribosome function, targeting both the 30S (tetracycline family and aminocyclitol family) and 50S (macrolide family and chloramphenicol) ribosome subunits.

With regard to other classes of bactericidal antibiotics, quinolones target DNA replication and repair by binding DNA gyrase complexed with DNA, which drives double-strand DNA break formation and cell death.

Cell-wall synthesis inhibitors (such as β-lactams), which interact with penicillin-binding proteins.

and glycopeptides that interact with peptidoglycan building blocks, interfere with normal cell-wall synthesis and induce lysis and cell death.

We have recently shown that bacterial gyrase inhibitors, including synthetic quinolone antibiotics and the native proteic toxin CcdB, induce a breakdown in iron regulatory dynamics, which promotes formation of reactive oxygen species that contribute to cell death.

Hydroxyl radical formation utilizing internal iron and the Fenton reaction appears to be the most significant contributor to cell death among the reactive oxygen species formed. The Fenton reaction leads to the formation of hydroxyl radicals through the reduction of hydrogen peroxide by ferrous iron. We chose to investigate whether hydroxyl radical formation also contributes to antibiotic-induced cell death in bacteria among the other classes of antibiotics. Here we report that the three major classes of bactericidal antibiotics, regardless of drug-target interaction, stimulate hydroxyl radical formation in bacteria. Furthermore, we demonstrate that hydroxyl radical generation contributes to the killing efficiency of these lethal drugs. We also show, in contrast, that bacteriostatic drugs do not produce hydroxyl radicals. We demonstrate that all bactericidal drug classes utilize internal iron from iron-sulfur clusters to promote Fenton-mediated hydroxyl radical formation and show that these events appear to be mediated by the tricarboxylic acid (TCA) cycle and a transient depletion of NADH. We propose that there is a common mechanism of cellular death underlying all classes of bactericidal antibiotics whereby harmful hydroxyl radicals are formed as a function of metabolism-related NADH depletion, leaching of iron from iron-sulfur clusters, and stimulation of the Fenton reaction.