Supplementary Materials Supplemental material supp_197_23_3708__index. is reversed upon the addition of antioxidants, which restores bacterial growth, suggesting that the cells are inhibited or killed by excessive free radicals. A model is proposed in which peptidoglycan-targeting antibiotics facilitate MAPK1 the entry of lethal levels of iron-complexed DFP into the bacterial cytoplasm, a process that drives the generation of ROS. This new finding suggests that, in addition to restriction of access to iron as a general growth-restricting strategy, targeting of cellular pathways or networks that selectively disrupt normal iron homeostasis can have potent bactericidal outcomes. IMPORTANCE The prospect that common bacteria will become resistant to all antibiotics is challenging the medical community. In addition to the development of next-generation antibiotics, new bacterial targets that display cytotoxic properties when altered need to be identified. Data presented here demonstrate that combining subinhibitory levels of both iron chelators and certain antibiotics kills pathogenic as well. INTRODUCTION Antibiotics are compounds that inhibit or kill SCH 727965 manufacturer bacteria and may have saved more lives than any other medical intervention, aside from vaccination (1). However, the development of strains resistant to antibiotics is precipitating a medical crisis. It is estimated that each year in the United States there SCH 727965 manufacturer are 900,000 cases of antibiotic-resistant infections, with an estimated cost of over 20 billion U.S. dollars (2). Several factors contribute to resistance, including the over- and misuse of these drugs (which generates evolutionary pressure that selects for resistant strains), horizontal gene transfer (which allows elements that confer resistance to be transferred among species or genera), and the high level of genetic diversity generated from mutation (which creates more fit members when they are faced with antibiotics [3, 4]). Numerous strategies have been employed to combat the resistance problem, including the reduced use of antibiotics in livestock, the development of next-generation antibiotics with little established resistance, the use of SCH 727965 manufacturer biologics such as phage to kill bacteria or probiotics to stimulate the host immune system, and the combination of different antibiotics into a type of killing SCH 727965 manufacturer cocktail (3, 5). Most antibiotics function by disrupting one of three critical cellular functions, including the inhibition of DNA replication (e.g., quinolones), the inhibition of protein biosynthesis (e.g., aminoglycosides), and the inhibition of cell wall biosynthesis (e.g., -lactams) (5). In addition to finding new compounds, there is also a great need to discover new targets and mechanisms to kill bacterial cells that differ from traditional approaches. Nutritional immunity is the term used to describe the host’s sequestration of critical nutrients to prevent the growth and replication of bacteria during an active infection. A component of nutritional immunity is the sequestration of metals, especially iron. Bacterial replication is absolutely dependent on the acquisition of iron from host sources. The disruption of bacterial iron metabolism has dramatic negative consequences on virulence and (6,C9). Because an estimated 30% of all enzymes require metals as a cofactor and iron is critical for such cellular events as DNA biosynthesis, the trichloroacetic acid cycle, oxidative stress defense, and energy transduction (7, 9, 10), targeting of iron-dependent processes represents a viable strategy for antimicrobial development. Indeed, there are a growing number of studies evaluating the use of iron chelators as antibacterials, with efficacy demonstrated in some cases (11,C13) but not others (14, 15). Inspired by the way mammals restrict bacterial growth to prevent infection (16, 17), we report here that the combined use of iron chelators and sublethal concentrations of some antibiotics generates a potent response that kills the cells of a model Gram-negative blood pathogen (extraintestinal pathogenic [ExPEC]). Investigation of the mechanism behind this response links it to a supraphysiologic elevation in the levels of cellular iron content coupled to an iron starvation response. This state, in turn, promotes the development of high cellular levels of reactive oxygen SCH 727965 manufacturer species (ROS) that ultimately kill the cell. MATERIALS AND METHODS Bacterial strains, media, and reagents. ExPEC CP9 (18) and methicillin (MET)-resistant TCH1516 (19) were isolated from hospitalized patients and were kind gifts.