A-056. Mistranslation of Membrane Proteins and Two-Component System Activation Trigger Aminoglycoside-Mediated Hydroxyl Radical Formation and Cell Death

M. A. Kohanski, D. J. Dwyer, J. Wierzbowski, G. Cottarel, J. J. Collins;
Boston Univ., Boston, MA.

Background: Bactericidal antibiotic-mediated free radical formation occurs via fluctuations in metabolism culminating in the formation of lethal hydroxyl radicals. It remains unclear how cidal antibiotic classes that target completely different cellular functions can all stimulate a common cellular death pathway. Here we investigate the pathway whereby aminoglycosides trigger hydroxyl radical formation in E. coli. Methods: To work out the aminoglycoside trigger, we utilized gene expression microarrays and high-throughput screening of the single-deletion knockout library following aminoglycoside treatment to focus on the pathways and genes likely related to radical formation. To confirm pathways and genes specifically related to aminoglycoside-mediated hydroxyl radical formation, we examined deletion strains for changes in radical formation, membrane depolarization and survival following antibiotic treatment. Results: Using systems-level approaches, we identified three key networks related to aminoglycoside lethality: an ArcA-regulated metabolic network, a protease network related to protein misfolding, and a network of membrane-bound proteins related to transport and the electrochemical gradient. We found, using high-throughput screening, that knocking out hflK, hflC or secG, which are involved in regulating membrane proteins, led to decreased growth in the presence of gentamicin. We show by disabling these systems that mistranslation and misfolding of membrane proteins are central for aminoglycoside-induced hydroxyl radical formation and cell death. We further demonstrate that aminoglycoside-induced mistranslation of membrane proteins leads to membrane depolarization, alteration of metabolism, hydroxyl radical formation and ultimately cell death via signaling through the envelope stress response and redox-responsive two-component systems. Conclusions: Our results indicate that aminoglycoside-induced hydroxyl radical formation is triggered by two-component system sensing of misfolded envelope-bound proteins resulting in hydroxyl radical formation and cell death, and that these systems are viable targets for enhancing aminoglycosides.