Nitric oxide potentiates hydrogen peroxide-induced killing of Escherichia coli

Previously, we reported that nitric oxide (NO) provides significant protection to mammalian cells from the cytotoxic effects of hydrogen peroxide (H₂O₂). Murine neutrophils and activated macrophages, however, produce NO, H₂O₂, and other reactive oxygen species to kill microorganisms, which suggests a paradox. In this study, we treated bacteria (Escherichia coli) with NO and H₂O₂ for 30 min and found that exposure to NO resulted in minimal toxicity, but greatly potentiated (up to 1,000-fold) H₂O₂-mediated killing, as evaluated by a clonogenic assay. The combination of NO/H₂O₂ induced DNA double strand breaks in the bacterial genome, as shown by field- inverted gel electrophoresis, and this increased DNA damage may correlate with cell killing. NO was also shown to alter cellular respiration and decrease the concentration of the antioxidant glutathione to a residual level of 15-20% in bacterial cells. The iron chelator desferrioxamine did not stop the action of NO on respiration and glutathione decrease, yet it prevented the NO/H₂O₂ synergistic cytotoxicity, implication metal ions as critical participants in the NO/H₂O₂ cytocidal mechanism. Our results suggest a possible mechanism of modulation of H₂O₂-mediated toxicity, and we propose a new key role in the antimicrobial macrophagic response for NO.