Catalase inhibition by nitric oxide potentiates hydrogen peroxide to trigger catastrophic chromosome fragmentation in Escherichia coli

Abstract

Hydrogen peroxide (H2O2, HP) is a universal toxin that organisms deploy to kill competing or invading cells. Bactericidal action of H2O2 presents several questions. First, the lethal H2O2 concentrations in bacterial cultures are 1000x higher than, for example, those calculated for the phagosome. Second, H2O2-alone kills bacteria in cultures either by mode-one, via iron-mediated chromosomal damage, or by mode-two, via unknown targets, but the killing mode in phagosomes is unclear. Third, phagosomal H2O2 toxicity is enhanced by production of nitric oxide (NO), but in vitro studies disagree: some show NO synergy with H2O2 antimicrobial action, others instead report alleviation. To investigate this “NO paradox,” we treated Escherichia coli with various concentrations of H2O2-alone or H2O2+NO, measuring survival and chromosome stability. We found that all NO concentrations make sublethal H2O2 treatments highly lethal, via triggering catastrophic chromosome fragmentation (mode-one killing). Yet, NO-alone is not lethal, potentiating H2O2 toxicity by blocking H2O2 scavenging in cultures. Catalases represent obvious targets of NO inhibition, and catalase-deficient mutants are indeed killed equally by H2O2-alone or H2O2+NO treatments, also showing similar levels of chromosome fragmentation. Interestingly, iron chelation blocks chromosome fragmentation in catalase-deficient mutants without blocking H2O2-alone lethality, indicating mode-two killing. In fact, mode-two killing of WT cells by much higher H2O2 concentrations is transiently alleviated by NO, reproducing the “NO paradox.” We conclude that NO potentiates H2O2 toxicity by promoting mode-one killing (via catastrophic chromosome fragmentation) by otherwise static low H2O2 concentrations, while transiently suppressing mode-two killing by immediately lethal high H2O2 concentrations.

Nitric oxide precipitates catastrophic chromosome fragmentation by bolstering both hydrogen peroxide and Fe(II) Fenton reactants in E. coli

“…1A, left). We have previously demonstrated that the nature of this phenomenon is not simply redundancy of two toxic treatments overwhelming anti-toxicity mechanisms of the cells, but potentiation of H2O2 toxicity by NO to cause catastrophic chromosome fragmentation (22) (Fig. 1A, right).…”

“…While direct electrochemical detection of ROS and RNI in phagosomes within macrophages with nano-electrodes is still being developed (25), and the outside microelectrode J o u r n a l P r e -p r o o f measurements suggesting their extremely high (micromolar) concentrations (26)(27)(28) not being widely appreciated, researchers still operate with reasonable calculations, that suggest H2O2 concentrations inside the macrophage and neutrophil phagosome are on the order of 10 µM (29,30). At the same time, as mentioned above, the minimal H2O2 concentrations that kill WT E. coli in culture are 10 mM (7,21,22). To explain how such low H2O2 concentrations can be effective against bacteria in the phagosome, H2O2 was proposed and shown to be potentiated by other substances (reviewed in (31)), notably by nitric oxide (NO), abundantly produced by immune cells.…”

“…In addition, the Fenton-induced DNA damage is repaired by base-excision repair and recombinational repair (18)(19)(20). These multiple systems in E. coli ensure that acute H2O2 doses up to 5 mM are only bacteriostatic in E. coli, although H2O2 concentrations ≥10 mM apparently self-potentiate to cause chromosomal fragmentation and loss of viability even in this quite resistant bacterium (7,21,22).…”

“…Generally, in cells with intact H2O2 scavenging, low millimolar H2O2 concentrations kill by mode-one; mode-one killing is blocked by iron chelators and affects mostly the DNA repair mutants, defining mode-one as IF-iron-dependent killing via DNA damage. When WT E. coli is killed by 10 mM H2O2, it is still mode-one (22). In contrast, higher concentrations of H2O2, starting with 20-25 mM in E. coli, kill by modetwo, which is insensitive of iron-chelation, active metabolism or of DNA repair capacity, defining mode-two as killing of unknown nature that does not depend on IF-iron or DNA damage.…”

“…In contrast, higher concentrations of H2O2, starting with 20-25 mM in E. coli, kill by modetwo, which is insensitive of iron-chelation, active metabolism or of DNA repair capacity, defining mode-two as killing of unknown nature that does not depend on IF-iron or DNA damage. The additional distinction between the two H2O2 killing modes relevant for this work is that, while mode-one killing is potentiated by NO, mode-two killing is inhibited by NO treatment (22).…”