Cyanide enhances hydrogen peroxide toxicity by recruiting endogenous iron to trigger catastrophic chromosomal fragmentation

Abstract

Hydrogen peroxide (HP) or cyanide (CN) are bacteriostatic at low-millimolar concentrations for growing Escherichia coli, whereas CN+HP mixture is strongly bactericidal. We show that this synergistic toxicity is associated with catastrophic chromosomal fragmentation. Since CN-alone does not kill at any concentration, while HP-alone kills at 20 mM, CN must potentiate HP poisoning. The CN+HP killing is blocked by iron chelators, suggesting Fenton’s reaction. Indeed, we show that CN enhances plasmid DNA relaxation due to Fenton’s reaction in vitro. However, mutants with elevated iron or HP pools are not acutely sensitive to HP-alone treatment, suggesting that, in addition, in vivo CN recruits iron from intracellular depots. We found that part of the CN-recruited iron pool is managed by ferritin and Dps: ferritin releases iron on cue from CN, while Dps sequesters it, quelling Fenton’s reaction. We propose that disrupting intracellular iron trafficking is a common strategy employed by the immune system to kill microbes.

Bactericidal Antibiotics Induce Toxic Metabolic Perturbations that Lead to Cellular Damage

“…It only takes a single unrepaired double-stand break (DSB) to kill a bacterial cell (Bonura and Smith, 1975; Kouzminova and Kuzminov, 2012); consequently, it can take very little 8-oxo-dG in DNA and/or chromosomal fragmentation to kill a cell (Mahaseth and Kuzminov, 2015). For example, recent work has provided evidence that even the very low endogenous levels of 8-oxo-dG in DNA can prove lethal if cells hyper-initiate DNA replication (Charbon et al, 2014).…”

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

“…When both double-strand break repair and linear DNA degradation are inactivated in cells with runaway overinitiation, subchromosomal DNA fragments are indeed detected by pulsed-field gels (Figure 4, C and D). Interestingly, the levels of chromosomal fragmentation induced by runaway overinitiation are moderate compared to some DNA-damaging treatments (Khan and Kuzminov 2013;Mahaseth and Kuzminov 2015), suggesting circular chromosomes with multiple linear tails as the main chromosome state ( Figure 5G, III). Another factor likely diminishing the actual fragmentation levels is new initiation on subchromosomal fragments (Figure 5G,II), which will block their entrance into pulsed-field gels.…”

“…Indeed, we found modest levels of chromosomal fragmentation [reaching 15% over the background after 5-6 hr of IPTG induction ( Figure S5)] to develop in the IOC recBC mutant, compared to ,5% in the wild-type cells, recA, ruvABC, and seqA mutants (Figure 4, C and D). The modest levels of this fragmentation were far from the immediately lethal catastrophic fragmentation levels observed with some DNA-damaging treatments (Khan and Kuzminov 2013;Mahaseth and Kuzminov 2015) and predicted an equally modest effect on the actual survival. At the same time, the IPTG-induced IOC recD mutant (reduced for linear DNA degradation, but enhanced for repair of double-strand breaks) showed a 23 higher fragmentation (.30%) (Figure 4, C and D), suggesting that, in the case of runaway CRC, linear DNA Black lines, the circular domains of the chromosome; blue lines, the (partially) linear domains that would not enter pulsed-field gels; the pink lines, subchromosomal fragments that would enter pulsed-field gels.…”

Potentiation of hydrogen peroxide toxicity: From catalase inhibition to stable DNA-iron complexes

“…Both are rare examples of diffusion-limited enzymes — the fastest enzymes possible (39) — capable of scavenging up to low millimolar concentrations of hydrogen peroxide during acute exposures without adverse consequences for the cell, once H 2 O 2 is removed. Even though 3 mM concentrations of H 2 O 2 will eventually kill during prolonged exposures (40), the H 2 O 2 concentrations that kill cells within minutes start around 30 mM (24, 41). This creates a classic engineering problem: besides the obvious caveat that such high H 2 O 2 concentrations will be dangerous to the producing cell itself, no known cells are actually capable of producing a short burst of 30 mM H 2 O 2 , or of maintaining a several-hour 3 mM levels of H 2 O 2 (perhaps with the exception of lactobacilli (42)).…”

“…Catalase poisoning has been offered to explain potentiation of H 2 O 2 toxicity by other chemicals (Fig. 1), however the inefficiency of this poisoning limits the power of this explanation (41, 50–52). Important for our discussion, though, is the concept of catalase inhibition by a separate agent, which highlights a general strategy for solving the in vivo hydrogen peroxide concentration problem, the strategy of potentiated toxicity .…”