. However, when cells were cultured with poor sulfur sources and then exposed to cystine, they transiently exhibited a greatly increased susceptibility to H 2 O 2 , with <1% surviving the challenge. Cell death was due to an unusually rapid rate of DNA damage, as indicated by their filamentation, a high rate of mutation among the survivors, and DNA lesions by a direct assay. Cell-permeable iron chelators eliminated sensitivity, indicating that intracellular free iron mediated the conversion of H 2 O 2 into a hydroxyl radical, the direct effector of DNA damage. The cystine treatment caused a temporary loss of cysteine homeostasis, with intracellular pools increasing about eightfold. In vitro analysis demonstrated that cysteine reduces ferric iron with exceptional speed. This action permits free iron to redox cycle rapidly in the presence of H 2 O 2 , thereby augmenting the rate at which hydroxyl radicals are formed. During routine growth, cells maintain small cysteine pools, and cysteine is not a major contributor to DNA damage. Thus, the homeostatic control of cysteine levels is important in conferring resistance to oxidants. More generally, this study provides a new example of a situation in which the vulnerability of cells to oxidative DNA damage is strongly affected by their physiological state.

Since the discovery of catalase, it has been postulated that aerobic organisms generate enough oxidants to threaten their own fitness and, in particular, their genetic stability. An alternative is that these enzymes exist to defend the cell against more-abundant oxidants imposed by external sources. These hypotheses were tested directly through study of Hpx ؊ (katG katE ahpCF) mutants of Escherichia coli, which lack enzymes to scavenge hydrogen peroxide (H 2O2). These strains grew well in anaerobic medium but poorly when they were aerated. The Hpx ؊ bacteria formed filaments and exhibited high rates of mutagenesis, both indicators of DNA damage. An additional recA mutation caused Hpx ؊ cells to die rapidly upon aeration, even though the intracellular H 2O2 was <1 M. Spin-trap experiments detected substantial hydroxyl radicals, and cell-permeable iron chelators eliminated both the phenotypic defects and hydroxyl-radical formation, confirming that the Fenton reaction was responsible. An Hpx ؊ oxyR strain exhibited even more DNA lesions than did the Hpx ؊ mutant, indicating that the OxyR stress response induced protein(s) that suppressed DNA damage. One critical protein was Dps, an iron-sequestration protein, because Hpx ؊ dps mutants exhibited sensitivity similar to that of the Hpx ؊ oxyR mutant. These results reveal that aerobic E. coli generates sufficient H 2O2 to create toxic levels of DNA damage. Scavenging enzymes and controls on free iron are required to avoid that fate. The rate constant of the Fenton reaction measured at physiological pH was much higher than under the acidic conditions that were used to determine the commonly cited value.Fenton ͉ oxidative DNA damage ͉ Dps

Aerobic growth of Streptococcus pneumoniae results in production of amounts of hydrogen peroxide (H 2 O 2 ) that may exceed 1 mM in the surrounding media. H 2 O 2 production by S. pneumoniae has been shown to kill or inhibit the growth of other respiratory tract flora, as well as to have cytotoxic effects on host cells and tissue. The mechanisms allowing S. pneumoniae, a catalase-deficient species, to survive endogenously generated concentrations of H 2 O 2 that are sufficient to kill other bacterial species is unknown. In the present study, pyruvate oxidase (SpxB), the enzyme responsible for endogenous H 2 O 2 production, was required for survival during exposure to high levels (20 mM) of exogenously added H 2 O 2 . Pretreatment with H 2 O 2 did not increase H 2 O 2 resistance in the mutant, suggesting that SpxB activity itself is required, rather than an H 2 O 2 -inducible pathway. SpxB mutants synthesized 85% less acetyl-phosphate, a potential source of ATP. During H 2 O 2 exposure, ATP levels decreased more rapidly in spxB mutants than in wild-type cells, suggesting that the increased killing of spxB mutants was due to more rapid ATP depletion. Together, these data support the hypothesis that S. pneumoniae SpxB contributes to an H 2 O 2 -resistant energy source that maintains viability during oxidative stress. Thus, SpxB is required for resistance to the toxic by-product of its own activity. Although H 2 O 2 -dependent hydroxyl radical production and the intracellular concentration of free iron were similar to that of Escherichia coli, killing by H 2 O 2 was unaffected by iron chelators, suggesting that S. pneumoniae has a novel mechanism to avoid the toxic effects of the Fenton reaction.

SummaryIn aerobic environments, mutants of Escherichia coli that lack peroxidase and catalase activities (Hpx -) accumulate submicromolar concentrations of intracellular H2O2. We observed that in defined medium these strains constitutively expressed members of the Fur regulon. Iron-import proteins, which Fur normally represses, were fully induced. H2O2 may antagonize Fur function by oxidizing the Fur:Fe 2+ complex and inactivating its repressor function. This is a potential problem, as in iron-rich environments excessive iron uptake would endanger H2O2-stressed cells by accelerating hydroxyl-radical production through the Fenton reaction. However, the OxyR H2O2-response system restored Fur repression in iron-replete Luria-Bertani medium by upregulating the synthesis of Fur protein. Indeed, when the OxyR binding site upstream of fur was disrupted, Hpx -mutants failed to repress transporter synthesis, and they exhibited high levels of intracellular free iron. Mutagenesis and bacteriostasis resulted. These defects were eliminated by mutations or chelators that slowed iron import, confirming that dysregulation of iron uptake was the root problem. Thus, aerobic organisms must grapple with a conundrum: how to monitor iron levels in oxidizing environments that might perturb the valence of the analyte. The induction of Fur synthesis by the OxyR response comprises one evolutionary solution to that problem.

Foot and ankle infections are the most common cause of hospitalization among diabetic patients, and Staphylococcus aureus is a major pathogen implicated in these infections. Patients with insulin-resistant (type 2) diabetes are more susceptible to bacterial infections than nondiabetic subjects, but the pathogenesis of these infections is poorly understood. C57BL/6J-Lepr db /Lepr db (hereafter, db/db) mice develop type 2 diabetes due to a recessive, autosomal mutation in the leptin receptor. We established a S. aureus hind paw infection in diabetic db/db and nondiabetic Lepr +/+ (+/+) mice to investigate host factors that predispose diabetic mice to infection. Nondiabetic +/+ mice resolved the S. aureus hind paw infection within 10 days, whereas db/db mice with persistent hyperglycemia developed a chronic infection associated with a high bacterial burden. Diabetic db/db mice showed a more robust neutrophil infiltration to the infection site and higher levels of chemokines in the infected tissue than +/+ mice. Blood from +/+ mice killed S. aureus in vitro, whereas db/db blood was defective in bacterial killing. Compared with peripheral blood neutrophils from +/+ mice, db/db neutrophils demonstrated a diminished respiratory burst when stimulated with S. aureus. However, bone marrow-derived neutrophils from +/+ and db/db mice showed comparable phagocytosis and bactericidal activity. Our results indicate that diabetic db/db mice are more susceptible to staphylococcal infection than their nondiabetic littermates and that persistent hyperglycemia modulates innate immunity in the diabetic host.