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

Immune cells kill invading microbes by producing reactive oxygen and nitrogen species, primarily hydrogen peroxide (H2O2) and nitric oxide (NO). We previously found that NO inhibits catalases in Escherichia coli, stabilizing H2O2 around treated cells and promoting catastrophic chromosome fragmentation via continuous Fenton reactions generating hydroxyl radicals. Indeed, H2O2-alone treatment kills catalase-deficient (katEG) mutants similar to H2O2+NO treatment. However, the Fenton reaction, in addition to H2O2, requires Fe(II), which H2O2 excess instantly converts into Fenton-inert Fe(III). For continuous Fenton when H2O2 is stable, a supply of reduced iron becomes necessary. We show here that this supply is ensured by Fe(II) recruitment from ferritins and Fe(III) reduction by flavin reductase. Our observations also concur with NO-mediated respiration inhibition that drives Fe(III) reduction. We modeled this NO-mediated inhibition via inactivation of ndh and nuo respiratory enzymes responsible for the step of NADH oxidation, which results in increased NADH pools driving flavin reduction. We found that, like the katEG mutant, the ndh nuo double mutant is similarly sensitive to H2O2-alone and H2O2+NO treatments. Moreover, the quadruple katEG ndh nuo mutant lacking both catalases and efficient respiration was rapidly killed by H2O2-alone, but this killing was delayed by NO, rather than potentiated by it. Taken together, we conclude that NO boosts the levels of both H2O2 and Fe(II) Fenton reactants, making continuous hydroxyl-radical production feasible and resulting in irreparable oxidative damage to the chromosome.

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

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.

Autoantibodies targeting TLR and SMAD pathways define new subgroups in systemic lupus erythematosus.

OBJECTIVES: The molecular targets of the vast majority of autoantibodies in systemic lupus erythematosus (SLE) are unknown. We set out to identify novel autoantibodies in SLE to improve diagnosis and identify subgroups of SLE individuals. METHODS: A baculovirus-insect cell expression system was used to create an advanced protein microarray with 1543 full-length human proteins expressed with a biotin carboxyl carrier protein (BCCP) folding tag, to enrich for correctly folded proteins. Sera from a discovery cohort of UK and US SLE individuals (n = 186) and age/ethnicity matched controls (n = 188) were assayed using the microarray to identify novel autoantibodies. Autoantibodies were validated in a second validation cohort (91 SLE, 92 controls) and a confounding rheumatic disease cohort (n = 92). RESULTS: We confirmed 68 novel proteins as autoantigens in SLE and 11 previous autoantigens in both cohorts (FDR<0.05). Using hierarchical clustering and principal component analysis, we observed four subgroups of SLE individuals associated with four corresponding clusters of functionally linked autoantigens. Two clusters of novel autoantigens revealed distinctive networks of interacting proteins: SMAD2, SMAD5 and proteins linked to TGF-ß signalling; and MyD88 and proteins involved in TLR signalling, apoptosis, NF-κB regulation and lymphocyte development. The autoantibody clusters were associated with different patterns of organ involvement (arthritis, pulmonary, renal and neurological). A panel of 26 autoantibodies, which accounted for four SLE clusters, showed improved diagnostic accuracy compared to conventional antinuclear antibody and anti-dsDNA antibody assays. CONCLUSIONS: These data suggest that the novel SLE autoantibody clusters may be of prognostic utility for predicting organ involvement in SLE patients and for stratifying SLE patients for specific therapies.