H2O2 Stress Research Articles

The mitochondrial glutathione peroxidase GPX3 is essential for H2O2 homeostasis and root and shoot development in rice

Glutathione (GSH) peroxidases (GPXs: EC 1.11.1.9 and EC1.11.1.12) are non-heme thiol peroxidases that catalyze the reduction of H2O2 or organic hydroperoxides to water, and they have been identified in almost all kingdoms of life. The rice glutathione peroxidase (OsGPX) gene family is comprised of 5 members spread throughout a range of sub cellular compartments. The OsGPX gene family is induced in response to exogenous H2O2 and cold stress. In contrast, they are down regulated in response to drought and UV-B light treatments. Transgenic rice plants have been generated that lack mitochondrial OsGPX3. These GPX3s plants showed shorter roots and shoots compared to non-transformed (NT) plants, and higher amounts of H2O2 mitochondrial release were observed in the roots of these plants cultivated under normal conditions. This accumulation of H2O2 is positively associated with shorter root length in GPX3s plants compared to NT ones. Moreover, GPX3 promoter analysis indicated that it is mainly expressed in root tissue. These results suggest that silencing the mitochondrial OsGPX3 gene impairs normal plant development and leads to a stress-induced morphogenic response via H2O2 accumulation.

Astaxanthin decreases inflammatory biomarkers associated with cardiovascular disease in human umbilical vein endothelial cells

As a potent antioxidant, astaxanthin may downregulate inflammation‐associated factors involved in endothelial dysfunction and cardiovascular disease progression. We studied the possible protective effects of astaxanthin against endothelial dysfunction in human umbilical vein endothelial cells (HUVEC) induced with H2O2. Cells were pre‐incubated with 0, 0.01, 0.1, or 1.0 μmol/L astaxanthin for 48 h, then oxidative stress induced with 100 μmol/L H2O2 overnight. Uptake kinetics showed a time‐dependent accumulation of astaxanthin (1.0 μmol/L) by HUVEC over the 48 h period. Oxidative stress induction with H2O2 decreased intracellular antioxidant activity (SOD and GPx) and increased the production of reactive oxygen species (ROS), inflammatory cytokines and mediators. In contrast, pre‐incubation of HUVEC with astaxanthin prior to H2O2 stress increased (P < 0.05) SOD, but not GPx activity, and decreased the production of ROS, PGE2, LTB4, NO, IL‐8, and IFN‐γ. Pre‐treatment with astaxanthin also downregulated transcriptional activation of NF‐κB and activator protein‐1, thereby inhibiting downstream production of inflammatory cytokines and mediators. Therefore, astaxanthin inhibits inflammatory biomarkers upregulated by H2O2, likely by scavenging ROS required for transcriptional activation. Supported by the University of Washington and Washington State University.

Reduction of oxidative stress during recovery accelerates normalization of primary cilia length that is altered after ischemic injury in murine kidneys

The primary cilium is a microtubule-based nonmotile organelle that extends from the surface of cells, including renal tubular cells. Here, we investigated the alteration of primary cilium length during epithelial cell injury and repair, following ischemia/reperfusion (I/R) insult, and the role of reactive oxygen species in this alteration. Thirty minutes of bilateral renal ischemia induced severe renal tubular cell damage and an increase of plasma creatinine (PCr) concentration. Between 8 and 16 days following the ischemia, the increased PCr returned to normal range, although without complete histological restoration. Compared with the primary cilium length in normal kidney tubule cells, the length was shortened 4 h and 1 day following ischemia, increased over normal 8 days after ischemia, and then returned to near normal 16 days following ischemia. In the urine of I/R-subjected mice, acetylated tubulin was detected. The cilium length of proliferating cells was shorter than that in nonproliferating cells. Mature cells had shorter cilia than differentiating cells. Treatment with Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP), an antioxidant, during the recovery of damaged kidneys accelerated normalization of cilia length concomitant with a decrease of oxidative stress and morphological recovery in the kidney. In the Madin-Darby canine kidney (MDCK) cells, H(2)O(2) treatment caused released ciliary fragment into medium, and MnTMPyP inhibited the deciliation. The ERK inhibitor U0126 inhibited elongation of cilia in normal and MDCK cells recovering from H(2)O(2) stress. Taken together, our results suggest that primary cilia length reflects cell proliferation and the length of primary cilium is regulated, at least, in part, by reactive oxygen species through ERK.

Structure of the N-terminal domain of human thioredoxin-interacting protein

Thioredoxin-interacting protein (TXNIP) is one of the six known α-arrestins and has recently received considerable attention owing to its involvement in redox signalling and metabolism. Various stress stimuli such as high glucose, heat shock, UV, H2O2 and mechanical stress among others robustly induce the expression of TXNIP, resulting in the sequestration and inactivation of thioredoxin, which in turn leads to cellular oxidative stress. While TXNIP is the only α-arrestin known to bind thioredoxin, TXNIP and two other α-arrestins, Arrdc4 and Arrdc3, have been implicated in metabolism. Furthermore, owing to its roles in the pathologies of diabetes and cardiovascular disease, TXNIP is considered to be a promising drug target. Based on their amino-acid sequences, TXNIP and the other α-arrestins are remotely related to β-arrestins. Here, the crystal structure of the N-terminal domain of TXNIP is reported. It provides the first structural information on any of the α-arrestins and reveals that although TXNIP adopts a β-arrestin fold as predicted, it is structurally more similar to Vps26 proteins than to β-arrestins, while sharing below 15% pairwise sequence identity with either.

Adaptive evolution of Corynebacterium glutamicum resistant to oxidative stress and its global gene expression profiling

Corynebacterium glutamicum was adapted in a chemostat for 1,900 h with gradually increasing H2O2 stress to understand the oxidative stress response of an industrial host. After 411 generations of adaptation, C. glutamicum developed the ability to grow under stress of 10 mM H2O2, whereas the wild-type did not. The adapted strain also showed increased stress resistance to diamide and menadione, SDS, Tween 20, HCl, NaOH, and ampicillin. A total of 1,180 genes in the RNA-seq transcriptome analysis of the adapted strain were up-regulated twice or higher (corresponding to 38.6 % of the genome), and 126 genes were down-regulated half or less (4.1 % of genome) under 10 mM H2O2-stress conditions compared with those of the wild-type under a no stress condition. Especially the aromatic compound-degrading gene clusters (vanRABK, pcaJIRFLO, and benABCDRKE) were more than threefold up-regulated. Plausible reasons for the H2O2-stress tolerance of the adapted strain are discussed as well as the potential strategy for development of oxidative stress-tolerant strain.

Reversible thiol oxidation in the H2O2-dependent activation of the transcription factor Pap1

Reversible thiol oxidation is both a mark of hydrogen peroxide (H2O2) toxicity and an initiator of signalling events. H2O2 sensors contain exposed and reactive cysteine residues, which become transiently oxidized as an activation mechanism. In fission yeast, the Pap1 (pombe AP-1) transcription factor is normally cytosolic, and upon H2O2 stress it undergoes post-translational modifications impairing its nuclear export; genetic evidences suggested the formation of a disulphide bond in Pap1 as a triggering activation event. Nuclear Pap1 is then recruited to about 50-80 promoters and induces an adaptation response. We have now dissected the role of all seven cysteine residues in Pap1 using genetic and proteomic techniques, and we show that four of them are required for Pap1 to be activated by H2O2 stress. Thus, mutants lacking each one of these cysteine residues display sensitivity to peroxides. Furthermore, these mutant proteins do not become oxidized by H2O2 and cannot bind to promoters or trigger the Pap1-dependent gene expression program. We also demonstrate, by proteomic analysis of reduced and oxidized Pap1, that these four cysteine residues are reversibly oxidized upon H2O2 stress. Our study suggests that not just one but probably two disulphide bonds are required to promote the important conformational changes that trigger Pap1 activation and nuclear accumulation.

Comparison of Efficacy and Safety Evaluation of Latanoprost Formulations with and without Benzalkonium Chloride

Background: This study investigated the safety (cytotoxicity in vitro) and pharmacological effects (ocular hypotensive effects and aqueous humor concentrations in normotensive monkeys in vivo) of latanoprost formulations with benzalkonium chloride (latanoprost with BAK) and without BAK (NP). Methods: A bioequivalence study of latanoprost with BAK and NP was also conducted on human healthy volunteers. Cytotoxicity and the protective effect against H2O2 stress in vitro were evaluated using human corneal epithelial cells. The ocular hypotensive effects in normotensive monkeys were measured by pneumatonometer and the aqueous humor concentrations of latanoprost free acid were determined by liquid chromatography/mass spectrum (LC/MS) methods. The bioequivalence study of latanoprost with BAK and NP was carried out as a single eye drop, two-sequence, crossover randomized study. Results: Cytotoxicity tests in vitro revealed that NP was less toxic than latanoprost with BAK and significantly inhibited H2O2 induced cell damage while latanoprost with BAK did not. The hypotensive efficacy and the latanoprost free acid concentrations in aqueous humor of each formulation were not significantly different in monkeys. In the bioequivalence study, NP was bioequivalent to latanoprost with BAK. NP was safer than latanoprost with BAK with respect the results obtained in the in vitro cytotoxicity test. There was no difference observed between latanoprost with BAK and NP in the IOP lowering effect in monkeys and healthy volunteers. Conclusion: Taken together, these results indicate that NP is as effective as latanoprost with BAK, and is more likely to maintain ocular surface health than latanoprost with BAK.

Astaxanthin Decreases Inflammatory Biomarkers Associated with Cardiovascular Disease in Human Umbilical Vein Endothelial Cells

Oxidative stress and inflammation are strongly linked to the development of cardiovascular disease (CVD). As a potent antioxidant, astaxanthin may downregulate inflammation-associated factors involved in endothelial dysfunction and CVD progression. We studied the possible protective effects of astaxanthin against endothelial dysfunction in human umbilical vein endothelial cells (HUVEC) induced with hydrogen peroxide (H2O2). Cells were pre-incubated with 0, 0.01, 0.1, or 1.0 μmol/L astaxanthin for 48 h, then oxidative stress induced with 100 μmol/L H2O2 overnight. Uptake kinetics of astaxanthin showed a time-dependent uptake of astaxanthin (1.0 μmol/L) by HUVEC over the 48 h incubation period. Oxidative stress induction with H2O2 in HUVEC decreased intracellular antioxidant activity and increased the production of inflammatory biomarkers and reactive oxygen species (ROS). In contrast, pre-incubation of HUVEC with astaxanthin prior to H2O2 stress increased (P < 0.05) superoxide dismutase activity and decreased ROS, prostaglandin E2, leukotriene B4, NO, IL-8, and IFN- production. Pre-treatment with astaxanthin also downregulated the transcriptional activation of NF-κB and activator protein-1, thereby inhibiting downstream production of inflammatory mediators and cytokines. Therefore, astaxanthin protects against factors initiated by H2O2 addition, likely by scavenging ROS required for transcriptional activation and inhibiting the production of inflammatory biomarkers involved in endothelial dysfunction and CVD. Abbreviations: AP-1: activator protein-1; CVD: cardiovascular disease; CS: culture supernatant; EGM-2: endothelial growth medium-2; GPx: glutathione peroxidase; H2O2: hydrogen peroxide; HUVEC: human umbilical vein endothelial cell; LTB4: leukotriene B4; PGE2: prostaglandin E2; ROS: reactive oxygen species; SOD: superoxide dismutase; THF: tetrahydrofuran.

Haberlea Rhodopensis has Potential as a New Drug Source Based on its Broad Biological Modalities

ABSTRACTHaberlea rhodopensis—a Balkan endemite belonging to the group of resurrection plants, was studied as a potential source of novel cancer modulating drugs. Human embryonic (HEK293 p53++/+) and prostate cancer cell lines—LNCaP (p53+/+) and PC3 (p53−/-) were used as a model to follow the reaction to oxidative and genotoxic stress after pre-treatment with H. rhodopensis extract. Oxidative stress was estimated by flow cytometry (FCS) andfluorescent plate reader (FPR) using reactive oxygen speciesfluorescent dye (H2DCFDA). UV induced DNA damage was assessed by FCS PUMA (p53 upregulated modulator of apoptosis) expression. Inflammatory pathways were challenged using synthetic peptidoglycan by FCS Act1 expression and NFkB reporter stable cell line. Pre-treated-cells were assessed using DCF dye (FPR and FCS) for ROS (Reactive Oxygen Species) generation after dose-dependent H2O2 stress and decreased signal compared to non-treated cells. H. rhodopensis had cumulative effect on cell death in PC3 cells. UV-inducedgenotoxic stress resulted in FCS-detected PUMA upregulation only in PC3. Haberlea treated cells had better vitality and their challenge using a bacterial peptidoglycan resulted in an upregulation of Act1, but only in LNCaP cells. The NFkB reporter vector revealed transcription factor activation upon treatment with only peptidoglycan, while Haberlea pre-treatment resulted in negative modulation of the NFkB induction.Our data show that H. rhodopensis extracts have anti-oxidative effect in cancer vs. normal cell lines and differentially modulate distinct cell lines in genotoxic and inflammatory stress, favoring NFKB activation in p53+/+ cells, while suppressing its signaling in p53−/- cells.

Hydrogen peroxide를 처리한 rat 배아심근 H9c2 세포에서 CLA의 oxidative stress 완화 효과

Conjugated linoleic acid (CLA) exhibits several beneficial biological activities including anticarcinogenesis and body-fat reduction. Now, we report that CLA ameliorated the oxidative stress in rat cardiomyoblast cells, H9c2, treated with hydrogen peroxide (H2O2). Cells were cultured in DMEM/F-12 media at 37℃ with humidified atmosphere of 5% CO2. The cells, cultured for 48 hrs, were seeded at a density 3.5×103 cell/well in a 24 well-plate and incubated for 24 hr. Using these cells, two experiments were performed: the cytotoxicity test of CLA (10, 20, 30, 40, and 50 μMs), and the oxidative stress amelioration test of CLA (20 and 50 μMs) against cells treated with H2O2 (10 and 50 μMs) for 1 and 2 hrs. CLA enhanced the growth of H9c2 cells at any concentrations of CLA and at any incubation times (up to 6 days), indicating that CLA acts as a growth stimulant. No protective effect of CLA (20 and 50 μMs) was seen in cells treated 50 μM H2O2 for 1 and 2 hr, but these CLA concentrations ameliorated (p<0.05) the adverse effect of 10 μM H2O2 in cells treated for 1 hr. These CLA concentrations significantly (p<0.05) reduced the proportion of apoptotic cells, relative to control cells. These results suggest that CLA protected H9c2 cells from the oxidative stress of H2O2 through the suppression of cell apoptosis and could be a useful compound for the prevention of cardiac diseases caused by oxidative stress.

L‐carnitine improves hydrogen peroxide‐induced impairment of nuclear maturation in porcine oocytes

We investigated the effect of oxidative stress induced by hydrogen peroxide (H2 O2 ) on lipid peroxide (LPO) level and nuclear maturation in porcine oocytes cultured with or without cumulus cells. After 22 h of pre-culture, oocytes with attached cumulus cells (COC group) or denuded oocytes (DO group) were cultured with H2 O2 , and intra-oocyte H2 O2 and LPO levels were quantitatively analyzed using immunofluorescence. This is the first report evaluating LPO levels in porcine oocytes. After H2 O2 supplementation, the DO group showed severe accumulation of H2 O2 and LPO in the oocytes. Similarly, while inhibition of progression of nuclear maturation was observed in both groups, the effect was more severe in the DO group. These results demonstrate that cumulus cells reduce the accumulation of H2 O2 stress in oocytes. Furthermore, we attempted to reduce the oxidative stress by H2 O2 with L-carnitine, a H2 O2 scavenger. L-carnitine decreased H2 O2 and LPO levels in the oocytes in both groups, and improvement in the progression of impaired nuclear maturation was observed. These effects were different by the presence of cumulus cells. Our results provide that L-carnitine is useful for alleviating H2 O2 -induced oxidative stress by reducing LPO levels and improving the progression of nuclear maturation.

Comparative cytological responses of lung epithelial and pleural mesothelial cells following in vitro exposure to nanoscale SiO2

Due to unique surface chemistries and the ability to easily functionalize their surface, amorphous silica nanoparticles are being assimilated into medicinal and consumer products at an increasing rate. Subsequently, there is an emergent need to understand the interactions of these particulates with biological systems in an attempt to mitigate toxicity. The identification of susceptible or resistant cell types of the pulmonary system remains a critical step in the development of toxicity assessments for nanoparticle-based platforms. Specific to this study, the cellular responses of A549 lung epithelial and MeT-5A pleural mesothelial cell lines as a means of detecting nanoparticle-induced oxidative stress were examined. Basal expression and cellular antioxidant activity, including SOD, CAT, and GSH, were examined prior to H2O2 and ∼30nm SiO2 (0.01–100mg/L) exposures. Dose–response observations were made regarding oxidant production, cytotoxicity, GSH depletion and NRF2 transcription factor activation. Results indicated that, while both cell types exhibited susceptibility to H2O2 and SiO2-induced oxidative stress and damage, the A549 cell line was relatively more resilient.

Quantification and purification of mangiferin from Chinese Mango (Mangifera indica L.) cultivars and its protective effect on human umbilical vein endothelial cells under H(2)O(2)-induced stress.

Mangiferin is a natural xanthonoid with various biological activities. Quantification of mangiferin in fruit peel, pulp, and seed kernel was carried out in 11 Chinese mango (Mangifera indica L.) cultivars. The highest mangiferin content was found in the peel of Lvpimang (LPM) fruit (7.49 mg/g DW). Efficient purification of mangiferin from mango fruit peel was then established for the first time by combination of macroporous HPD100 resin chromatography with optimized high-speed counter-current chromatography (HSCCC). Purified mangiferin was identified by both HPLC and LC-MS, and it showed higher DPPH• free-radical scavenging capacities and ferric reducing ability of plasma (FRAP) than by l-ascorbic acid (Vc) or Trolox. In addition, it showed significant protective effects on human umbilical vein endothelial cells (HUVEC) under H2O2-induced stress. Cells treated with mangiferin resulted in significant enhanced cell survival under of H2O2 stress. Therefore, mangiferin from mango fruit provides a promising perspective for the prevention of oxidative stress-associated diseases.

SpxA1 Involved in Hydrogen Peroxide Production, Stress Tolerance and Endocarditis Virulence in Streptococcus sanguinis

Streptococcus sanguinis is one of the most common agents of infective endocarditis. Spx proteins are a group of global regulators that negatively or positively control global transcription initiation. In this study, we characterized the spxA1 gene in S. sanguinis SK36. The spxA1 null mutant displayed opaque colony morphology, reduced hydrogen peroxide (H2O2) production, and reduced antagonistic activity against Streptococcus mutans UA159 relative to the wild type strain. The ΔspxA1 mutant also demonstrated decreased tolerance to high temperature, acidic and oxidative stresses. Further analysis revealed that ΔspxA1 also exhibited a ∼5-fold reduction in competitiveness in an animal model of endocarditis. Microarray studies indicated that expression of several oxidative stress genes was downregulated in the ΔspxA1 mutant. The expression of spxB and nox was significantly decreased in the ΔspxA1 mutant compared with the wild type. These results indicate that spxA1 plays a major role in H2O2 production, stress tolerance and endocarditis virulence in S. sanguinis SK36. The second spx gene, spxA2, was also found in S. sanguinis SK36. The spxA2 null mutant was found to be defective for growth under normal conditions and showed sensitivity to high temperature, acidic and oxidative stresses.

Mitochondrial mutation impairs cytoplasmic male sterility rice in response to H2O2 stress

Cytoplasmic male sterility (CMS) is a phenomenon widely observed in various plant species characterized with disrupted anther development caused by mitochondrial mutation. CMS is becoming a model system for the investigations of nucleus–cytoplasmic interaction. To reveal the possible effects of CMS genes on plant growth in adverse environment, plant development and biochemical characters of mitochondria from Honglian (HL)-CMS line Yuetai A and maintainer Yuetai B treated with H2O2 were analyzed. Results showed that 40–60mM H2O2 significantly inhibits rice seedling development and growth. When treated with H2O2, ATP content and mitochondrial membrane potential in Yuetai A decreased significantly faster than those of Yuetai B. These biochemical changes were accompanied by the severe nuclear DNA fragmentation and the release of mitochondrial cytochrome c in the leaf cells of Yuetai A. In addition, the antioxidative enzyme activities and mitochondrial electron transfer chain complexes were significantly down-regulated. Disturbance of the biochemical indexes indicate that HL-CMS line is more susceptible to H2O2 stress than the maintainer line, the deleterious effects caused by the CMS-related ORFH79 peptide compromises the adaptability of HL-CMS line to the adverse environment.

Yap1-dependent oxidative stress response provides a link to riboflavin production in Ashbya gossypii

Ashbya gossypii is a natural overproducer of riboflavin. Overproduction of riboflavin can be induced by environmental stress, e.g. nutritional or oxidative stress. The Yap-protein family has a well-documented role in stress response. Particularly, Yap1 has a major role in directing the oxidative stress responses. The A. gossypii YAP-family consists of only three genes in contrast to its closest relative Eremothecium cymbalariae, which has four YAP-homologs. Gene order at Eremothecium YAP-loci is conserved with the reconstructed yeast ancestor. AgYap1p is unique amongst Yap-homologs as it lacks the cysteine-rich domains (CRDs). AgYAP1 expression is inducible and GFP-AgYap1 localizes to the nucleus. Agyap1 mutants displayed higher sensitivity against oxidative stress – H2O2 and menadione - and are strongly reduced in riboflavin production. High levels of cAMP, which also reduce riboflavin production, show a synergistic effect on this sensitivity. AgYAP1 and a chimera of AgYAP1 (with the DNA-binding domain) and ScYAP1 (with the CRDs) can both complement the Scyap1 oxidative stress sensitivity. This suggests that the DNA-binding sites of ScYap1 are conserved in A. gossypii. Expression of AgRIB4, which contains three putative Yap1-binding sites, assayed via a lacZ-reporter gene was strongly reduced in an Agyap1 mutant suggesting a direct involvement of AgYap1 in riboflavin production. Furthermore, our data show that application of H2O2 stress leads to an increase in riboflavin production in a Yap1-dependent manner.

New Role for the ibeA Gene in H2O2 Stress Resistance of Escherichia coli

ibeA is a virulence factor found in some extraintestinal pathogenic Escherichia coli (ExPEC) strains from the B2 phylogenetic group and particularly in newborn meningitic and avian pathogenic strains. It was shown to be involved in the invasion process of the newborn meningitic strain RS218. In a previous work, we showed that in the avian pathogenic E. coli (APEC) strain BEN2908, isolated from a colibacillosis case, ibeA was rather involved in adhesion to eukaryotic cells by modulating type 1 fimbria synthesis (M. A. Cortes et al., Infect. Immun. 76:4129-4136, 2008). In this study, we demonstrate a new role for ibeA in oxidative stress resistance. We showed that an ibeA mutant of E. coli BEN2908 was more sensitive than its wild-type counterpart to H(2)O(2) killing. This phenotype was also observed in a mutant deleted for the whole GimA genomic region carrying ibeA and might be linked to alterations in the expression of a subset of genes involved in the oxidative stress response. We also showed that RpoS expression was not altered by the ibeA deletion. Moreover, the transfer of an ibeA-expressing plasmid into an E. coli K-12 strain, expressing or not expressing type 1 fimbriae, rendered it more resistant to an H(2)O(2) challenge. Altogether, these results show that ibeA by itself is able to confer increased H(2)O(2) resistance to E. coli. This feature could partly explain the role played by ibeA in the virulence of pathogenic strains.

Stress influenced increase in phenolic content and radical scavenging capacity of Rhodotorula glutinis CCY 20-2-26.

Rhodotorula glutinis CCY 20-2-26 when grown under controlled stress of either NaCl (1–5 %) or H2O2 (1–5 mM) on basal media exhibited a twofold increase in its total phenolic contents. The radical scavenging capacities (RSCs) as determined by ABTS test were found to be highest in 4 mM H2O2 (1.44 mM TEAC mg−1) and 4 % NaCl (1.13 mM TEAC mg−1) as compared to control samples (0.41 mM TEAC mg−1). Similarly, the RSCs as determined by DPPH test were also highest in 4 % NaCl (1.83 mM TEAC mg−1) and 4 mM H2O2 (1.78 mM TEAC mg−1) compared to control (0.48 TEAC mg−1). The relative RSCs from EPR spin-trapping assay for H2O2-stressed cultures were highest in 1 mM H2O2 (56.1 μM TEAC g−1) whereas in NaCl-stressed cultures it was highest in 5 % NaCl (44.6 μM TEAC g−1) as compared to control (30.9 μM TEAC g−1). Five phenolic compounds (gallic acid, benzoic acid, catechin, caffeic acid and ferulic acid) were detected for the first time in R. glutinis CCY 20-2-26.

A Fur-like protein PerR regulates two oxidative stress response related operons dpr and metQIN in Streptococcus suis

BackgroundMetal ions are important micronutrients in cellular metabolism, but excess ions that cause toxic reactive oxygen species are harmful to cells. In bacteria, Fur family proteins such as Fur, Zur and PerR manage the iron and zinc uptake and oxidative stress responses, respectively. The single Fur-like protein (annotated as PerR) in Streptococcus suis has been demonstrated to be involved in zinc and iron uptake in previous studies, but the reports on oxidative stress response and gene regulation are limited.ResultsIn the present study, the perR gene deletion mutant ΔperR was constructed in Streptococcus suis serotype 2 strain SC-19, and the mutant strain ΔperR exhibited less sensitivity to H2O2 stress compared to the wild-type. The dpr and metQIN were found to be upregulated in the ΔperR strain compared with SC-19. Electrophoretic mobility shift assays showed that the promoters of dpr and metQIN could be bound by the PerR protein. These results suggest that dpr and metQIN are members of the PerR regulon of S. suis. dpr encodes a Dps-like peroxide resistance protein, and the dpr knockout strains (Δdpr and ΔdprΔperR) were highly sensitive to H2O2. MetQIN is a methionine transporter, and the increased utilization of methionine in the ΔperR strain indirectly affected the peroxide resistance. Using a promoter–EGFP gene fusion reporting system, we found that the PerR regulon was induced by H2O2, and the induction was modulated by metal ions. Finally, we found that the pathogenicity of the perR mutant was attenuated and easily cleared by mice.ConclusionsThese data strongly suggest that the Fur-like protein PerR directly regulates dpr and metQIN and plays a crucial role in oxidative stress response in S. suis.

Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.